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1 -18 Plenary lectures and concurrent symposia
Abstract
Virus-specific CD8+ T cell-mediated immunity will be reviewed from the aspect of CD8+ T cell effector function in different anatomical sites, and in the context of the nature and limitations of immune memory and the recall response. Particular attention will be given to recent experiments that use reverse genetics technology to explore the interaction between responses to bmajorQ and bminorQ epitopes. The relative importance of quantity versus quality of response will be considered, together with the characteristics of immunodominance hierarchies. Factors determining the nature of T cell receptor (TCR) repertoires will be explored using approaches that combine single cell PCR with mutational and structural biology analysis. We are beginning to understand the chemistry of the TCR/epitope interaction in ways that will hopefully inform both our fundamental insights and practical considerations like vaccine design. The molecular diversity of CD8+ T cell response profiles will also be discussed.
Primed individuals not only can mount secondary immune responses that are more rapid and effective than primary responses, but also maintain, in the absence of further boosting, a certain level of effector T cells and antibodies for a lifetime. These aspects of immunological memory have a distinct cellular basis. Recall responses are mediated in secondary lymphoid organs by central memory T cells and memory B cells, while immediate protection is mediated in peripheral tissues by effector memory T cells and by antibodies produced by long-lived plasma cells. I will review the experimental evidence supporting a bstem cell modelQ of immunological memory. Central memory T cells and memory B cells are intermediates of a progressive differentiation process, which have acquired the capacity to proliferate and differentiate in response to polyclonal stimuli such as cytokines, microbial products or bystander T cell help. While self renewing, central memory T cells and memory B cells continuously spill out effector T cells and plasma cells, thus replenishing those that turn over. I will describe in detail the mechanisms that sustain serum antibody levels following vaccination and discuss the implications of these findings for vaccine design.
The finding that the central nervous system (CNS) embodies neurogenetic regions enriched of neural stem cells (NSCs) has spurred a flurry of studies on both their basic biology and their perspective application for the therapy of many neurological disorders. NSCs are multipotential precursors that grow and selfrenew for extensive time in culture. It has recently been argued that NSCs undergo rapid, spontaneous transformation, which would represent an obstacle to their use both in therapeutic applications for neurological disorders as well as in basic research. In my talk, I shall present an overview of the state of the art in this field and discuss recent findings that demonstrate the extreme functional stability of NSCs, particularly of human origin, their actual capacity to act as therapeutic entities in the damaged CNS and their involvement in the formation and propagation of the most malignant adult human brain tumor, i.e., the glioblastoma multiforme.
CNS-reactive T cell clones are present in the healthy immune repertoire. They attack their target tissue only upon full activation. Invasion of the CNS by inflammatory cells occurs in two waves. Shortly after transfer, some few activated T cells pass through the intact BBB. They reside in the CNS for 72-96 h without causing disease. Then, a second, much larger wave of cells invades the CNS and apparently precipitates neurological defects and loss of weight. We have traced the migratory pathways of encephalitogenic T cells before, during and after clinical EAE in the Lewis rat. We study retrovirally transduced GFP expressing (bgreenQ) myelin specific T cells combining serial 2-photon imaging with real-time assays of gene transcription and protein expression. We find that activated encephalitogenic T cells radically re-program their gene expression pattern during a first 72 h prodromal period while sitting in peripheral immune organs, by down-regulating activation markers and upregulating chemokine receptors. After 72-96 h, large numbers of these T cells invade the CNS via the BBB. Two-photon imaging reveals that many of the cells rapidly cruises through the brain parenchyma, while the others seem to be arrested. We shall present evidence that the arrested cells recognize locally presented autoantigen. Intra-parenchymal T cell activation seems to underlie the pathogenic inflammatory response responsible for clinical EAE. Shortly after re-activation, most, if not all of the green T cells undergo apoptotic cell death. Their depletion from the CNS coincides with termination of the clinical EAE episode.
Neural and immune synaptic relations
Inflammation is a local, protective response to microbial invasion or injury. It must be fine-tuned and regulated precisely because deficiencies or excesses of the inflammatory response cause morbidity and shorten life span. The discovery that cholinergic neurons inhibit acute inflammation has qualitatively expanded our understanding of how the nervous system modulates immune responses (Tracey KJ, Nature 420: 853-859, 2002 ). The nervous system reflexively regulates the inflammatory response in real time, just as it controls heart rate and other vital functions. The vagus nerve transmits anti-inflammatory cholinergic signals to the reticuloendothelial system, inhibiting acute cytokine release during the response to injury and infection. This is a selective and reversible dhard-wiredT neural system that is a critical control point, and it can be manipulated to therapeutic advantage in animals.
Pattern recognition receptors: innate immunity, inflammation and more A. Mantovani Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy
Pentraxins are a group of proteins conserved in evolution. Long pentraxins represent a discrete subfamily, the prototype of which is the first cloned member PTX3. In addition to PTX3 other long pentraxins include neuronal pentraxin 1 and neuronal pentraxin 2, as well as the NP receptor. The prototypic long pentraxin PTX3 is a multifunctional pattern recognition receptor produced by diverse cell types and tissues, including the central and peripheral nervous system, in response to diverse signals. Evidence suggests that PTX3 plays a non redundant role in innate immunity against selected pathogens, female fertility and inflammation/ repair in the central nervous system. Progress in the definition of ligands recognized by PTX3 and in vitro as well as in vivo function will be discussed.
The role of TREM-2 receptor in myeloid cell development and function M. Colonna Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Triggering receptors expressed on myeloid cells (TREM) belong to a rapidly expanding family of receptors that activate myeloid cells by signaling through the adaptor protein DAP12. While TREM-1 triggers phagocyte secretion of proinflammatory chemokines and cytokines, TREM-2 activates monocyte-derived dendritic cells. Remarkably, TREM-2 deficiency leads to a severe disease characterized by dementia and bone cysts, which implies that under homeostatic conditions TREM-2 may also regulate the function of brain microglial cells and bone osteoclasts. To address TREM-2 function in vivo, we have generated TREM2À/À mice and compared them with DAP12À/À mice. Results demonstrate a critical function of TREM-2/DAP12 in the development of microglia and osteoclasts and in the function of dendritic cells in immune responses.
Innate immunity: neuroprotection or neurodegeneration? S. Rivest CHUL Research Center and Laval University, Quebec City, Quebec, Canada
Innate immune response is initiated by the binding of pathogen-associated molecular patterns (PAMPs) to their cognate Toll-like receptors (TLRs) expressed on the surface of antigen-presenting cells (APCs). Because of the blood-brain barrier (BBB) that restricts the access of immune mediators and cells from the blood, the mechanisms leading to proinflammatory signaling in the brain are different from those of systemic organs. For this reason, the term bimmune-privileged organQ has long been used to describe the reduced exposure of the brain to inflammatory events and its inability to mount an immune response and process antigens. Contrary to this notion, there is an elegant innate immune response that takes place in a very well organized and coordinated manner in the central nervous system (CNS) during systemic infection and cerebral insults. Like systemic APCs, brain microglia react to PAMPs in a TLR-dependent manner. Emerging evidence suggests that innate immunity plays beneficial roles in host defense and recovery, although microglial reactivity is becoming a hallmark of neurodegenerative processes. This lecture will present both sites of the story and highlight the mechanisms involved in control of the inflammatory response in the brain that either protect neurons or is the direct cause of some neurodegenerative disorders.
Activation and deactivation of regulatory T cell function E. Shevach Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Naturally occurring regulatory CD4+CD25+ T cells serve a vital function as cellular attenuators of immune responses. In most instances, suppression does not dominate raising the important question as to the nature of the factors that control the maintenance and activation of suppressor cell function. Although the main function of CD4+CD25+ T cells is to suppress IL-2 production by CD4+CD25Àeffector cells, suppression is still manifest in the presence of exogenous IL-2. Moreover, the addition of anti-IL-2 completely abrogates suppressor function. Thus, IL-2 must first be produced by the responder cells prior to initiation of suppressor cell function. Non-activated CD4+CD25+ regulatory T cells constitutively express GITR, a TNFR-family member, whose engagement was thought to abrogate regulatory T cell-mediated suppression. However, using GITRÀ/À mice, the capacity of anti-GITR to abrogate suppression could be shown to be mediated by its action on CD25À, not CD25+ T cells. Mouse antigen presenting cells constitutively express GITR-L, which is downregulated following Toll-like receptor signaling. Thus, GITR-L provides an important signal for CD25À T cells, rendering them resistant to CD25(+)-mediated regulation at the initiation of the immune response. The downregulation of GITR-L by inflammatory stimuli may enhance the susceptibility of effector T cells to suppressor activity during the course of an infectious insult. Manipulation of GITR/GITR-L interactions may prove to be an effective way of manipulating regulatory T cell function in vivo.
Regulatory T cells: a privileged target for the treatment of autoimmune diseases L. Chatenoud Hopital Necker, Paris, France Autoimmune responses are controlled by CD4+ regulatory T cells that, depending on the model, may express distinct phenotypic and functional characteristics. In the thymus these regulatory cells are CD25+ like CD4+ cells shown to control physiologic organ-specific autoimmunity. In contrast, in the periphery, both CD4+CD25+ and CD4+CD25À cells exhibit regulatory capacities. Antibodies to CD3 are potent immunosuppressants now generally applied as non Fc-receptor (FcR) binding monoclonals. Data from our laboratory were the first to show that CD3 antibodies could reverse autoimmune diabetes in NOD mice by restoring self-tolerance to b cell antigens. This led to clinical trials, presently ongoing, in recent onset type 1 diabetic patients using non Fc-receptor (FcR) binding monoclonals antibodies to CD3. Concerning mechanistic aspects CD3 antibodies promote immediate clearance of insulitis, followed by bresettingQ of specialized subsets of immunoregulatory CD4+ T cells mediating active tolerance. In treated mice regulatory T cells concentrate in the CD4+CD62L+ compartment and some share the CD25 marker, their proportion increase in pancreatic and mesenteric lymph nodes and they are TGF-beta-dependent both in vivo and in vitro. Antibodies to CD3 appear thus as the first representatives of a novel category of immunotherapeutic agents that provide a cure for established autoimmunity through their capacity of boosting immunoregulatory T cells. It is foreseeable that this proof of concept in autoimmune diabetes could be extended in the near future to other autoimmune diseases.
Vaccination against beta-amyloid in Alzheimer's disease R.M. Nitsch and C. Hock Division of Psychiatry Research, University of Zurich, Zurich, Switzerland Immunization against beta-amyloid can reduce amyloid plaque load and improve impaired behavior in transgenic mice. To determine whether antibodies against beta-amyloid are also effective in human patients with Alzheimer's disease (AD), we participated in the Wyeth/Elan AN-1792 Phase 2A clinical trial, and included 30 patients with mild to moderate dementia who received two subsequent doses of pre-aggregated Abeta42 and QS-21 as an adjuvant in a randomized, placebo-controlled study design. We observed the generation of antibodies against beta-amyloid in the patient sera, and found that the antibodies were characterized by remarkable specificity for bona fide beta-amyloid plaques in brain tissue, and by absent detectable cross-reaction with full-length APP or monomeric forms of Abeta. Therefore, we developed a tissue amyloid plaque immunoreactivity (TAPIR) assay to quantify the ability of the antibodies to react with brain beta-amyloid plaques in brain tissue of transgenic mice expressing ADcausing mutations. Patients who generated TAPIR-positive antibodies over a 1-year period, showed significantly slower rates of decline of cognitive functions and activities of daily living, and scored better on hippocampusdependent memory tests as compared to patients without such antibodies. The beneficial clinical effects were also present in patients who had experienced transient episodes of immunization-related aseptic meningoencephalitis as an unwanted side effect of immunization. These data show that the generation of antibodies against beta-amyloid in response to immunization can slow cognitive decline in patients with AD. Provided that the tolerability can be improved, they suggest that vaccination against betaamyloid is a valuable option for the treatment of AD.
Prospects and limitations in the genetic analysis of common autoimmune diseases F. Cucca a and M.G. Marrosu b a Dipartimento di Scienze Biomediche e Biotecnologie, University of Cagliari, Cagliari, Italy; b Centro Sclerosi Multipla, Dipartimento di Neuroscienze, University of Cagliari, Cagliari, Italy
The genetic analysis of multifactorial traits such as multiple sclerosis and type 1 diabetes is complicated by many factors. These include interlocus and allelic heterogeneity, interactions between different disease loci, and interactions between disease loci and environmental factors. Further complications are caused by low penetrance which may be compounded by the small individual genetic effects of most of the disease loci. Some of the aforementioned difficulties could be alleviated by concentrating our research on an isolated and relatively homogenous population such as that from the Mediterranean island of Sardinia. This population has one of the highest incidences of both type 1 diabetes and multiple sclerosis in the world. Moreover, there is evidence for the co-inheritance of these two disorders in Sardinia. This is consistent with the suggestion that there are highly penetrant gene(s) present, with a central role in general autoimmunity in this population. The Sardinians represent an ancient genetic isolate of accepted pre-Neolithic origin in which the substantial lack of population sub-structure reduces the risk of artifacts due to population admixture. The present-time Sardinian population is the result of a fixation of genetic variants which are rare or absent in other European derived populations and which are particularly useful for pinpointing the etiological polymorphisms. The results obtained from this population so far, clearly illustrate the advantages of using genetic isolates for the genetic analysis of multifactorial disorders. The relative importance of population-demographic and chromosome-region specific variables as well as the relevance of the interaction between loci in the genetic analysis of complex traits will be discussed.
Genetic analysis of multiple sclerosis: linkage and association A. Compston Department of Clinical Neurosciences, Cambridge, UK Using linkage and association, several candidate genes show some reproducibility for an effect on susceptibility, resistance, and the clinical course and phenotype but most are resolutely negative. The best supported are effects on susceptibility at HLA-DR/DQ, TNFalpha, IL-1Ra, IL-4, and CTLA4. Protection may be provided by Fas-670 and IL-12p40. The course may be influenced by polymorphisms for IL-10, CNTF, and ApoE4. The phenotype of multiple sclerosis is determined by mutations of mitochondrial DNA. The whole genome linkage and association screens suggest that effects may be encoded at 2p, 5p, 6p, 10p, 11p, 16p, 17q, 19q, 22q, and Xp. Eventually, six main categories of susceptibility gene can be predicted: genes which determine susceptibility to the process of inflammation across a range of disorders-the autoimmune genes; those which determine the specificity of that process for the development of multiple sclerosis-the ubiquitous genes; those which are relevant for the pathogenesis in isolated populations-the domestic genes; those which determine particular phenotypes-the pleotropic genes; those which determine variations in the clinical course-the modifying genes; and those which cluster to provide specifically different (heterogenous) contributions to the pathogenesis-the epistatic genes. A major part of future studies will be to resolve the question of disease heterogeneity in multiple sclerosis. The HLA complex is associated with a large number of diseases including Multiple Sclerosis. In the case of MS, there is a strong association with the DR15 haplotype. It remains uncertain exactly how this translates into risk. The commonly believed notion is that this is in some way analogous to IR genes in experimental animal models and relates to differential presentation by MHC molecules to T cells. There are however significant difficulties with this interpretation, not the least of which is that there is equal evidence for linkage through non DR 15 haplotypes for sibling pairs. We report a comprehensive analysis of MAC Class 2 typing in a thousand families with MS covering some 5000 individuals. This locus is complex being characterised by cis and trans effects and a hierarchical susceptibility. These are interpreted in terms of the known genetic analysis of other loci.
Animal models as a genetic tool to enlight multiple sclerosis complexity R. Holmdahl Medical Inflammation Research, Lund University, Lund, Sweden Inbred animals are useful for studies of the identification of genes associated with complex diseases like MS. Many but not all of the basic disease pathways leading to MS is likely to be shared with other autoimmune chronic inflammatory disease like rheumatoid arthritis (RA). We have used several different models for MS and RA to identify the underlying gene regions and genes that contribute to disease. This is in principal done by analysis of genetically segregating crosses of inbred strains with varying disease susceptibility followed by linkage of the genotypic and phenotypic information. By this method, we and others have isolated the most important gene regions and bred these into congenic strain in which each loci can be studied separately and through each contributing gene can eventually be cloned. Such congenic strains have been shown to be useful for studies on the MHC region genes but also genes outside the MHC region and some examples have been reported; the MHC class II Ab gene in the mouse and the Ncf1 gene in the rat. The Ncf1 protein is a part of the NADPH oxidase complex involved in generation of the inducible oxidative burst. The discovery of the Ncf1 polymorphism led to a new proposed pathway in which oxygen radicals modify antigen presentation and the resulting activation of autoreactive T cells. New data will be presented on the role of the Ncf1 gene in models for MS and also the importance of identifying genetic and environmental interactions to identify genes in complex diseases like MS.
Molecular mechanisms driving transendothelial migration of leukocytes across the blood-brain barrier B. Engelhardt Theodor Kocher-Institute, University of Bern, Bern, Switzerland Diapedesis of encephalitogenic T lymphocytes across the blood-brain barrier is unique. By applying intravital fluorescence microscopy that allows us to visualize the interaction of circulating encephalitogenic T lymphoblasts within the CNS microvasculature in vivo, we demonstrated that encephalitogenic T cell blasts interact with the spinal cord white matter microvasculature without rolling and that a4-integrin mediates the Gprotein independent capture and subsequently the G-protein-dependent adhesion strengthening of T cell blasts to microvascular VCAM-1. LFA-1 was found to be involved in transendothelial migration of T cell blasts across spinal cord microvessels in vivo. Endothelial ICAM-1 and ICAM-2 are both ligands for LFA-1. To define their exact role during T cell extravasation we used mouse T cells and ICAM-1À/ÀICAM-2À/À brain endothelioma cells. ICAM-1À/ÀICAM-2À/À brain endothelioma cells did not support transendothelial migration of T cells in vitro. Re-expression of different ICAM-1 mutants in the ICAM-1À/ÀICAM-2À/À endothelioma cells demonstrated that the extracellular domain of ICAM-1 suffices to support T cell adhesion while presence of the cytoplasmic tail was strictly required for TEM and Rho GTPase activation within the endothelial cells. Surprisingly, tyrosine-phosphorylation of endothelial ICAM-1 was not necessary for TEM of T cells nor for RhoGTPase activation. The cytoplasmic tail of endothelial ICAM-1-independent from tyrosine phosphorylation-is therefore essential for supporting TEM of T lymphocytes probably by activating Rho-signalling in endothelial cells. It is generally believed that this signal is required to open endothelial junctions for T cell diapedesis. At the blood-brain barrier, endothelial cells are interconnected by complex tight junctions. By serial section electron microscopy, we found that in experimental autoimmune encephalomyelitis mononuclear cells traverse cerebral microvessels by a transcellular pathway leaving the endothelial tight junctions intact whereas morphological evidence for the involvement of tight junctions in the diapedesis of mononuclear cells across the inflamed BBB could not be obtained in any case. The unique molecular mechanisms driving transendothelial migration of leukocytes across the blood-brain barrier will be discussed.
The Jun/JNK axis in regulation of neurodegeneration and neuroinflammation T. Herdegen Institute of Pharmacology, Kiel, Germany
The c-Jun N-terminal kinases (JNK) are essential mediators of the proinflammatory actions of peripheral immune cells. In striking contrast, the actions of JNKs in the immune cells of the nervous system remains to be elucidated. We have demonstrated for the first time that microglia express the JNK1, JNK2 and JNK3 isoforms in primary brain microglial cultures from neonatal mice and rats [Hidding et al., Biochem. Pharm. 65: 781-788 (2002) ]. Here, we present data on the activation and function of JNKs. Lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFalpha) and thrombin induced a rapid and lasting activation of JNKs, which are not activated in resting microglia. Stimulation with LPS increased the total amount of nuclear JNKs, whereas the pool of activated JNKs rapidly decreased, however, the phosphorylation of the high affinity substrate and transcription factor c-Jun was dramatically enhanced. Analysis of nuclear JNK isoforms revealed that the amount of JNK1 declined, while JNK2 increased and JNK3 did not vary. This observation suggests that JNK2 is mainly responsible for the activation of transcription factors. Upstream of JNKs, LPS induced a lasting activation of the constitutively present MKK4.
Interleukin signaling pathways and role in autoimmune inflammation of the CNS C.L. Langrish, Y. Chen and D.J. Cua DNAX Research Inc., Palo Alto, CA, USA Interleukin-23 is a heterodimeric cytokine comprised of a unique p19 subunit and a common p40 subunit shared with IL-12. IL-23 signals through a receptor complex consisting of IL-12R-beta-1 and the newly discovered IL-23R which activates Jak-stat DNA-binding complexes. Engagement of IL-23 receptor complex is required for the expansion of a highly encephalitogenic T cell population that produces IL-6, IL-17, and TNF but not IFN-gamma. Thus, IL-23 plays an important role in the development and expansion of an alternative T cell subset-distinct from the classical Th1-and Th2-type responses, that are critical for the pathogenesis of CNS autoimmune inflammation.
Detrimental and beneficial cytokine signaling in Alzheimer's disease M.P. Mattson Laboratory of Neurosciences, National Institute on Aging IRP, Baltimore, MD, USA Alzheimer's disease (AD) is a progressive neurodegenerative disorder in which synapses become dysfunctional, neurites degenerate and neurons die resulting in memory impairment and psychiatric problems. Excessive production and accumulation of cytotoxic forms of amyloid beta peptide (Abeta) is believed to be a pivotal abnormality in AD. Deposits of Abeta are associated with increased levels of pro-inflammatory cytokines and activated microglial cells and astrocytes, and Abeta induces the production of the same cytokines in cultured microglia and astrocytes. The mechanisms responsible for the inflammatory responses involve increased oxidative stress and perturbed cellular calcium homeostasis. Data suggest that cytokines may either promote or protect against neuronal degeneration, depending upon the cell type and signaling pathway(s) they activate. For example, tumor necrosis factor (TNF) can activate receptors on neurons that are coupled to the activation of the transcription factor NF-kB which activates genes that encode proteins that can prevent neuronal death. On the other hand, TNF stimulates microglia to produce damaging reactive oxygen species (nitric oxide) and excitotoxins. Studies of transgenic mice and cultured neural cells that express mutant forms of amyloid precursor protein and presenilin-1 that cause inherited forms of AD have shown that the mutations result in enhanced production of TNF, IL-1beta and IL-6 in response to immune challenge. Thus, activation of innate immune responses may contribute to the demise of neurons in AD and drugs that suppress the damaging cytokine cascades may have therapeutic benefit. On the other hand, recent studies have demonstrated striking beneficial effects of immunization of bAD miceQ with Abeta, suggesting that it may be possible to prevent AD by stimulating a humoral immune response to Abeta. We developed an infection-induced molecular mimicry model of CNS autoimmune disease induction by engineering a non-pathogenic variant of Theiler's virus (TMEV) to encode a 30-39mer peptides encompassing the immunodominant myelin PLP139-151 epitope or epitope mimics. SJL mice infected with TMEV encoding either the native PLP139-151 determinant or a molecular mimic, H. influenzae protease IV (HI574-586) which shares only 6/13 amino acids, develop an earlyonset demyelinating disease mediated by PLP139-151-specific Th1 cells. The autoimmune nature of the disease was shown by the fact that tolerance to either the native mouse PLP139-151 epitope or the HI574-586 mimic epitope prevented clinical disease and induction of associated autoreactive PLP139-151-specific Th1 responses. Disease and PLP139-151-specific cross-reactive responses could also be induced by infection with a virus engineered to express a 39-mer peptide encompassing the HI574-586 mimic epitope (HI39-TMEV) indicating that the mimic peptide is a natural mimic epitope capable of being processed from its flanking residues. However, mice immunized multiple times with the core HI574-586 core peptide in complete Freund's adjuvant (CFA) exhibited no clinical disease symptoms due to failure to secrete high levels of IFN-gamma in response to in vitro re-challenge with PLP139-151 indicating the importance of virus-induced innate immune signals in triggering autoimmune disease via molecular mimicry.
Viral infections and autoimmunity-a double-edged sword M. von Herrath La Jolla Institute for Allergy and Immunology, San Diego, USA Intuitively one tends to associate viral infections that frequently induce profound inflammation in multiple organs with enhancement of autoimmune processes and diseases such as type 1 diabetes and multiple sclerosis. There is substantial evidence that viral infections can increase the incidence of autoimmune diseases (i.e., NOD mouse and Coxsackie B3 infection) or precipitate inflammatory disorders with autoimmune components (Theiler's virus). This can occur either by antigennonspecific bystander events or through cross-reactivity between viral and self-components. We will show data for the second scenario (mimicry) and illustrate that molecular mimicry is more likely to enhance an already ongoing autoimmune process rather than precipitating disease de novo. However, recently, it has become increasingly clear that the opposite situation, prevention of autoimmunity by viral infections, might be more common. There is accumulating evidence that viruses such as Coxsackie B or LCMV can prevent diabetes. How can viral infections be good for us? We will show recent findings that demonstrate how chemokine gradients established by viral infections can lure autoaggressors away from the site of organ or tissue inflammation. The reversal of disease appears in most cases to be permanent. This finding fits with the recent increasing trend in autoimmune diseases, as well as asthma, in industrialized nations (bhygiene hypothesisQ).
Coronavirus-induced immunopathology M. Buchmeier a and J. Rempel b a The Scripps Research Institute, La Jolla, CA, USA; b University of Manitoba, Winnipeg, Canada Differences in disease outcome between the highly neurovirulent MHV-JHM and mildly neurovirulent MHV-A59 have been attributed to variations within the spike (S) glycoprotein. We found that MHV-JHM neurovirulence was marked by diminished expression of IFNgamma mRNA and recruitment of CD8 T cells into the CNS concomitant with heightened MIP-1 transcript levels and greater macrophage infiltration relative to MHV-A59 infection. Here, the ability of the S and non-spike genes to regulate these immune responses was evaluated using chimeric viruses. Chimeric viruses, WTR13 and S4R22, were made on MHV-A59 variant backgrounds and, respectively, contained the S gene of MHV-A59 and MHV-JHM. Unexpectedly, genes other than S appeared to modulate events critical to viral replication and survival. Unlike unresolving MHV-JHM infections, the clearance of MHV-A59, WTR13 and S4R22 infections coincided with strong IFNgamma transcription and increased CD8 T cells infiltration. Even in the absence of viral replication, approximately 40% of S4R22 infected mice succumbed within 3 weeks indicating that the enhanced mortality following S4R22 infection was not associated with viral fitness. Rather, similar to the MHV-JHM infection, reduced survival following S4R22 infection was observed in the presence of elevated MIP-1alpha and MIP-1beta mRNA accumulation and enhanced macrophage numbers within infected brains. These observations suggest that the S protein of MHV-JHM influences neurovirulence through the induction of MIP-1alpha and MIP-1beta driven macrophage immunopathology. Supported by NIH grants AI43103 and AI25913. HHV-6 is a b-herpesvirus characterized by a distinct neurotropism, which has been implicated in various neurologic disorders, including seizures, meningitis, encephalitis, and multiple sclerosis. We previously demonstrated that the cellular receptor for HHV-6 is CD46, a ubiquitous complement-regulatory protein that prevents the cytolytic attack of autologous complement and that also serves as a receptor for other human infectious agents. Evidence suggests that the viral use of CD46 does not merely represent a cell surface anchoring mechanism, but may also have profound implications for pathogenesis. We developed a novel in vitro experimental model to investigate the mechanisms of HHV-6-mediated neuropathology. Long-term cultures of primary neural stem cells derived from human fetal brain were differentiated and studied for their susceptibility to infection by HHV-6 and subsequent complement-mediated damage. These cultures typically consist of astrocytes (80-85%), neurons (15-20%), and oligodendrocytes (b1%). Productive infection was detected with HHV-6 A, but not with HHV-6 B, as determined by quantitativecalibrated real-time PCR, and was associated with giant multinucleated cell formation. Expression of HHV-6 antigens was observed in both astrocytes and neurons, although infection was associated with a loss of typical lineage-associated markers (GFAP for astrocytes, beta-tubulin for neurons). Treatment of neural cells with purified HHV-6 virions or anti-CD46 antibodies followed by exposure to human complement resulted in a massive deposition of opsonic C3 fragments, as detected by confocal microscopy, and widespread cell death. These results suggest that the cytopathic effects associated with HHV-6 replication in the CNS may be amplified by the direct engagement of CD46, which results in a dramatic impairment of its complement-regulatory function.
Self representation in the thymus: an extended view B. Kyewski German Cancer Research Center, Heidelberg, Germany
The prevailing notion that tolerance to peripheral self-antigens is solely covered by post-thymic mechanisms has been revised by the recent finding that tissue-specific antigens are expressed by thymic stromal cells, a phenomenon termed promiscuous gene expression. Promiscuous expression of tissue-antigens in the thymus is a property of thymic epithelial cells rather than bone marrow-derived antigen presenting cells, implying cell-type specific regulation. Thymic medullary epithelial cells express a unique range of tissue antigens at the mRNA level when compared to other antigen presenting cells or epithelial cells of other tissues. This set of genes (N400) includes many tissue-specific antigens, clinically relevant auto-antigens, and cancer-germ line genes without sharing apparent structural or functional commonalities. Antigen expression, when restricted to this cell type, is sufficient to confer specific T-cell tolerance attesting to its functional role. While the overall contribution of promiscuous gene expression to central tolerance induction can be inferred from the range and level of self-antigens expressed in this manner, the tolerance state for each antigen has to be analyzed individually. Implications of these results for the regulation of promiscuous gene expression and the scope of central tolerance will be discussed.
Making and breaking tolerance to myelin basic protein J. Goverman a , S. Cabbage a , T. Brabb a , E.S. Huseby b and D. Liggitt a a University of Washington, Seattle, WA, USA; b National Jewish Medical Centre, Denver, CO, USA
The activation of immune responses to myelin antigens is believed to be one of the early steps in the pathogenesis of multiple sclerosis. This process has been extensively studied using the animal model experimental allergic encephalomyelitis (EAE), which is induced by immunization with myelin antigen in complete Freund's adjuvant. The ability to trigger myelin-specific T cells in the periphery indicates that these T cells escaped immune tolerance mechanisms that normally eliminate self-reactive T cells. Our interests focus on defining the role of tolerance mechanisms in shaping the repertoire of peripheral myelin-specific T cells and on determining how a breakdown of these mechanisms contributes to CNS autoimmunity. To pursue these studies, our laboratory generated T cell receptor transgenic models of CD4+ T cell responses to different epitopes of myelin basic protein (MBP). We found that clonal deletion in the thymus was an important tolerance mechanism only for T cells undergoing high avidity interactions with their MBP epitope on antigen-presenting cells while T cells undergoing low avidity interactions with their MBP epitope escaped deletion. Our results also showed that regulatory T cells can prevent autoimmunity mediated by both high and low avidity MBP-specific T cells. How regulatory T cells function in both the CNS and the periphery will be discussed.
Expression of myelin antigens in the thymus: a little goes a long way V.K. Kuchroo, Z. Illes, A. Anderson and J. Reddy Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Using Class II PLP 139-151/IAs tetramers, we have determined the frequency of PLP 139-151 reactive T cells in the naRve repertoire of the SJL mice. Although frequency varies with age, approximately 0.5% of CD4+ cells in the peripheral repertoire of SJL mice bind specifically to the tetramer. The EAE-resistant B10.S mice, also show a similar frequency of the PLP 139-151 reactive T cells in the peripheral repertoire but these cells cannot be expanded following specific immunization. We now find that while the tetramer reactive T cells in the naRve peripheral repertoire of the SJL mice are in the CD4+, CD25À compartment, the vast majority of PLP 139-151 tetramer reactive cells in the resistant B10.S mice are in the CD4+, CD25+ gate. This may be one of the reasons why the PLP 139-151-specific T cells cannot be expanded in the resistant B10.S mice. Deletion of CD25+ T cell, prior to immunization with PLP 139-151 results in expansion of T cells and induction of disease in the EAE-resistant B10.S mice. Preliminary data suggest that expression of myelin PLP vs. DM20 isoform in the thymus and peripheral lymphoid tissue may play a key role in affecting susceptibility and resistance to EAE by affecting the generation of regulatory T cells.
Antigen recognition patterns of autoreactive T cells in multiple sclerosis R. Martin Neuroimmunology Branch, NINDS, NIH, Bethesda, MD, USA MS is considered a T cell-mediated autoimmune disease with central nervous system (CNS) inflammation, demyelination and varying degrees of axonal damage. While the effector mechanisms of CNS damage include antibodies, complement, cytokines, CD8+ T cell lysis and others as well as factors intrinsic to CNS tissue, e.g., vulnerability to insult, most evidence indicates that CD4+ T cells induce and perpetuate of the disease. The specificity spectrum of autoreactive CD4+ T cells in MS for self-and foreign antigens is not completely understood, but a number of myelin antigens are among the targets. We have recently addressed two questions related to the biological role of autoreactive CD4+ T cells in MS: (1) do the specificity and function of high avidity myelin-specific T cells in MS differ from controls, and is there a correlation between specificity and clinical phenotype? (2) Can we identify the specificity of CSF-infiltrating T cells in an unbiased way using combinatorial peptide chemistry as well as expression libraries employing cDNAs derived from MS brain tissue? Both projects have lead to numerous novel observations. In brief, high avidity myelin-specific T cells are enriched in MS patients, are more frequently of Th1 phenotype, and specific myelin peptides clearly are more relevant than others. The specificity and phenotype of CD4+ myelin-specific T cells is related to clinical phenotypes. Furthermore, our unbiased approaches identified viral antigens as triggers for CSFinfiltrating, clonally expanded T cells. CSF-infiltrating T cells recognize a number of different self antigens, among them brain-specific as well as ubiquitous autoantigens.
Oligodendrogenesis and neurogenesis in multiple sclerosis brain A. Chang, S.M. Staugaitis and B.D. Trapp The Cleveland Clinic Foundation, Cleveland, OH, USA Multiple Sclerosis (MS) is an inflammatory disease of the CNS that destroys myelin, oligodendrocytes and axons. We and others have described the generation of new oligodendrocytes in chronically demyelinated MS lesions. Here, we present evidence that the MS brain is also generating new neurons. MAP2 immunohistochemistry highlights a population of cells with mature neuronal morphology in the subcortical and deep white matter of the human brain. These cells have pyramidal or fusiform cell bodies and one to three major processes that branch several times. Their cell bodies measure up to 25 mm and their processes, up to 5 mm long. MAP2-positive neurons are seen throughout normal white matter of control and MS cerebral hemispheres. Cells with neuronal morphologies are also PSA-NCAM-positive and some white matter neurons express neuronal NOS and somatostatin. All white matter neurons are NeuN-positive. When compared to normal appearing white matter, the number of white matter neurons is significantly decreased within MS plaques. NeuN-positive neurons, however, are increased around MS plaques. Interestingly, bipolar NeuN and PSA-NCAMpositive cells are also detected in the subventricular zone (SVZ) of MS lesions but not in the SVZ of aged-matched control brains. The identification of PSA-NCAM/NeuN-positive cells with an immature morphology in the SVZ and an increase in NeuN-positive cells at the lesion periphery raises the possibility that there is neurogenesis within demyelinated lesions of MS. Numerous oligodendrocyte precursor cells and premyelinating oligodendrocytes could be preserved within multiple sclerosis lesions attesting that, in this disease, myelin repair is not only limited by the depletion of oligodendroglial cells. Neurotrophic factors could be of therapeutical interest in MS as they might promote either oligodendrocyte survival, myelin formation or axonal protection. Using a recently developed index of myelination, we have shown that synthetic neuroprotective sigma agonists could strongly enhance myelination. We further analyzed whether some endogenous neurotrophic factors could also stimulate myelin formation. We found that CNTF-related factors activating LIFR/gp130 receptors are able to stimulate myelination via the activation of the JAK/ STAT pathway. In addition CNTF-related cytokines are also able to promote oligodendrocyte survival by the activation of a distinct signaling pathway involving the kinases PI3-K and AKT. This pathway appears to be crucial in regulating oligodendrocyte death and survival as most of the factors aimed at protecting oligodendroglial cells from apoptosis (either neurotrophic factors or antioxydant molecules) require the direct or indirect activation of PI3-K. We have further analyzed the oligodendroglial expression of two G-protein coupled receptor named Edg2/LPA1 and Edg-8/S1P5 activated respectively by lysophosphatidic acid and sphingosine-1-phosphate. These receptors are specifically expressed by oligodendrocytes in the post-natal brain, and we have observed that their activation could enhance survival of mature oligodendrocyte by activating the PI3-K/AKT pathway. Therefore this new family of receptors might also be of therapeutical interest in MS.
The role of inflammation in creating a pro-remyelination signalling cascade M.R. Kotter a , W.-W. Li a , F. Molina-Holgado a , C. Zhao a , A. Setzu a,b , C. ffrench-Constant b and R.J. Franklin a a Cambridge Centre for Brain Repair, Department of Vetenirary Medicine, University of Cambridge, Cambridge, UK; b Cambridge Centre for Brain Repair, Department of Vetenirary Pathology, University of Cambridge, Cambridge, UK Remyelination is one the few spontaneous regenerative process in the adult mammalian CNS, restoring saltatory conduction to demyelinated axons and potentially exerting an axon-protective effect. This process is mediated by oligodendrocyte precursor cells (OPCs), recruited to areas of demyelination and differentiating into myelinating oligodendrocytes. The environmental factors that orchestrate this process have attracted much recent interest. A picture is emerging of a complex matrix of factors that changes with time, initially promoting OPC recruitment and then inducing differentiation. Several studies indicate that the inflammatory response associated with demyelination is critical for triggering a sequence of events involving cross-talk between inflammatory cells and reactive astrocytes that lead to the creation of a pro-remyelination environment. These studies have mainly used toxin-models of demyelination, where inflammation is a result of oligodendrocyte injury and not its primary cause. For example, slow remyelination that occurs in old adult animals is associated with a delayed inflammatory response and intrinsic changes in the properties of ageing microglia. Furthermore, suppressing the macrophage response to toxin-induced demyelination by monocyte depletion or using the microglia activation inhibitor minocycline lead to an impaired OPC response and impaired remyelination. Conversely, promoting an inflammatory response substantially enhances myelination by OPCs injected into the adult rat retina. In acute experimental autoimmune encephalitis (EAE), Th2/3 cell-derived signals may not only downregulate the immune response but also induce central nervous system (CNS) repair. Remyelination is initiated by recruitment and differentiation of oligodendrocyte precursor cells (OPC) in demyelinated CNS lesions. Our data show that the Th2/3 cytokine transforming growth factor (TGF)-beta induces microglia to secrete a chemotactic factor for OPC, identified here as hepatocyte growth factor (HGF). Gene microarray analysis and functional studies show that (i) TGF-beta-1-2-3 and also interferon (IFN)-beta, a cytokine used to treat multiple sclerosis (MS) patients, induce HGF secretion by microglia and (ii) antibodies to the HGF receptor cMET abrogate OPC chemotaxis induced by TGF-beta-2-treated microglia. In EAE, HGF expression is seen in macrophages and microglia in the recovery phase of EAE, but not in the acute stage of disease. Taken collectively, the Th2/3 cytokine TGFbeta may play a pivotal role in remyelination by inducing microglia to release HGF which is both a chemotactic and differentiation factor for OPC.
Antibodies at the neuromuscular junction-effects on gene expression S. Berrih-Aknin CNRS UMR 8078, UPS, IPSC, Hopital Marie Lannelongue, Le Plessis Robinson, France
Myasthenia gravis (MG) is a neuromuscular disorder mediated by antibodies directed against the acetylcholine receptor (AChR) found in about 85% of patients (SPMG). The remaining 15% patients have either autoantibodies to Musk or to a not yet defined antigen (SNMG). To analyze the molecular mechanisms involved at the muscular target site, we studied the expression of selected genes by quantitative PCR, in muscle biopsies from MG patients. We found that the levels of AChR subunit transcripts were increased in severely affected MG patients, independently of the level of anti-AChR antibodies. We also observed an upregulation of utrophin, ErbB2, ErbB3, ErbB4, and Musk while rapsyn and MCK were unchanged in most patients. Interestingly, these changes were similarly observed in SNMG and SNPG patients. To complete these results with an overview of the events occurring in the patient's muscle, we compared the transcriptome of muscles from SNMG and SPMG to a reference normal muscle. In both groups of patients, the subcategories of upregulated genes were essentially involved in muscle development and in chaperone activity, while that of down regulated genes was extracellular matrix category. Altogether, these data suggest the existence of a compensatory mechanism regulating the expression of several genes involved in the neuromuscular junction. Most dysregulated genes are common between SNMG and SNPG patients, suggesting common mechanisms of autoimmune attack in these groups of patients.
Autoantibodies and the autonomic nervous system S. Vernino Mayo Clinic, Rochester, MN, USA Many cases of acute or subacute peripheral autonomic failure are associated with neurological autoimmunity. Antibodies specific for ganglionic acetylcholine receptors (nicotinic receptors that mediate fast synaptic transmission in autonomic ganglia) are found in about 50% of patients with idiopathic autoimmune autonomic neuropathy. Seropositive patients have a characteristic clinical profile with prominent parasympathetic failure (dry eyes, dry mouth and impaired pupillary responses), impaired bowel and bladder motility and orthostatic hypotension. Antibody levels correlate with severity of clinical symptoms, and the sera from some patients inhibit binding of agonist to the ganglionic receptor in vitro. In addition, a clinical response to intravenous immunoglobulin or plasma exchange and evidence in experimental animal models indicate that ganglionic AChR antibodies are a direct cause of autonomic failure. Other cases of autoimmune autonomic failure or severe gastrointestinal dysmotility occur in association with small-cell lung carcinoma (paraneoplastic autonomic neuropathy). These patients may have one or more serum autoantibody markers including ANNA-1 (anti-Hu) and may manifest symptoms of other paraneoplastic neurological disorders.
Paraneoplastic antibodies-Novel targets J. Dalmau University of Pennsylvania, Philadelphia, PA, USA It has been 40 years since the first antibody to neuronal antigens was identified in the serum of patients with paraneoplastic neurologic disorders (PND). Since then, additional antibodies have been reported in association with PND, 12 of them currently considered clinically relevant markers of paraneoplasia (PND-Ab). The ability to test for these markers and an increased awareness of clinicians for PND is resulting in early recognition and management of these disorders with preliminary evidence that this is impacting the neurologic outcome. On a more basic level, PND-Ab are used to isolate the target antigens that often play critical roles in neuronal biology and neurogenesis. Overall,~50% of patients with PND of the CNS harbor one or more of the 12 wellcharacterized PND-Ab. Among the other 50% of patients, many show signs of inflammation in the CNS or CSF suggesting immune mechanisms. Several strategies that have been applied to these patients have lead to the identification of novel PND-Ab. These include single case observations (i.e., antibodies to Zic4); identification of groups of patients with similar neuro-oncologic/immunologic findings (i.e., antibodies to Ma proteins), or grouping patients with similar syndromes that have previously been considered PND-Ab negative. In the latter group, changes in tissue processing or probing techniques (i.e., immunoprecipitation or modified cDNA library screening) have uncovered novel immunities and the identity of the target antigens, some of which will be discussed.
Antibodies in demyelinating diseases C. Linington Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK There is increasing evidence that antibody-dependent mechanisms are involved in the pathogenesis of demyelination in multiple sclerosis, but the identity of the antigens targeted by this pathogenic response remains uncertain. Studies using myelin oligodendrocyte glycoprotein (MOG) as a model antigen reveal that antibody-mediated demyelination must be viewed as an epitope-, rather than antigen-specific phenomenon. The demyelinating antibody response to MOG selectively targets discontinuous MOG epitopes, whilst the responses to linear MOG epitopes are generally benign. The solution of the crystal structure of MOG and its complex with a Fab fragment of a demyelinating antibody demonstrates the importance of structural constraints in determining the balance between autoaggression and benign autoimmunity, which is further influenced by genetic and environmental factors. These results will be discussed with respect to the relevance of MOG as a target for antibody mediated demyelination in human disease, and the use of animal models and proteomic/bioinformatic-based methodologies to identify pathophysiologically relevant autoantibody responses to novel autoantigens of low physical abundance in MS.
Gangliosides as autoantibody targets at the neuromuscular junction in immune-mediated neuropathies H. Willison University of Glasgow, Glasgow, Scotland Progress in our understanding of the Guillain-Barré syndromes has been greatly accelerated by the identification of humoral responses to glycolipids in a significant proportion of cases, and the identification of molecular mimicry with preceding infections as a key mechanism of disease induction. Using in vivo and ex vivo mouse neuromuscular junction preparations as a model system to test for effects of antiglycolipid antibodies at nerve terminals, we have shown complementdependent, alpha latrotoxin-like effects comprising a block of evoked ACh release and destruction of the terminal. Studies on GM2/GD2 synthase and GD3 synthase null-mutant mice that lack different gangliosides provide insights into the role of ganglioside composition and density in neuronal membranes that predisposes them to injury. The terminal attack complex of complement forms membrane pores that allow free ionic movement, and is critical to the development of the structural disorganisation of the nerve terminal. This talk will review our current understanding of this complex field and address new approaches to testing novel treatments for these debilitating diseases.
Molecular mimicry and disease model of Guillain-Barré syndrome N. Yuki Department of Neurology, Dokkyo University School of Medicine, Tochigi, Osaka, Japan Molecular mimicry between microbial and self components is postulated as the mechanism that accounts for the antigen and tissue specificity of immune responses in post-infectious autoimmune diseases. Little direct evidence exists, and research in this area has focused principally on T cell-mediated, anti-peptide responses, rather than on humoral responses to carbohydrate structures. Guillain-Barré syndrome, the most frequent cause of acute neuromuscular paralysis, occurs 1 to 2 weeks after various bacterial and viral infections, particularly Campylobacter jejuni enteritis. Carbohydrate mimicry between the bacterial lipo-oligosaccharide and human GM1 ganglioside is seen as having relevance to the pathogenesis of Guillain-Barré syndrome, and conclusive evidence is reported here. On sensitization with C. jejuni lipo-oligosaccharide, rabbits developed anti-GM1 IgG antibody and flaccid limb weakness. Paralyzed rabbits had pathological changes in their peripheral nerves identical to those present in Guillain-Barré syndrome. Immunization of mice with the lipooligosaccharide generated a monoclonal antibody that reacted with GM1 and bound to human peripheral nerves. The monoclonal antibody and anti-GM1 IgG from patients with Guillain-Barré syndrome blocked muscle action potentials in a muscle-spinal cord co-culture, indicative that anti-GM1 antibody can cause muscle weakness. These findings show that carbohydrate mimicry is an important cause of autoimmune neuropathy.
Cytokines and T-cell immunity in inflammatory neuropathies J. Zhu Karolinska Institute, Stockholm, Sweden Acute inflammatory demyelinating polyneuropathy (Guillain-Barré syndrome, GBS) and its animal model experimental autoimmune neuritis (EAN) are prototypes of T cell mediated autoimmune diseases affecting the peripheral nervous system (PNS). Perivascular accumulation of T lymphocytes and macrophages in the PNS, and high levels systemically of PNS myelin antigen-reactive T cells are characteristic features of both diseases, thereby suggesting a pathogenic role for specific T cell and immunoregulatory cytokines. Apoptosis of autoreactive myelin specific T cells may be an important mechanism to limit the immunoinflammatory response and to abort disease. Th1/Th2/Th3 cytokines are differently upregulated during various clinical phases of EAN and GBS. However, the role of cytokines in immune regulation and autoimmune inflammatory neuropathies is more complex than a simple Th1-Th2 dichotomy would suggest. New treatments may be searched for that augments T cell apoptosis and inhibits T cell responses towards peripheral myelin and that counteract this complex cytokine imbalance. Treatments with antibodies that selectively target certain proinflammatory cytokines, as well as with immunomodulatory preparations that promote cytokines that beneficially influence the disease course should be in focus of future therapeutic trials.
The Rockefeller University, New York, NY, USA Dendritic cells (DCs) are a distinct lineage of hematopoietic cells that control several facets of immunity and tolerance. It is now feasible to directly study and modify a major reservoir of DCs in peripheral lymphoid tissues of mice. To do so, antigens are incorporated into antibodies that selectively target to DCs, currently antibodies to an endocytic receptor, DEC-205/CD205. With these engineered antibodies, there is greatly enhanced presentation in vivo on MHC class I and II products. Simultaneously, it is important to control DC maturation state. Appropriate stimuli can differentiate DCs and render them immunogenic, whereas in the steady state, most DCs are immature and can induce peripheral tolerance. These in vivo antigen targeting strategies are now being pursued to better control the induction of tolerance to autoantigens, and the induction of immunity to microbial and tumor antigens. DC function can also be studied with ex vivo approaches. A broadly recognized mechanism for peripheral tolerance induction involves regulatory or suppressor T cells. It has become apparent that DCs induce and/or expand these regulators, including CD4+ CD25+ suppressor T cells. We find that DCs serve as presenting cells to expand antigen-specific suppressors ex vivo, and that the expanded cells efficiently control the development of diabetes in NOD mice. Although this field is just beginning, we would like to suggest that more direct consideration be given to DCs in the design of therapies for autoimmune disease.
Traffic of antigen presenting cells to and from the brain T. Newman, R. Carare-Nnadi, M. Bernades-Silva, R. Weller and V.H. Perry The University of Southampton, Southampton, UK
In the normal healthy rodent brain, there are no dendritic cells. However, following acute brain injury, and in immune mediated lesions, small numbers of OX62+ve dendritic cells appear in the rat brain parenchyma. To investigate the origin of these cells, rats with acute excitotoxic lesions were subjected to a number of different manipulations. Depletion of the ED2+ perivascular macrophages by intracerebroventricular injection of chlodronate liposomes did not prevent the appearance of OX62+ve cells in the parenchyma, but bone marrow depletion by whole body irradiation abrogated their appearance. Thus, the OX62+ve cells are derived from blood and not transformation of resident cell populations. To investigate whether potential antigen presenting cells in the brain parenchyma, such as the perivascular cells, traffic from brain to the peripheral immune system fluorescent microspheres approximating the size of a virus (0.02 micrometre) or a bacterium (1.00 micrometre) were microinjected into distinct brain compartments. Injection into the meninges or ventricles led to the appearance of the fluorescent microspheres in the cervical lymph nodes and the liver. In contrast no fluorescent microspheres were found peripherally following injections restricted to the parenchyma. The microspheres were observed within perivascular macrophages. A local inflammatory response within the brain resulted in a dramatic alteration of the distribution of microspheres delivered to the parenchyma.
On the function of dendritic cells in CNS disease T. Suter a , G. Biollaz a , D. Gatto a , L. Bernasconi a , C. Cretton a , T. Herren a , W. Reith b and A. Fontana a a University Hospital, Zurich, Switzerland; b University of Geneva Medical School, Geneva, Switzerland Dendritic cells (DC) are unique in their ability to prime naive T cells and initiate adaptive immunity. In recent years, DC were identified in the inflamed central nervous system (CNS), but their role in the initiation or regulation of the tissue specific immune response is unknown. We report that DC isolated from mice with experimental autoimmune encephalomyelitis (EAE) exhibit a maturational phenotype similar to immature bone marrow-derived DC or splenic DC as characterized by intermediate surface MHC class II and low expression of the costimulatory molecule CD80. However, they are unable to prime naive T cells. Moreover, they inhibit T cell proliferation stimulated by mature bone marrow-derived DC. TGF-beta, IL-10 and TRAIL were found to significantly contribute to the CNS-DC-mediated inhibition of T cell proliferation. Thus, CNS-DC may be the key responsible for maintaining immune privilege within the inflamed CNS. The molecular basis of cell-cell interactions that regulate the function and activation of myeloid lineage cells is now coming into focus. Foremost are activating receptors, such as the TREM family, usually associated with the ITAM-expressing DNAX Adapter Protein (DAP)-12, that signals cellular activation, while inhibitory receptors commonly express ITIMs that trigger cellular inhibitory pathways. OX2 (CD200) is a very broadly expressed Ig-superfamily molecule, including neurons of the CNS. CD200 has a very short cytoplamic tail devoid of any obvious signaling domains. Gene targeting of CD200 resulted in enhanced macrophage and microglial activation in situ, suggesting that CD200 may be a ligand that normally engages an inhibitory receptor on myeloid cells. The CD200 receptor (CD200R) has been cloned, extensively characterized, and shown to transduce potent inhibitory activities in a range of myeloid cells in vitro and in vivo despite the absence of a classic ITIM. Fresh insights into the role of CD200R in regulating microglial cell function will be described as will data from gene expression profiling of adult microglial cells and other brain macrophages. These studies have revealed a number of molecules, but especially certain activating receptors that characterize a unique molecular fingerprint for microglial cells within the adult CNS. That these molecules normally regulate microglial cells in situ is likely.
Effect of GM-CSF on microglial cells L. Santambrogio Albert Einstein College of Medicine, New York, NY, USA Microglial cells derive from bone marrow precursors which colonize the brain parenchyma. They have important functions in CNS inflammatory, degenerative, traumatic conditions as well as in brain homeostasis. In contrast with other organs where terminally differentiated dendritic cells and macrophages are the resident population, the rather undifferentiated state of microglial cells in the brain could contribute to its bimmunoprivileged stateQ. A global gene expression analysis on the extent and consequences of GM-CSF and M-CSF treatment in microglial cells revealed substantial expression reprogramming upon cytokine stimulation. Primary murine microglial cells stimulated with M-CSF-induced transcription of genes involved in tissue organization and remodeling, maintenance and regulation of brain homeostasis. On the other handed, GM-CSF stimulation induced transcription of genes important in antigen processing, T cell stimulation, chemotaxis, innate immunity and immunosuppression. This GM-CSF promotion of microglial participation in adaptive immune responses is distinct from its known function in cell proliferation and survival.
Non-neuronal cells in ALS neurodegeneration: mechanisms and therapies J.D. Rothstein Johns Hopkins University, Department of Neurology, Baltimore, MD, USA Amyotrophic lateral sclerosis is an adult onset neurodegenerative disease characterized clinically by muscle wasting and weakness. Traditionally, the disease is characterized by loss of cortical and spinal motor neurons. However, recent studies have strongly suggested that disease progression and death of motor neurons depends upon participation of non-neuronal cells including focal inflammatory responses and dysfunction of astroglia. Chimeric animal studies and multiple transgenic mouse studies provide evidence for the critical participation of non-neuronal cells in the ultimate degeneration of spinal motor neurons. In addition, human and animal model studies demonstrate multiple abnormalities in astroglial function in ALS. These abnormalities are detectable even in astroglia and neuronal stem cells derived from transgenic models. Using these data, attempts at therapies include the use of neuronal and astroglial stem cells and successful engraftment of motor neuron stem cells as well as astroglial stem cells have been achieved. Finally, pharmacological means to modulate CNS inflammation (e.g., COX2 inhibition) and astroglial dysfunction (e.g., glutamate transporter enhancement) will be presented in both animal models and human clinical trials.
Activation of complement in the central nervous system: roles in neurodegeneration and neuroprotection P. Gasque Department of Medical Biochemistry, Brain Inflammation and Immunity Group, University of Wales College of Medicine, Cardiff, UK
The vertebrate brain has developed its own innate defence and clearance systems against pathogens and apoptotic cells based upon the activation of resident glia. The complement inflammatory cascade is an essential component of the phylogenetically ancient innate immune response and is crucial to our natural ability to ward off infection. For instance, complement is involved in host defence by triggering the generation of a membranolytic complex at the surface of the pathogen. Complement fragments (named opsonins; i.e., C1q, C3b, iC3b) interact with complement cell-surface receptors (C1qRp, CR1, CR3 and CR4) to promote phagocytosis and a local pro-inflammatory response that, ultimately, contributes to the protection and healing of the host. Moreover, complement biosynthesis and activation also occurs in neurodegenerative disorders such as Alzheimer's, Huntington's as well as Pick's disease and the cytolytic/ cytotoxic activities of complement are thought to contribute to neuronal loss and brain tissue damage. However, recent data suggest that at least some of the complement components have the ability to contribute to neuroprotective pathways. The emerging paradigm is that complement is involved in the clearance of toxic cell debris (e.g., amyloid fibrils) and apoptotic cells, as well as promoting tissue repair through the antiinflammatory activities of C3a. Knowledge of the unique molecular and cellular innate immunological interactions that occur in the development and resolution of pathology in the brain should facilitate the design of effective therapeutic strategies.
Protective autoimmunity as a defense mechanism against destructive self-compounds: role of CD4+CD25+ regulatory T cells and prospects for therapeutic vaccination M. Schwartz Weizmann Institute of Science, Rehovot, Israel Slowly progressing neurodegenerative diseases of the central nervous system (CNS), caused by numerous factors in addition to the primary trigger, can potentially benefit from neuroprotective therapy. We suggest that all self-compounds that participate in the progression of neurodegenerative diseases should be viewed as potential enemies of endogenous origin, some of which cause inappropriate activation of the resident microglia (e.g., TNF-alpha, NO, COX-2). It was found that the adaptive arm of the immune response, in the form of T cells directed against CNS self-antigens that reside in the site of injury (but not against the threatening compounds themselves), shape the phenotype of microglia. Naturally occurring CD4+CD25+ regulatory T cells were found to be the cells that restrict the spontaneous ability to elicit beneficial autoimmunity. Brainassociated neuropeptides/neurotransmitters were shown to act as signaling molecules through which the inhibitory activity and trafficking of the regulatory T cells can be switched on or off. Boosting of autoimmunity without invoking autoimmune disease can be achieved by weakening the activity of regulatory cells or by vaccinating with a weak agonist of selfantigens. These findings have far-reaching implications for risk-free therapy of neurodegenerative diseases in general, and in particular when the same individual is suffering from more than one such disorder.
Beta-amyloid vaccination and Alzheimer's disease T. Tabira National Institute for Longevity Sciences, Aichi, Japan Alzheimer's disease (AD) is a disorder characterized by senile plaques with amyloid deposits and primitive immune responses due to activated microglia/macrophages. It was shown that immunization of amyloid precursor protein (APP) gene transgenic mice with aggregated amyloid beta protein (A-beta)1-42 reduced the amyloid burden in the brain. The clinical trial of this vaccination therapy in AD patients was halted because of acute meningoencephalitis as a side effect, which was thought to be induced by CD4+ T cells reactive to A-beta. However, the clinical decline was found much less in patients who developed antibodies to beta amyloid than those who did not. It is interesting to know that an autopsy case who had received the vaccine suggested the disappearance of senile plaques. Thus, development of a better vaccine for AD is warranted. We have developed a safe vaccine using adeno-associated virus vector carrying Abeta cDNA. Administration of this vaccine once by an oro-gastric tube induced antibodies to beta amyloid, which lasted for more than 6 months. We found that this vaccine was quite effective and safe in APP transgenic mice (tg2576), which might be explained by the shift from Th1 to Th2 in the gut immune system.
The neurotransmitters glutamate and dopamine activate their specific receptors in human T-cells and trigger key T-cell functions M. Levite and Y. Ganor Weizmann Institute of Science, Rehovot, Israel
Do T-cells express specific neurotransmitter-receptors? Can neurotransmitters by themselves trigger T-cell functions? Glutamate: we discovered for the first time that normal, cancer and autoimmune encephalitogenic Tcells, alike neurons and glia cells, express high levels of ion-channel (ionotropic) glutamate-receptors. The evidence for glutamate/AMPA receptor subtype-3 (GluR3) in human T-cells includes RT-PCR, western blot and immunostaining/flow-cytometry. Sequencing showed identity between the T-cell's GluR3 and the brain's GluR3. Further, glutamate on it own (~10 nM) potently triggers several T-cell functions among them:
(1) integrin-mediated-adhesion to laminin and fibronectin, and 2) CXCR4mediated chemotactic-migration towards the key chemokine SDF-1. Dopamine: We discovered for the first time that normal human T-cells express on their outer membranes functional dopamine receptors of the D3 and D2 subtypes, and that dopamine (~10 nM) or selective dopaminereceptor agonists trigger various T-cells functions among them: (1) differential cytokine-secretion of either inflammatory (TNFalpha) or anti-inflammatory (IL-10) cytokines, different from the robust nonselective cytokine-secretion btriggered by classicalQ TCR-activation, (2) integrin-mediated adhesion to extracellular glycoproteins, (3) unique denovo gene-expression. The ability of glutamate and dopamine on their own to trigger T-cell functions and gene-expression could be of substantial scientific and clinical importance for numerous conditions in health and disease, among them: (a) transmigration of bgoodQ T-cells into the brain for different patrolling/protecting tasks, (b) Detrimental activation and maintenance of bbadQ T-cells in the CNS, e.g., in multiple-sclerosis.
The presentation of self in the thymus network: a novel way for prevention and cure of autoimmune diseases V. Geenen Liege University Center of Immunology, Liege-Sart Tilman, Belgium
The thymus ensures the establishment of self-tolerance by deleting selfreactive T cells and generating self-antigen specific regulatory T cells. A repertoire of neuroendocrine genes is transcribed by thymic stromal cells. Thymic neuroendocrine precursors engage two types of interactions with pre-T cells either as ligands that bind with high affinity to neuroendocrine receptors expressed by thymocytes, or after processing as self-antigens presented by the thymic MHC machinery. Thus, in the thymus, a confrontation occurs throughout life between previously emerged neuroendocrine families and a recently evolved system characterized by stochastic generation of its response diversity. All members of the insulin gene family are transcribed in the thymus: IGF2 (TEC/TNC)NIGF1 (macrophages)JINS (medullary TEC and/or dendritic cells). IGF2 transcription is defective in the thymus of more than 80% of diabetesprone BB rats. The sequence Ins B9-23 is a dominant h-cell autoantigen presented by DQ8, a MHC-II allele conferring genetic susceptibility to type 1 diabetes (T1D). The homologous sequence IGF-2 B11-25 competes with Ins B9-23 for binding to DQ8. However, compared to Ins B9-23, presentation of IGF-2 B11-25 to PBMCs purified from DQ8+ T1D adolescents elicits a suppressive/regulatory cytokine profile. With regard to T1D prevention, administration of IGF-2 derived self-antigen(s) constitutes an innovative approach that combines antagonism for binding to a major susceptibility MHC-II allele, as well as promotion of a specific antigen tolerogenic response.
Receptor mechanisms of IL-1-induced sickness behavior R. Dantzer, A. Nadjar, R.M. Bluthé, P. Parnet and J.P. Konsman Integrative Neurobiology, Bordeaux, France
The signaling pathways that mediate the behavioral effects of interleukin-1 (IL-1) during the acute phase reaction have not yet been studied. The behavioral effects of IL-1 are mediated by the interaction of this cytokine with the type I IL-1 receptor (IL-1RI) and its accessory protein as demonstrated by the use of mice in which the gene coding for IL-1R1 or for IL-1RacP has been deleted. Immunoneutralization experiments with an antibody specific of IL-1RI confirm the role of this receptor. IL-1 receptors are expressed at low abundance in the central nervous system of the rat and mouse brain. Immunohistochemistry techniques reveal a predominant expression of IL-1RI at the level of the blood-brain interface in the rat brain. Double immunolabeling techniques show that this expression is restricted to endothelial cells of venules within the brain parenchyma and circumventricular organs. Activation of IL-1 receptors is associated with nuclear factor kappa B (NF-kappaB) nuclear translocation and a robust transcriptional activation of inhibitor of kappa B alpha (IkappaB). NF-kappaB translocation is a better marker of IL-1R activation than IkappaB. Prevention of NF-kappaB activation by intracerebroventricular administration of a NEMO-binding domain peptide that selectively inhibits the IKKgamma/IKKbeta interaction abrogated sickness behavior induced by intraperitoneal but not by intracerebroventricular injection of IL-1, confirming the predominant role of IL-1 receptors at the blood-brain interface in the neural transduction of the peripheral immune message.
Stress-induced augmentation of skin immunity F. Dhabhar Health Sciences Center, The Ohio State University, Columbus, OH, USA Cell-mediated immune (CMI) responses exert important immunoprotective (resistance to infectious agents) or immunopathological (allergic or autoimmune hypersensitivity) effects. We have utilized the skin CMI response as an in vivo model for studying neuro-endocrine-immune interactions. We initially hypothesized that just as an acute stress response prepares the cardiovascular and musculoskeletal systems for fight or flight, it may also prepare the immune system for challenges that may be imposed by a stressor. The skin CMI model enabled us to examine the effects of stress at the time of primary and secondary exposure to antigen. Studies showed that acute (2 h) stress experienced before either primary (innate immune response) or secondary (adaptive immune response) antigen exposure induces a large and long-lasting enhancement of skin CMI. Adrenalectomy eliminates the stress-induced enhancement of CMI. Acute administration of physiological (stress) concentrations of corticosterone and/or epinephrine to adrenalectomized animals enhances skin CMI. Compared with controls, CMI sites from acutely stressed animals show significantly greater erythema and induration, numbers of infiltrating leukocytes, and levels of cytokine gene and protein expression. In contrast to acute stress, chronic stress is immunosuppressive and chronic exposure to corticosterone, or acute exposure to dexamethasone, significantly suppress skin CMI. These results suggest that during acute stress, endogenous stress hormones enhance skin immunity by increasing leukocyte trafficking and cytokine gene expression at the site of antigen entry. Basic mechanistic experiments as well as clinical studies will be discussed.
Inflammatory stress G.P. Chrousos PREB, NICHD, NIH and University of Athens, Athens, Greece
Like the stress response, the inflammatory reaction of an individual is crucial for survival of the self and species. Also like the stress response, inflammation is tailored to the stimulus and time-limited. A fully fledged systemic inflammatory reaction consists of activation of immune and immune accessory cells and resultant stimulation of four major programs:
(1) the acute phase reaction, (2) the sickness syndrome, (3) the pain program, mediated by the afferent sensory and autonomic systems, and (4) the stress program, mediated by the stress system, i.e. the hypothalamicpituitary-adrenal (HPA) axis and the locus ceruleus-norepinephrine / sympathetic system. The main effector substances of the systemic inflammatory response are inflammatory cytokines, such as TNFalpha, IL-1 and IL-6, chemokines,, and other mediators of inflammation; the acute phase reactants, such as C-reactive protein (CRP), cell adhesion molecules, fibrinogen and plasminogen activator inhibitor 1; the effectors of the sensory afferent system, such as substance P; and, of the stress system, namely hypothalamic CRH and vasopressin, cortisol, norepinephrine and epinephrine, and peripheral neuronal CRH. The sickness syndrome consists of anorexia/nausea, fatigue and/or depressed affect, somnolence, hyperalgesia, sleep disturbances, elevated temperature and an increased metabolic rate, all manifestations suppressed by glucocorticoids. Yet, peripheral neuronal CRH activated by stress or the inflammatory reaction, and substance P, activated by the inflammatory reaction potentiate inflammation. We recently found that during a systemic inflammatory reaction, as in ARDS or sepsis, there is inadequate activation of cortisol secretion and significant cytokine-induced glucocorticoid resistance, both suggesting beneficial actions of added glucocorticoids.
Corticotropin-releasing hormone (CRH) and the immune/inflammatory response: lessons from the knockout mice K. Karalis Division of Endocrinology, Children's Hospital, Harvard Medical School and IIBEAA, Institute for Biomedical Research, Academy of Athens, Athens, Greece CRH, a neuropeptide isolated from the hypothalamus, is the main mediator of the stress response in mammals. CRH is expressed in inflamed human and rodent peripheral tissues and exerts potent proinflammatory effects in experimental models of inflammation. We have used the CrhÀ/À mouse to characterize the immunomodulatory properties of CRH and identify the pathways mediating its effects. The CrhÀ/À mice respond to inflammatory stimuli with a compromised ACTH response and a delayed, although finally similar to that of the Crh+/+ mice, rise in corticosterone. This response differs from that of the Crh receptor 1À/À suggesting that other receptor(s) may also mediate the effects of CRH on the pituitary following an immune stressor. It should be highlighted that immune activation is the only stressor that can induce a pituitary-adrenal response in the CrhÀ/À mice. We have found that CrhÀ/À mice have reduced inflammatory responses to local stimuli, such as carrageenin, confirming the proinflammatory effects of CRH. These experiments revealed proinflammatory effects of epinephrine unmasked by Crh deficiency and suggested the coordinated action of epinephrine and CRH in the regulation of the acute inflammatory response. Systemic inflammatory responses in the CrhÀ/À mice were also of significantly reduced magnitude, as well as autoimmune diseases such as experimental allergic encephalomyelitis. The pathways mediating the proinflammatory responses of CRH were studied and demonstrated specific CRH-mediated induction of the NFÀkB DNA binding activity in immune cells. Interestingly, CrhÀ/À mice had profound anorexia and weight loss despite their reduced inflammatory response. The latter suggests that in cases of Crh deficiency the pathways mediating the inflammation-induced anorexia are not dependent on the level of inflammation.
Identification of polymorphic genes regulating inflammatory diseases may unravel crucial pathogenic mechanisms. Initial steps to map such genes using linkage analysis in F2 intercross or backcross populations, however, result in broad quantitative trait loci (QTLs) containing hundreds of genes. In the present study, an advanced intercross line (AIL), in combination with congenic strains, was used to fine-map Eae18 on rat chromosome 10 in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE). MOG-EAE is a chronic relapsing disease that closely mimics key features of multiple sclerosis (MS). Congenic DA.ACI rat strains localized Eae18 to an approximately 30 Mb large region. Fine-mapping was then performed in an AIL consisting of a (DAÂPVG.1AV1) F7 intercross, resulting in two adjacent EAE-regulating QTLs designated Eae18a and Eae18b. The two QTLs span 5.5 and 3 Mb, respectively, and Eae18b contains as few as 10 genes, including a cluster of chemokine genes. Eae18a and Eae18b are syntenic to human chromosome 17p13 and 17q11, respectively, which both display linkage to MS. Thus, Eae18 consists of at least two EAE-regulating genes, providing additional evidence that clustering of disease-regulating genes in QTLs is an important phenomenon. The overlap between Eae18a and Eae18b with previously identified QTLs in humans and mice further supports the notion that susceptibility alleles in inflammatory disease are evolutionary conserved between species.
T cell gene expression profiling identifies molecularly and clinically distinct subgroups of multiple sclerosis J.-I. Satoh, M. Nakanishi, H. Onoue, T. Aranami and T. Yamamura National Institute of Neuroscience, Tokyo, Japan Objective: To clarify the molecular background underlying the phenotypic variability of multiple sclerosis (MS). Methods: Using a 1258 cDNA microarray, a comprehensive gene expression profile of T cells of 72 MS patients (65 RR and 7 SP) with various MRI lesion distributions before and after IFN-beta treatment was compared with that of 22 age-and sexmatched healthy subjects. Results: Hierarchical cluster analysis of 286 genes expressed differentially between IFN-untreated MS patients and controls as a discriminator identified four distinct subgroups of MS designated A, B, C, and D, among which the subgroup A showed the expression pattern most similar to the control group. The patients presenting with the lesion distribution restricted in the cerebrum were clustered in the subgroup C. When the 286 genes were categorized into five different classes numbered 1 to 5, the subgroup B showing the highest upregulation of class #5 genes, including nine chemokines, was associated with the most severe clinical activity. Furthermore, the IFNbeta responder was clustered in subgroups A and B, and was characterized by a persistent induction of IFN-responsive genes during IFN-beta treatment. Conclusions: These results indicate that microarray analysis of the gene expression profile of T cells could classify a heterogenous population of MS into four molecularly distinct subgroups closely associated with the disease activity, lesion distribution, and therapeutic response to IFN-beta.
A clinical-genetic study of multiple sclerosis in a Dutch genetically isolated community R. Hintzen, E. Croes, C. van Duijn and I. Hoppenbrouwers Erasmus MC, Department of Neurology, Rotterdam, The Netherlands
Background: Next to large genome screens, the study of genetically isolated populations is an alternative approach in order to identify the genetic factors involved in multiple sclerosis (MS) . Objectives: To identify MS cases within a Dutch genetically isolated population and attempt to link them to a common ancestor. Secondly, to assess familial association of type I diabetes mellitus (DM), autoimmune thyroid disease and MS. Thirdly, to perform a genome screen. Methods: MS patients were traced and an extensive family history was taken. Special emphasis was paid to the co-occurrence of autoimmune disease. Genealogical data extended up until 14 generations (around the year 1680). Results: Thirty-five female and 13 male MS patients were identified. Mean age at onset of symptoms was 32 years and at diagnosis 38 years. Forty patients had a relapsing-remitting course and 8 a primary progressive course. Twenty of the 48 patients could be linked to a common ancestor in 12-14 generations. The clinical characteristics of these 20 patients were not different from those not linked. Familial co-occurrence of MS was observed in 21% of the 48 patients. Thyroid disease in 17% and type I DM in 10%. HLA-Dr2 was not found significantly more often in the MS patients as in controls. Conclusions: Familial aggregation of MS is high in this Dutch genetic isolate, with a high co-occurrence of other autoimmune diseases. HLA-Dr2 was not specifically linked to the MS cases. Several studies have shown that multiple sclerosis (MS) is associated with the HLA class II specificity HLA-DR15. In Japan, an association has been reported between HLA-DR4 and a subpopulation of conventional-MS patients that lack oligoclonal bands (OCB) in the CSF. Though DR15 increases the risk for OCB-positive conventional MS in Japan, it is not a risk factor for OCB-negative MS. To investigate whether the OCBnegative and OCB-positive subpopulations of MS patients from Sweden show HLA-DR frequencies similar to those seen in Japan, patients were diagnosed according to the criteria of McDonald et al. HLA-DR genotypes were determined, for all patients and controls, with a resolution corresponding to serological typing. OCB were detected by isoelectric focusing and IgG-specific immunofixation of CSF proteins. DR15 and DR4 phenotype frequencies were compared in 55 OCB-negative patients, 228 OCB-positive patients and 284 controls. In OCB-negative patients, DR15 was significantly less common, and DR4 significantly more common, than in OCB-positive patients. The frequency of DR15 was significantly higher in OCB-positive patients than in controls, but did not differ between OCB-negative patients and controls. The frequency of DR4 was significantly higher in OCB-negative patients than in controls, but did not differ between OCB-positive patients and controls. The present study, performed on Swedish MS patients, confirms results first reported in Japan. Thus, it appears that OCB-positive MS and OCB-negative MS may be distinct disease subtypes, each associated with a particular predispositional HLA class II specificity. Objective: Iron misregulation, oxidative stress and inflammation play interacting roles in Alzheimer's disease (AD). We therefore examined polymorphisms in two genes of iron metabolism in the risk of AD: the C2 variant of the transferrin (TF) gene and the C282Y allele of the haemochromatosis (HFE) gene. Methods: We genotyped 269 elderly controls, 191 AD cases and 69 with mild cognitive impairment (MCI) from the OPTIMA cohort. Results: Each of the two variants was associated with an increased risk of AD only in the presence of the other. Carriers of both variants were at 5 times greater risk of AD compared with all others. The interaction was significant by logistic regression and by synergy factor analysis. Carriers of these two alleles plus apolipoprotein E4 (APOE4) were at exceptionally high risk of AD. Conclusions: We suggest that the combination of TF C2 and HFE C282Y may lead to an excess of redoxactive iron and the induction of oxidative stress in neurones, which is exacerbated in carriers of APOE4. As 4% of Northern Europeans carry both iron-related variants and since iron overload is a treatable condition, these results merit replication.
Age, parent, and season at immunization influence susceptibility to EAE E. Blankenhorn a , C. Teuscher b , J. Bunn b and P. Fillmore a a University of Illinois at Urbana-Champaign, Urbana, IL, USA; b University of Vermont School of Medicine, Burlington, Vermont, USA Experimental allergic encephalomyelitis (EAE), the principal animal model of multiple sclerosis (MS), is a complex trait influenced by both genetic and environmental factors. We examined the role of age and season at immunization, sex, and parent-of-origin (POO) in N1800 (B10.SÂSJL) F2 mice. Logistic regression analyses indicated that the odds of being affected increase by 4% for each increasing week of age, and were 90% greater for animals injected in the summer, compared to the winter or spring. The odds also differed significantly depending on the POO, where male F2 progeny of SJL maternal granddams and B10.S paternal grandsires were more than twice as likely to show EAE than males from reciprocal grandparents. Surprisingly, CNS inflammation in male mice was not affected by POO, and female progeny were not influenced by POO effects at all. Linkage analysis to eae1(H2)/eae5 using 6-to 12-week-old and 13-to 18-week-old cohorts of F2 mice failed to detect significant linkage to chromosome 17 marker loci in the older and winter/spring populations whereas significant linkage was detected with the younger and summer populations. Our results provide a new model for environmental effects in MS, and may help explain the fact that although linkage to HLA is the most consistent finding in MS genetics studies, the HLA-linked MS susceptibility locus may be influenced by the effects of age, season, or other non-Mendelian genetic factors. Background: Several studies demonstrated the involvement of the myelin oligodendrocyte glycoprotein (MOG) protein in multiple sclerosis (MS). We wanted to verify the presence of betiologicQ polymorphisms of the MOG gene in Sardinian MS patients. Methods: MOG gene was fully sequenced in 21 healthy Sardinians. The MOG51 micros and all SNPs showing a frequency N5% in sequencing were typed in a first data set of 249 trios families (one affected and both parents) and examined by transmission disequilibrium test (TDT). MOG variants significantly ( Pb0.05) associated with MS and 3 SNPs not associated used as controls were again tested in a second set of 152 families. Final significant values were set at Pb0.01. Results: Sequencing of MOG gene revealed 37 novel and 82 previously found SNPs. TDT showed evidence of association ( Pb0.05) in 12/40 SNPs and MOG51. TDT in the whole set of 401 families confirmed association with the G16457A ( P=0.001), A15643G ( P=0.004), C11544T ( P=0.003), C10495G ( P=0.0001) variants and MOG51 allele 8 ( P=0.01). Conclusions: MOG gene is involved in susceptibility to MS in Sardinians. An extension of the study in a large set of sporadic patients to confirm these data is ongoing.
MHC class II molecules (MHCII) present antigens to CD4+ T cells, which is a key feature of adaptive immune responses. We have previously identified the Vra4 locus on rat chromosome 10 with major influence on nerve injury-induced MHC II expression on microglia. The F8 generation of an advanced intercross line (AIL; DAxPVG.1AV1) was used for genetic fine mapping of Vra4, revealing linkage to MHCII expression with a LOD score of 26.4. MHC II immunolabeling was measured in spinal cord after ventral root avulsion (VRA). A microsatellite-based haplotype map over Vra4 in a panel of inbred rat strains identified two haplotypes extending 2.7 Mb. This fragment contains the mhc2ta gene encoding CIITA, a known regulator of MHC II expression. Sequencing of mhc2ta revealed two distinct haplotypes segregating inbred strains with high and low MHC II expression. Analysis of the expression of CIITA and the CIITA-induced invariant chain in spinal cord tissue revealed lower levels of both transcripts in PVG.1AV1 rats compared to DA, both under normal conditions and after VRA. There was also a strong correlation between CIITA and invariant chain in a group of F8 animals stratified for parental allele at the max marker, which is located within the mhc2ta gene. These results identify genetic heterogeneity in the mhc2ta gene to modulate inflammatory processes in the nervous system.
Identification of QTL controlling cortical motor evoked potentials in experimental autoimmune encephalomyelitis: correlation with incidence, onset and severity of disease I. Mazon a , S. Vogler a , U. Strauss b , P. Wernhoff a , A. Rolfs b and S. Ibrahim a a Institute of Immunology, University of Rostock, Rostock, Germany; b Department of Neurology, University of Rostock, Rostock, Germany Experimental autoimmune encephalomyelitis (EAE) is a polygenic chronic inflammatory demyelinating disease model of multiple sclerosis. Previous studies have identified multiple quantitative trait loci (QTL) controlling different aspects of disease pathogenesis. However, direct genetic control of motor-evoked potentials as a measure of demyelination was not addressed. In this study, we examined the genetic control of different traits of EAE in a F2 intercross population generated from the EAE susceptible SJL/J and EAE resistant C57Bl/10.S mouse strains involving 460 animals. All animals were genotyped with 150 microsatellite markers and interval mapping was used to identify QTLs controlling disease severity, onset and Motor Evoked Potentials. Six new QTL were identified. Three QTL mapped to chromosome 2, 10 and 18 controlled the severity of the disease, whereas QTL on 1, 8 and 15 were associated with the latency in the corticomotor evoked potentials (CMEP). Two of the loci were influenced by sex. One QTL on Chr. 8 cM 15-53(EAE31) controlled CMEP latencies before immunizations and correlated with disease onset suggesting a possible role of myelination patterns and or the structure of central motor pathways in susceptibility to EAE. Mapping and functional characterization of rat Eae4 R. Nohra, M. Jagodic and T. Olsson Karolinska Institute, Stockholm, Sweden Eae4, a locus regulating disease chronicity and cytokine production in experimental autoimmune encephalomyelitis, was initially mapped in (DAxBN)F2 rats. A congenic rat strain, DAc9BN-Eae4, has been established and a phenotypic effect of the isolated locus has been confirmed. Eae4 is being further narrowed down in recombinant congenic strains and the locus is currently mapped to a 3-Mb region. The EAE chronicity phenotype has so far been linked to the cytokine production phenotype (mainly measured as high or low TNF-alpha production in ConA stimulated naive splenocytes) in all currently analyzed recombinant strains, suggesting that identical or closely localized gene(s) regulate these two phenotypes. In parallel with the production of still smaller recombinant strains, high-resolution mapping is attempted in a (DAxPVG.AV1)F10 advanced intercross line (AIL). Furthermore, preliminary immunological data map the cytokine production phenotype to the CD4+ T cell lineage and not to CD8+ T cells, monocytes/macrophages or NK cells. Eae4 is, thus, a locus with effects in EAE and possibly also in other inflammatory conditions where cytokine dysbalance is implicated and positional cloning is ongoing.
High-resolution mapping of quantitative trait loci that influence an experimental model for multiple sclerosis in the rat M. Marta, M. Jagodic, T. Olsson and J.C. Lorentzen Karolinska Institutet, Stockholm, Sweden Inbred rat strains show varying susceptibility or resistance to myelin oligodendrocyte glicoprotein (MOG) induced encephalomyelitis (EAE), which closely mimics multiple sclerosis in humans. We previously reported that transfer of a 10-cM rat chromosome 4 region (C4R3) from resistant PVG.1AV1 rats to susceptible DA rats affects the susceptibility to MOG-EAE. Objective. To genetically dissect the influence of C4R3 on MOG-EAE. Methods. A 10th generation of an advanced intercross line (AIL) of DA and MHC identical PVG.1AV1 was injected with MOG. Clinical disease was followed and individuals in the test-cross were genotyped using microsatellite markers. Genotype-phenotype analysis was performed within C4R3 using R/qtl to detect quantitative trait loci. In addition, subcongenic rats with a 0.8 cM PVG.1AV1 region (C4R11) inserted on DA background were compared with DA rats for MOG-EAE susceptibility. Results. The AIL analysis revealed two narrow and closely situated QTLs in C4R3. The smaller C4R11 interval overlaps with one of the QTLs, and C4R11 congenic rats were also less susceptible than DA to MOG-EAE. Conclusion. The C4R3 interval harbors at least two genes that regulate MOG-EAE. One of these genes lies within the C4R11 interval previously reported to regulate oil-induced arthritis (QTL designated Oia2) . This gene(s) may be a general regulator of autoimmune diseases.
An advanced intercross line localizes Eae5 to a region containing Ncf-1 K. Becanovic a , M. Jagodic a , J.R. Sheng a , I. Dahlman a , E. Wallstrfm a , F. Aboul-Enein b , P. Olofsson c , R. Holmdahl c , H. Lassmann b and T. Olsson a a Karolinska Institute, Stockholm, Sweden; b University of Vienna, Vienna, Austria; c Lund University, Lund, Sweden Eae5 in rats was originally identified in two F2 intercrosses (DAÂBN) and (DAÂE3), displaying linkage to CNS inflammation and EAE severity, respectively. This region overlaps with Pia4, which was also identified in the (DAÂE3) cross. Two congenic strains, BN.DA-Eae5 and BN.DA-Eae5.R1, encompassing the previously described Eae5 and Pia4, were established. DA alleles within the chromosome 12 fragment conferred an increase in disease susceptibility as well as increased inflammation and demyelination in the CNS compared to BN alleles. To enable a more precise fine-mapping of EAE regulatory genes, we utilized a rat advanced intercross line (AIL) between the susceptible DA and the resistant PVG.1AV1 strain. Linkage analysis performed in the advanced intercross line considerably narrowed down the MOG-EAE regulatory locus (Eae5) to a 1.4-Mb region with a defined number of candidate genes. We here demonstrate a regulatory effect of Eae5 on MOG-EAE, using both congenic strains as well as fine-mapping these effects to a region containing Ncf-1, a gene with reported effects also in experimental arthritis. Studies of animal models of MS have provided important insight into the immunopathogenesis of disease. Genetic analyses of these models overcome certain obstacles encountered when studying human patients. MOG-EAE like MS is complex genetic disease with contributions from major histocompatibility complex (MHC) genes and multiple unknown non-MHC genes. Linkage analysis have been used to map EAE-linked loci on rat chromosome in an F2 cross between susceptible DA and resistant ACI rats, a region with disease regulating influences on MOG-EAE has been found on chromosome 15. In the present study, a direct examination of this loci was undertaken in congenic strains by transferring this region from resistant ACI rats to the susceptible DA background, phenotypic comparing among DA and congenic DA.ACI-D15Rat6-D15Rat 71 (25cM) and subcongenic recombinants 1(R1)-D15Rat 6-D15Rat13 (10 cM) show the congenic and R1 are resistant to MOG-EAE with low level of anti-MOG antibodies tendency in the sera and low inflammation or demyelination index in the central nervous system. It is very promising to keep the retained phenotypic difference by creating congenic strain and collecting recombinants in different congenic segments and permit rapid fine mapping within a few centiMorgans. Thus, the results indicate that this region harbors EAE regulating genes contributing to the protection against EAE and may reflect both the T and B cell arms of MOG autoimmunity. In conclusion, this studies will provide the approach for testing the human homologous gene in MS and unveiling the mechanisms of genes regulating the MS like disease.
A novel SNP in the coding region of PSGL-1 in multiple sclerosis patients C. Fenoglio a , D. Galimberti a , R. Clerici a , M. Ronzoni a , M. De Riz a , L. Piccio a , A. Gatti a , E. Venturelli a , L. Berti a , G. Constantin b , N. Bresolin a and E. Scarpini a a Ospedale Maggiore, Milano, Italy; b University of Verona, Verona, Italy Lymphocyte recruitment into the brain is a critical event in the pathogenesis of multiple sclerosis (MS). Selectins are expressed by inflamed endothelium and participate in lymphocyte adhesion to the brain vessels. P-selectin glycoprotein ligand 1 (PSGL-1) is the main ligand of E-and P-selectins and has a crucial role during lymphocyte recruitment. Genomic DNA was isolated from whole blood and mutational scanning of PSGL-1 has been carried out in 100 Italian patients with MS as well as in 100 age-matched controls, to detect new polymorphisms. The frequency of new mutations has then been compared between patients and controls. A novel Single Nucleotide Polymorphism (SNP), involving a GRA substitution, which results in an aminoacidic change at position 62 (M62I) in the coding region of PSGL-1 gene was determined. A different frequency of the M62I SNP in MS patients compared with controls was observed. Stratifying by age at onset or gender, no differences in allele frequencies were observed. This is the first evidence of the presence of M62I in Caucasians.This SNP could have an effect on the function of PSGL-1 as it is located within the region involved in the binding with selectins.
Analysis of the Asp299Gly polymorphism in the Toll-like receptor 4 (TLR-4) gene in patients with multiple sclerosis A. Kroner a , B. Rosche b , A. Kolb-M7urer a , N. Kruse c , K. Toyka a , B. Hemmer b , P. Rieckmann a and M. M7urer a a University of Würzburg, Würzburg, Germany; b Heinrich Heine University, Düsseldorf, Germany; c University of Göttingen, Göttingen, Germany
In multiple sclerosis, disease deterioration often occurs in association with bacterial infections, particularly with lipopolysaccharide (LPS) containing gram negative bacteria. LPS is known to activate immune cells via the toll-like-receptor-4 (TLR-4). A recently described Asp299Gly polymorphism in the tlr-4 gene was shown to cause hyporesponsiveness to LPS. We therefore investigated the functional role and the allelic distribution of this TLR-4 polymorphism in a population of MS-patients. DNA was obtained from 890 patients and 350 healthy donors. The polymorphism was analysed with allelic discrimination(AD)-PCR and confirmed by PCR-restriction-fragment-length-polymorphism (RFLP) and automatic sequencing. PBMCs from donors with different genotypes were stimulated with LPS and inactivated gram-negative and gram-positive bacteria. Proliferation and production of IL-6 and TNF-alpha were measured. Genotyping revealed 809 (90.9%) wildtype individuals, 79 (8.9%) patients with heterozygous and two individuals (0.2%) with homozygous mutant genotype. No association of different genotypes with MS susceptibility, subtypes or disease severity could be found. Significantly lower proliferation rates ( p=0.023) in PBMCs derived from heterozygous individuals could be detected after stimulation with LPS. We could not find an association between Asp299Gly genotypes and disease course of multiple sclerosis in our MS population. However, we could confirm the functional relevance of the Asp299Gly polymorphism after exposure of immune cells with LPS.
Multiple sclerosis and autoimmune diseases. Risk and HLA-DR association in Northern-east Italy P. Gallo a , A. Laroni a , M. Calabrese a , P. Perini a , M.P. Albergoni b , F. Ranzato a and L. Battistin a a Multiple Sclerosis Center, Department of Neurological Sciences, University of Padova, Padova, Italy; b Blood Bank, University Hospital, Padova, Italy An autoimmune background is thought to characterize the families of multiple sclerosis (MS) patients, but disease patterns and HLA-DR association seem to vary considerably among different ethnic groups. We studied the occurrence of 37 autoimmune diseases in 250 MS patients and 250 age-/sex-matched normal controls (NC), original of and living in the Northern-east Italy, and in almost all their first-degree relatives (2800 subjects). HLA-DR expression was analysed in MS and NC. The risk of having autoimmune diseases was not increased in MS patients (22/250,9%) compared to NC (15/250,6%)(MS vs NC: x2=1.052, p=0.3), but 40 families with multiple autoimmune pathology (autoimmune families, AF) were identified in the MS group [16%; only 4 in the NC group (1.6%) (x2=29.470, p=0.000; OR=11.4] and a significant excess of autoimmunity was found in first-degree relatives of MS patients ( p=0.000). An association of MS with Type 1 Diabetes Mellitus was also noticed (T1DM) ( p=0.04). DR4 expression in MS patients from AF was significantly higher (32%) than in reference MS patients (9%; x2: p=0.001) and NC (14%) (x2:p=0.02). Fifty percent of DR4-positive MS patients expressed the allele DRB1*0401. Our data suggest a significant risk of autoimmunity in first-degree relatives of MS patients, a role for DR4 in autoimmune predisposition, and a preferential association of MS with T1DM, at least in Southern Europe.
The CD14 C(-260)T promoter polymorphism is associated with disease severity in multiple sclerosis patients A. Lutterotti a , R. Ehling a , H. Lassmann b , F. Deisenhammer a , T. Berger a and M. Reindl a a Clinical Department of Neurology, Innsbruck Medical University, Innsbruck, Austria; b Brain Research Institute, Vienna Medical University, Vienna, Vienna CD14 is a membrane receptor expressed on cells of the monocyte lineage known to be an essential mediator in innate host defense and in the phagocytosis of apoptotic cells. The aim of our study was to investigate a possible association of the -260 C-T promoter polymorphism in the CD14 gene with the susceptibility and disease severity in 225 patients with clinically definite multiple sclerosis (MS). Outcome measures were disease severity measured by the progression index (EDSS/disease duration), the MS severity score, and the time to reach the hallmark EDSS 6 as well as the annual relapse rate. We established a new PCR assay with sequencespecific primers to analyze CD14 genotypes in our patients. Furthermore, we analyzed CD14 expression within MS lesions of 31 patients by histopathology. Carriers of the CD14-260 C allele have a more severe disease course (progression index, MS severity score) and a significantly shorter time to reach the hallmark EDSS 6. We found no association between CD14-260 C-T polymorphism and susceptibility to MS. CD14 is upregulated in MS lesions, most prominently in acute lesions. This is the first study to describe an association of the -260 C-T polymorphism within the promoter region of the CD14 gene, a receptor of the innate immune system, with the longterm disease severity in MS patients.
Knowledge-base of interactions of candidate genes and susceptibility loci: a portal to multiple sclerosis S. Möller a , P. Serrano-Fernández a , U.K. Zettl b , P. Wernhoff a , H.-J. Thiesen a , D. Koczan a and S.M. Ibrahim a a Institute of Immunology and b Department of Neurology, University of Rostock, Rostock, Germany
Multiple sclerosis (MS) is a polygenic disease. Many unknown genetic factors lead to a set of clinical phenotypes of different intensity and periodicity. The scientific community applies a variety of approaches on tissues from patients and animal models of the disease in order to determine candidate genes for the phenotypic manifestation. With genetic linkage studies, transcriptomics and proteomics experiments on different tissues a wealth of information are now available. The sheer number of publications and genes described the interpretation of results from novel experiments a laborious and time consuming task. To help setting up new hypotheses, we here present a web interface to query a database we providing new evidences for the pathogenesis of MS. The database includes individual genes and chromosomal loci of the human supplemented with data from animal models of the disease and links to public resources. The interaction of genes and loci may be graphically browsed. The database is primarily presenting information from RNA and protein expression data, i.e., the differential expression in healthy vs. disease tissue. It includes data from blood, urin, spinal cord and brain. If available, the regulatory interactions are represented. Chromosomal susceptibility loci from genetic linkage analyses for the human disease and its animal models are interlinked with the genes via the EnsEMBL genome annotation. Intergenomic synteny of chromosomal loci is further applied both to characterise genes and to transfer data from interacting loci from the animal model of the disease (experimental autoimmune encephalomyelitis) to the human. Lymphocyte recruitment into the brain is a critical event in the pathogenesis of multiple sclerosis (MS), and is controlled by adhesion molecules. It involves: (1) initial contact (tethering) and rolling along the vessel wall mediated by selectins and integrins; (2) chemoattractantinduced integrin activation; (3) firm arrest; (4) diapedesis. Both E-and Pselectins are required for efficient tethering and rolling of neutrophils in mouse brain vessels. The distribution of G89T and A561C polymorphisms in E-selectin gene in a population of 200 patients with MS and in 238 age-matched controls was studied. A significantly increased frequency of the TT genotype of the G98T polymorphism in MS patients was observed. No differences in the distribution of A561C between patients and controls were shown, but patients bearing the wild-type allele had an increased probability to develop the disease after the age of 40 years. The presence of the TT genotype of the G98T polymorphism seems to increase the susceptibility to develop MS, possibly by up regulating Eselectin transcription rate and thus facilitating the recruitment of activated lymphocytes into the brain. Besides, A561C polymorphism does not seem to be a susceptibility factor for MS, although its presence could influence the age at onset. IFN-beta exerts its functionality by binding to the subunits IFNAR1 and IFNAR2 of its receptor. The aim of this work is to establish genotype frequencies for a SNP within the promoter region of the IFNAR1 gene (at À408bp relative to start of transcription), and 2 polymorphisms in coding regions (SNP18417 in exon 4 of IFNAR1 and SNP11876 in exon 2 of IFNAR2 gene), to evaluate the impact of these polymorphisms on the response to IFN-beta, and to assess the influence of SNP-408 on IFNAR1 mRNA expression. 147 MS patients on IFN-beta therapy and 205 controls were evaluated for these SNPs by RFLP analysis of PCR-amplified fragments. IFNAR1-mRNA was measured by real time RT-PCR. Significant differences on the genotype distribution between patients and controls for SNP18417 and for SNP11876 were found by Chi 2 analysis. SNP-408 does not seem to have an influence on the response to IFN-beta therapy, nor on the levels of expression of IFNAR1. No significant differences in the distribution of responders and non responders were observed when stratified by any of the SNPs. Whether these differences in the distribution of the genotypes for the SNPs in the coding regions between controls and patients have any functional significance need to be further studied.
Chromosome 2q33 and multiple sclerosis A. Bonetti University of Helsinki, Helsinki, Finland
Multiple sclerosis (MS) is a demyelinating disease of the central nervous system with suspected autoimmune pathogenesis. Both genetic and environmental factors play a role in its aetiology. MS is considered a genetically heterogeneous polygenic disease. In MS, there is evidence from linkage studies and allelic association studies suggesting that chromosome 2q33 would harbour a predisposing gene. We have performed a two-stage study to analyse the association of polymorphism on chromosome 2q33 with MS. In all, 17 markers were analysed in stage-1 in 134 Finnish MS families and the observed associations were tested in stage-2 in 186 MS families. We did not find previously reported allelic or haplotype associations with CTLA4. We obtained a weak signal of two distinct predisposing genes, one proximal the other distal of CTLA4. The putative proximal gene was associated with the marker rs3977 in families lacking HLA-DR2 ( P=0.02 and 0.02) and the other distal gene was associated with D2S1271 in families from a high-risk region in western Finland ( P=0.02 and 0.01). Based on the N3 cM distance and the lack of linkage disequilibrium between these loci, we conclude that the two association signals are independent. Our results provide preliminary evidence for two distinct MS susceptibility genes on 2q33 outside of CTLA4. Background: To evaluate correlation between HLA-DRB1-DQB1 haplotype and age-at-onset in a population of multiple sclerosis (MS) patients from Sardinia. Methods. A total of 1242 MS patients and 1918 healthy controls (C) were typed at the DRB1-DQB1 loci. The individual frequencies of the HLA class II genotypes observed in patients and in controls were compared by estimating odds ratio (OR) determined by genotype frequencies in patients with early onset (b16 years) vs. those with adult onset (N16 years) and vs, controls. Level of significance was set at Pb0.001. Results. We identified 76 (6.5%) individuals with early onset. Risk of MS increased in adult onset patients carrying the DR3/ DR13 (OR=6.0, P=1.2Â10 À9 ), DR3/DR2 (OR=5.7, P=4.8Â10 À6 ), DR3/ DR3 (OR=2.9, P=1.2Â10 À15 ), DR3/DR4 DQ3.5 (OR=2.9, P=3.9Â10 À6 ) genotype compared to C. In early onset patients, compared to C, risk was increased in carriers of DR3/DR13 (OR=10.3, P=6.1Â10 À4 ), DR3/ DR4DQ3.1 (OR=7.9, P=5.8Â10 À9 ) and DR3/DR4DQ3.2 (OR=6.2, P=4.8Â10 À7 ) genotypes. Comparison of adult and early onset patients showed that risk of early onset was specifically associated with the DR3/ DR4DQ3.2 (OR=3.9, P=3.8Â10 À4 ) genotype (genotype frequency 10.5% in early onset and 2.9% in adult onset patients). Conclusions. This results indicate that in Sardinians MS patients HLA-DRB1-DQB1 molecules contribute to early onset of the disease. Interestingly, risk of early onset is associated with the autoimmune diabetes high-risk genotype. Background: To analyze the distribution of men and women in a cohort of Sardinian MS patients, according to their HLA-DRB1-DQB1 haplotype and to examine the inheritance of the DR3 and DR4 MS-associated alleles according to their parental transmission. Methods. The study comprised 1097 (339 men and 758 women) MS patients and 329 healthy siblings. Typing of HLA-DRB1 and -DQB1 was performed as previously described. A logistic model was used to test the parent-of-origin (PO) and maternal genotype effects of the DR3 allele. Results. In the whole cohort of patients, the F/M ratio was 2.24, ranging from 2.88 in the DR3/DR3, to 2.52 in the DR3/DRX and 1.36 in the DR4/DRY (DRY#DR3) sub-category. An excess of paternally transmitted DR3 in affected (IM=0.61, P=0.05) but not in healthy offspring (IM=1.63, P=0.14) was found. The PO effect on DR3 transmission from fathers was evident in affected women (IM=0.55, P=0.04) but not in men (IM=0.77, P=0.57). An effect of two copies of the maternal non-transmitted DR3 genotype was found in DR3 females (S2=2.21, P=0.05) but not in males (S2=1.43, P=0.57). Conclusions. Present findings demonstrate that in Sardinia the relative risk of MS due to the HLA-DRB1-DQB1 locus is heterogeneous, with one etiological pathway associated with DR3 and possibly involving both paternal and maternal effects on disease risk. Based on the nationwide series of Italian twins with MS, we collected information about family aggregation; perinatal, early and late life events; physiological, medical and surgical history and other items that may represent nonheritable risk factors. Data from 70 twin pairs were prosecuted using a logistic regression model through stepwise forward selection procedure in order to identify only the best subset of covariates in terms of MS prediction. The selection process was based on a formal Likelihood Ratio test procedure that allowed the identification of a subset of covariables which seem to have a significant impact on the raise of the disease and on its concordance in twins. This logistic approach has been further generalized by adding a Gaussian random effect accounting for potential heterogeneity among twin pairs and dependence between twins in the same pair; this model has been estimated using both a GEE (generalized estimating equations) and a maximum likelihood approach. All analyses were performed with STATA (release 7.0, College Station, TX) and confirmed that early life infections, later life infections, breast feeding, mumps, herpes virus infection, red measles and the assumption of milk are covariables with a significant positive or negative association with the disease.
The role of herpesvirus entry receptors for the development of MS F. Vogel a , B. Rosche a , S. Cepok a , S. Stei b , V. Grummel a , A. Kroner c , M. M7urer c , P. Rieckmann c , N. Sommer b and B. Hemmer a a University of Düsseldorf, Düsseldorf, Germany; b University of Marburg, Marburg, Germany; c University of Würzburg, Würzburg, Germany
Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system (CNS). Although the cause of MS is still uncertain, it is well accepted that both genetic and environmental factors are important for the development of disease. Common viral infections have been implicated as environmental factors influencing onset and activity of disease. In this study, we focused on genes coding for common Herpes virus entry receptors (PRR1, PVR1, PRR2, CD21). Some of the genes were located in regions previously linked to MS in genome wide linkage studies. All the receptors are expressed in the brain and immune system and may play an important role for inter-cellular adhesion and entry of neurotropic viruses to the brain. The exons of the genes were sequenced in 15 MS patients and healthy controls. We identified a number of new and confirmed previously described polymorphisms. They were tested for their relevance in MS by a casecontrol (266 MS patients, 227 controls) and TDT study (60 MS families). So far, none of the polymorphisms was associated with MS. This observation was complemented by expression analyses of the receptors on peripheral mononuclear cells, which did not demonstrate differential expression between MS patients and controls. In summary, we did not find evidence that herpes virus receptor genes play a role in the development of MS. Complement activation is a central feature of inflammatory processes. Previously, we have demonstrated rat strain differences in complement C3 expression after lumbar ventral root avulsions (VRA), a model of nerve injury-induced neurodegeneration. Thus, in situ hybridization labeling for C3 mRNA after VRA was twice as high in DA rats compared to PVG. Furthermore, in a large scale microarray experiment, C3 mRNA levels significantly differed between the two strains both at baseline in naive rats, and 5 and 14 days after VRA. We have here extended these observations by genetic mapping of C3 mRNA expression in a F2(DAxPVG) intercross (n=186) by in situ hybridization and computer-based image analysis. All rats were genotyped with 177 microsatellites. This demonstrates a suggestive linkage (LOD score 3.0) of C3 expression to Vra1, a genetic locus on chromosome 8 that regulates neurodegeneration in the VRA paradigm. In addition, C3 mRNA expression in PVG and DA rats was studied in a brain contusion model. One week after unilateral brain contusion levels of C3 mRNA measured with real time PCR were more than twice as high in DA rats compared to PVG ( pb0.01). These results demonstrate substantial differences in the regulation of C3 expression after mechanical nerve-injuries as well as a potential link between genetically determined differences in nerve cell survival in the VRA paradigm and the expression of C3.
Cytogenetic and molecular-genetic markers of age changes of lymphocytes S. Kuznetsova Institute of Gerontology, Kiev, Ukraine Objective: Study on Y-chromosome polymorphism in three ethnic regions: Abkhasia and Azerbaijan (high) and Ukraine (medium longevity level) for establishing ethnic longevity variants. Methods: In 105 centenarians of Abkhasia and Azerbaijan, the Y chromosome polymorphism by C heterochromatin and Y chromosome length according to Y/F index were determined. Results: In long-lived of Abkhasia and Azerbaijan, the length of Y-chromosome was bigger than that in Ukraine (Y/F index is 1.16F0.04; 1.08F0.03 and 0.99F0.10, pb0.05, respectively). In middle-aged population segment, ethnic differences were more marked compared to those of centenarians. Sizes of the C band on Y-chromosome in centenarians of three study regions did not differ significantly: 65.39F1.19% in Abkhasia, 1.02F0.03, pb0.05 in Azerbaijan and 0.86F0.02, pb0.05 in Ukraine. However, there were ethnic differences of C band size on Y-chromosome in middle-aged group. The longest C band was registered in Azerbaijan nationality (70.51F1.81%); it was shorter in Abkhasians and Ukrainians: 51.28F2.93%, and 45.28F1.36% ( pb0.05), respectively. Conclusions: The absence of marked ethnic differences of Y-chromosome and C band sizes in long-lived and their presence in middle-aged groups point to the presence of general Y-chromosome polymorphism variants in long-lived of three regions. The sizes of Y-chromosome and C band in individuals with ethnic longevity (Abkhasia and Azerbaijan) are similar to indices of longlived. This fact suggests that Y-chromosomal polymorphism variants constitute the cytogenetic markers of both individual and probably ethnic longevity. Leber's hereditary optic neuropathy (LHON) is associated with point mutations in the mitochondrial DNA and characterized by bilateral, usually sequential, optic neuropathy that may co-occur with multiple sclerosis (MS)like white matter lesions. Despite of repeated clinical reports including magnetic resonance imaging as well as histopathological examination of the visual system, there is a paucity of histopathological descriptions of LHON associated with MS-like syndrome. We present here the case of a female patient with the T14484C mutation, who presented bilateral optic neuropathy and consecutively developed Hashimoto thyreoiditis as well as widespread demyelinating CNS lesions outside the visual system. She died of bronchopneumonia at the age of 44. Immunohistochemical analysis revealed a spectrum of neuropathological changes, including actively and inactively demyelinating plaques in the white matter and optic nerve, vacuolation and cystic necrosis with CD8+ T-cells in the frontal lobe, axonal damage and shadow plaques. Massive tissue destruction, cystic necrosis, respectively, was associated with immunohistochemically detectable inducible nitric oxide synthase (iNOS) in macrophages and microglia. This variable phenotype of extraoptic LHON disease suggests that mtDNA mutations may affect the nervous system on a common metabolic basis and occasionally may aggravate or initiate autoimmune pathology. Guillain-Barré syndrome (GBS) is a post-infectious polyradiculoneuropathy characterized by inflammatory demyelination and axonal degeneration. Macrophages and ganglioside-specific IgG have been implicated in the pathogenesis of GBS. We recently demonstrated the capacity of antiganglioside IgG to induce leukocyte activation via crosslinking of IgG receptors (FcgammaR), suggesting inflammatory potential of autoantibodies in GBS. Three FcgammaR family members, FcgammaRIIa, FcgammaRIIIa and FcgammaRIIIb, display functional bi-allelic polymorphisms (FcgammaRIIa: H131 vs. R131; FcgammaRIIIa: V158 vs. F158; FcgammaRIIIb: NA1 vs. NA2), and determine the efficacy of IgGmediated cellular inflammatory responses. FcgammaRIIa-H131 binds IgG2 and IgG3 immune complexes more efficiently than FcgammaRIIa-R131, whereas FcgammaRIIIa-V158 and FcgammaRIIIb-NA1 display increased binding capacity for IgG1-and IgG3-immune complexes. Therefore, FcgammaR polymorphisms could influence the vigour of antibody-induced immune responses. To assess the relevance of FcgammaR polymorphisms for susceptibility to and severity of GBS, we determined FcgammaR genotype distributions in 192 Dutch and 91 British patients with GBS, and 514 Dutch and 111 British controls by allele-specific PCR. The FcgammaRIIIb-NA2/2 genotype frequency was increased among Dutch patients with severe disease compared to patients with mild disease (OR=2.03 (1.06-3.90), p=0.03). A meta-analysis incorporating all previously published data, encompassing a total of 345 GBS patients and 714 controls, confirmed the association of the FcgammaRIIIb-NA2 allele with severe disease, and additionally showed an enrichment of the FcgammaR-IIIa-F/F158 genotype in patients with mild disease compared to patients with severe disease (OR=0.64 (0.40-1.02), p=0.06). FcgammaRIIIa and FcgammaRIIIb genotypes may represent disease-modifying factors in GBS.
No association of IL-10 and IL-12 polymorphisms with myasthenia gravis (MG) V. Yilmaz a,b , Y.G. Parman c , P. Serdaroglu c , F. Deymeer c and G. Saruhan-Direskeneli b a DETAE, Department of Immunology; b DETAE, Department of Physiology and c DETAE, Department of Neurology, Istanbul Medical Faculty, Istanbul, Turkey MG is characterized by autoantibodies (Ab) to acetylcholine receptor on the post-synaptic membrane of the neuromuscular junction. The production of these Ab is regulated by T cells via cytokines. Interindividual differences in cytokine profiles appear related to allelic polymorphisms of genes. Differences in the distribution of polymorphic alleles may have implications on genetic susceptibility to MG. To analyze the genetic susceptibility to MG, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was applied to screen single nucleotide polymorphisms at À3575(T/A) and À2763(C/A) positions of the IL-10 promoter and at +16974 (A/C) 3V UTR of the IL-12p40 genes in 106 patients and 152 healthy controls (HC). Among the MG group, 74% were seropositive and 30 of selected 63 patients had anti-titin Ab. Comparisons of allele, haplotype and genotype frequencies of IL10 and IL12p40 polymorphisms between MG and HC groups or subgroups of MG did not reveal any significant difference. However, an association of IL-12p40 3V UTR C allele with anti-titin antibody positivity is detected (OD=4.25, pc=0.02). These results implicate that the selected IL10 and IL12p40 polymorphisms do not contribute to genetic susceptibility in MG. This study is supported by the Istanbul University Research Fond (T-1125). Myasthenia gravis (MG) is an autoimmune disorder mediated by autoantibodies against the nicotinic acetylcholine receptor (nAChR) of neuromuscular junction. The production of autoanti-nAChR antibodies in MG is dependent on T-cell help. A polymorphism in the cytotoxic Tlymphocyte-associated antigen-4 (CTLA4) gene has been reported to alter T cell activation and confirm genetic susceptibility to several autoimmune diseases including MG. To determine the role of CTLA4 gene polymorphisms in predisposition to MG, we investigated 101 patients with MG and 135 ethnically matched healthy controls for allelic determinants at two polymorphic sites of this gene (in the promoter region and in the first exon at position +49) by MseI and BstEII restriction enzyme digest analyses correspondingly. Allele frequencies of C-318T gene polymorphism in patients and in healthy controls for allele C were found to be 86.74% and 88.15% correspondingly. The frequency of the allele A of A+49G polymorphism was 48.91% in the MG group compared with 50.0% in the control group. There was no significant difference in the allele frequencies distribution of these loci between patients and healthy controls. Our results did not show any evidence of association of C-318T and A+49G of CTLA4 gene polymorphisms with MG. Myasthenia gravis (MG) is an autoimmune disorder in which antibodies are directed against the human nicotinic acetylcholine receptor at the neuromuscular junction. An association with HLA class II antigens and the disease was described in other countries. In an ethnically mixed group of Brazilian MG patients, an association with HLA-DR5 was found in the global group (n=78) when compared with case-controls studies (n=237). Interestingly, HLA-DR11 and HLA-DR12, splits of HLA DR5, were significantly over-represented in male MG and in female patients (pc=0.01), when compared to sex-matched controls. Severe MG was associated with HLA-DR2 and HLA-DR3 ( p=0.04 for both). The co-occurance of HLA-DR1 with HLA-DR5 ( p=0.03) or HLA-DR5 homozigozity ( p=0.006) appears to have a multiplying effect on the risk of developing MG. In contrast, HLA DR6 appears to be involved in protection as well as HLA DR7, a common protective marker for male MG patients or nonthymomatous thymectomy. On the other hand, an strong association between HLA DR7 and early-onset disease was found in female MG patients ( p=0.0005). In summary, association with HLA-DR class II in Brazilian MG patients will contribute to understand the complexity of the disease and the HLA phenotype role on MG pathogenesis. Supported by FAPESP (00/11889-9). Age-related changes in the inflammatory response to cytokines in the brain S. Campbell and D.C. Anthony University of Southampton, Southampton, UK Adult rat brain is resistant to the action of pro-inflammatory mediators and to the recruitment of leukocytes following injury, but this resistant phenotype may be age-dependent. In this study, we examined whether age-related changes in the inflammatory response reflect altered responses to the pro-inflammatory cytokines interleukin-1h (IL-1h) or tumour necrosis factor-a (TNF-a). In response to equivalent brain microinjections of IL-1h or TNF-a, juvenile and 18-month-old aged rats displayed increased leukocyte recruitment to the brain and a loss of specificity of the leukocyte recruitment profile compared to the responses in 2-month-old adult rats. Neutrophil recruitment through vessel walls was associated with loss of the tight-junction protein claudin-1, and with blood-brain barrier breakdown. Leukocyte recruitment to the liver, a surrogate marker for the hepatic acute-phase response after brain injury, was also elevated in juvenile and aged rats as compared to adult rats. These studies suggest that windows of susceptibility exist in the rodent brain, at either end of the age spectrum, which should guide experimental design to assess anti-inflammatory strategies for age-related neuropathologies.
Chemokines modulate calcium dynamics and electrophysiological properties of CNS neurons D.L. Gruol a , K.L.I. van Gassen b , J.G. Netzeband a and P.N.E. de Graan b a The Scripps Research Institute, La Jolla, CA, USA; b Rudolf Magnus Institute for Neuroscience, Utrecht, The Netherlands CCL2 is one of a number of chemokines that belong to the cytokine superfamily and are involved in inflammatory and chemoattractive processes. CCL2 is produced at high levels in the central nervous system (CNS) during viral infection, injury, neuroinflammation and other neurodegenerative conditions. CNS cells including neurons and glia express receptors for CCl2 and other cytokines suggesting that pathologic conditions are communicated to CNS cells through cytokine signaling pathways. However, our understanding of the consequences of activation of cytokine signaling systems in the CNS is relatively limited, especially for CNS neurons. In many cell types, chemokine signaling alters intracellular calcium dynamics. Therefore, we have investigated the potential involvement of this mechanism in the neuronal signaling activated by CCL2. In addition, we have examined the effects of CCL2 on neuronal excitability. The studies focused on the cerebellar Purkinje neuron, an identified CNS neuronal type that expresses CCR2, the receptor for CCL2. The studies were carried out in a culture preparation and CCL2 was tested by acute bath application. High concentrations (25-100 nM) of CCL2 were used to reflect conditions during a disease state. Results show that CCL2 increased resting calcium levels, enhanced the calcium response evoked by a metabotropic glutamate receptor agonist and depressed excitability. These modulatory effects of CCL2 on neuronal properties are likely to contribute to alter CNS function associated with CNS disease and injury. Supported by MH63712 and HD0382310. Microglia and infiltrating macrophages are involved in phagocytosis of apoptotic cells and myelin debris in multiple sclerosis (MS) lesions in the central nervous system (CNS). The accumulation of ingested lipids is associated with a foamy appearance of these cells. It is generally assumed that foamy macrophages contribute to a local pro-inflammatory environment, but this assumption is based on surprisingly scant evidence. Since many lipid mediators are actually known to be mediators of immune suppression, we addressed the hypothesis that foamy macrophages demonstrate an anti-inflammatory phenotype in situ, and that this can be mimicked in vitro by myelin-lipid preparations. We demonstrate that the large majority of foamy macrophages in postmortem MS brain express markers characteristic for anti-inflammatory alternatively activated macrophages (aamf), including IL-1ra, CCL18 and CD163, and produce the antiinflammatory molecules IL-10 and TGF-beta. Expression patterns vary strongly and depend on the location within the lesion. Macrophages in perivascular infiltrates do express pro-inflammatory cytokines, such as TNF-alpha and IL-1beta. Preliminary in vitro data suggest that myelin-lipid preparations decrease LPS-induced TNF production and induce IL-10 by macrophages. Thus, during MS, foamy macrophages within CNS lesions resemble aamf, which may be caused by lipid mediators since myelinderived lipids interfere with pro-inflammatory mechanisms in macrophages in vitro. Our data suggest that phagocytic macrophages may display regulatory functions within MS lesions.
The surface unit of MSRV (multiple sclerosis associated retroviral element) envelope protein induces an immune bias in patients with MS that correlates with disease activity A. Rolland a , E. Jouvin-Marche a , M. Saresella b , P. The presence of MSRV, a retroviral element defining a novel family of human endogenous retroviruses (HERV-W) has now been confirmed in serum and cerebrospinal fluids (CSF) of patients with multiple sclerosis (MS) and a correlation between circulating MSRV virion load and MS evolution has been demonstrated. In an attempt to link the potential effects of MSRV with MS disease, the pro-inflammatory properties of the surface unit of MSRV envelope protein were evaluated in PBMC of MS patients and compared with healthy controls. We observed divergent reactivity to ENV-SU between MS and control PBMCs. This was reflected by a significant increase of IFN-gamma, IL-6 and IL-12p40 productions by the overall MS population when compared with the control group. TNF-alpha, IL-1-beta and IL-10 were similarly stimulated. Interestingly, the over productions of IL-6 and IL-12p40 were found to correlate with the Expanded Disability Status Scale (EDSS) of the patients. The surface unit of MSRV envelope protein can thus affect the immune system of patients with MS and the immune bias thus created appears to increase with disease evolution. The pro-inflammatory properties of ENV-SU could be compatible with a relationship between MSRV expression, MS immunopathology and disease evolution.
TRADD interacts with STAT-1alpha and influences IFN-gamma signaling in macrophages/microglia E.N. Benveniste, H. Qin, N. Kokorina and D. Wesemann University of Alabama at Birmingham, Birmingham, Alabama, USA Inflammatory events in the Central Nervous System (CNS) contribute to the pathology underlying diseases such as multiple sclerosis, Alzheimer's disease and HIV-1-associated dementia, and activated macrophages/ microglia are central to this response. Macrophages/microglia respond to cytokines upon engagement of cell surface receptors and activation of complex intracellular signaling pathways. IFN-gamma and TNF-alpha are potent activator of macrophages and microglia and are aberrantly expressed in the CNS in the diseases mentioned above. Tumor necrosis factor receptor-1 (TNFR1)-associated death domain protein (TRADD) plays an essential role in recruiting signaling molecules to the TNFRI receptor complex. IFN-gamma utilizes signal transducer and activator of transcription-1alpha (STAT-1 alpha) for signal transduction. Herein, we demonstrate that IFN-gamma induces the formation of a nuclear-localized TRADD-STAT-1alpha complex. IFN-gamma-mediated STAT-1alpha phosphorylation is prolonged in macrophages with reduced TRADD expression. Moreover, an increase in IFN-gamma-mediated STAT-1alpha DNA binding activity, nuclear presence and transcriptional potential is observed in the TRADD bknock-downQ cells. These data indicate that TRADD may play a role in IFN-gamma signaling by forming a complex with STAT-1alpha within the nucleus, and regulating IFN-gammamediated STAT-1alpha activation. Thus, TRADD has a functional role in regulating the response of macrophages/microglia to IFN-gamma, impacting on cellular activation and subsequent neuroinflammatory responses.
Identification of a complement 5a-like receptor from astrocytes: characterization of anti-inflammatory properties V. Gavrilyuk a , S. Kalinin a , B. Hilbush b , A. Middlecamp b , S. McGuire c and D.L. Feinstein a a University of Illinois, Chicago, IL, USA; b Digital Gene Technology, San Diego, CA, USA; c Loyola University, Chicago, IL, USA Brain inflammation is regulated by endogenous substances, including noradrenaline (NA) which can increase anti-inflammatory genes. To identify NA-regulated, anti-inflammatory genes, we used TOGA (total gene expression analysis) to screen astrocyte-derived RNA. NA-inducible cDNA clone DST11 encodes a form of the complement C5a receptor (C5aR), with 39% identity to rat C5aR, and 56% identity to human C5aR variant C5L2. Quantitative PCR shows that in astrocytes DST11 mRNA is increased by NA, whereas in vivo depletion of NA reduced DST11 levels. Western blot analysis demonstrated basal and NA-induced expression of DST11 in primary astrocytes. Immunocytochemical staining revealed DST11-immunoreactivity throughout brain, co-localized to neurons and astrocytes. In astrocytes, induction of nitric oxide synthase type 2 or IL-beta was increased by treatment with antisense oligonucleotides to DST11, and reduced in cells overexpressing DST11. Levels of human C5L2 andC5aR, were lower in MS and AD brains versus controls. These results demonstrate that DST11 is a C5aR isoform expressed by glia and neurons, regulated by NA, and exerts anti-inflammatory functions. Changes in DST11 levels in brain could contribute to the progression of inflammatory damage. This work was sponsored by a grant from NIH.
IL-1 activates interferon regulatory factor-3: implications for an antiviral response in human primary astrocytes M.A. Rivieccio a , G.R. John b , H.S. Suh a , X. Song a , S.C. Lee a and C.F. Brosnan a a Albert Einstein College of Medicine, New York, USA; b Mount Sinai School of Medicine, New York, USA
The cytokine interleukin-1 (IL-1) is a major activator of astrocytes, leading to the production of a wide range of cytokines and chemokines important for host defense against pathogens. IL-1, like LPS, signals via the MyD88/ IRAK-1 pathway linked to NF-kappaB and AP-1. Recent studies have shown that the TLR4 ligand LPS signals independently of MyD88 resulting in the activation of interferon regulatory factor-3 (IRF-3), a transcription factor required for induction of primary anti-viral response genes such as type 1 interferons and the chemokines RANTES/CCL5 and IP-10/ CXCL10. Considering the apparent similarities between IL-1 and TLR4 signaling, we hypothesized that IL-1 is also capable of activating IRF-3 in human primary astrocytes and that this activation would result in the expression of IRF-3-dependent type I interferons necessary for innate antiviral immunity. Using Q-PCR and ELISA, we found that IL-1-induced IFNbeta, RANTES/CCL5, and IP-10/CXCL10 in astrocytes. We also observed that IL-1 induced IRF3 nuclear translocation by immunofluorescence and by cell fractionation followed by immunoblotting. We confirmed the dependency of type I interferon expression on IRF3 activation by utilizing a dominant negative IRF3 expressing adenovirus. These data are the first to show that IL-1, in addition to TLR's, can stimulate IRF3 in human primary astrocytes, implicating this cytokine as an activator of genes involved in innate antiviral responses.
Downregulation of Abeta-induced cytokine expression in glial cells using NFkappaB decoy L. Fisher, M. Samuelsson, V. Cortes Toro, U. Soomets, J. Yang, Ü . Langel and K. Iverfeldt Department of Neurochemistry and Neurotoxicology, Stockholm University, Stockholm, Sweden Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with impairments in cognition and memory. The beta-amyloid (Abeta) plaques seen in AD are surrounded by activated astrocytes and microglia. It is believed that these activated glial cells contribute to neurotoxicity through the induction of oxidative damage and proinflammatory cytokines like interleukin-1beta (IL-1beta). The activation of glial cells is believed to be an early event in the development of AD, and both IL-1beta and interleukin-6 (IL-6) have been detected early in AD brains, suggesting that these cytokines may play an important role in the development of AD. For many inflammatory events, including the cytokine induction in glial cells, the transcription factor NFkappaB plays a key role. This suggests that therapeutical strategies aimed to control the development of AD could include administration of drugs that hinder NFkappaB activation. In this study, the effects mediated by IL-1beta and the Abeta (1-42) and Abeta (25-35) peptides on NFkappaB binding activity and cytokine expression in rat primary mixed glial cell cultures were examined. We also investigated the possibility to block the action of NFkappaB using double-stranded oligonucleotides containing the consensus sequences for NFkappaB binding. We investigated the effect of blocking the NFkappaB action on cytokine expression in these cells. In order to increase the cellular uptake of the NFkappaB decoy, we have used the cell penetrating peptide Transportan.
Role of TNF in axonal lesion-induced microglial activation in vivo C. Fenger, N. Drbjdahl, M. Meldgaard, R. Ladeby, K. Lambertsen and B. Finsen University of Southern Denmark, Odense C, Denmark TNF is known to exert strong auto-and paracrine effects on microglia in vitro and presumably in vivo. Here, we investigated the role of TNF and its receptors on the microglial response to Wallerian and terminal synaptic degeneration within the hippocampus. Quantitative PCR of microdissected, perforant path-denervated hippocampus showed a 22-fold upregulation of TNF mRNA, and a 2-to 3-fold upregulation of TNF-p55R and -p75R mRNA at days 2 and 5, respectively. The presence of TNF was confirmed by Western blotting. In situ hybridization showed microglial expression of TNF mRNA at day 2, and upregulation of TNF-p55R and -p75R mRNA in microglial-like cells starting at day 2, when microglia underwent proliferation. Lesioning induced a 3-fold increase in microglial number in wildtype mice at day 5 (251F20, lesioned n=11; 81F7, unlesioned n=5). Comparison of microglial numbers in wildtype (251F20, n=11; 81F7, n=5), TNF-KO (219F15, n=14; 85F5, n=7), and TNF-p55p75R-KO (221F11, n=13; 95F3, n=4) mice pointed towards a reduced lesion-induced increase in microglial number in TNF-and TNF-R-KO mice, which, however, was not statistically significant ( P=0.16, two-way ANOVA). The data suggest that endogenously produced TNF may be redundant in terms of lesion-induced expansion of the microglial population in vivo.
Production of interferon-gamma by microglia J. Kawanokuchi, T. Mizuno, H. Kato, H. Takeuchi, J. Wang, G. Zhang, N. Mitsuma, R. Kuno, S. Jin and A. Suzumura Department of Neuroimmunology, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan
Because of the lack of major histocompatibility complex (MHC) antigen expression, neural cells had been considered not to interact with immune cells in a specific manner. Interferon-gamma (IFN-gamma) enables the interaction via induction of MHC antigen expression in neural cells. IFNgamma also induces inflammatory mediators such as TNF-alpha or nitric oxide in glial cells. Thus, IFN-gamma is a critical cytokine in the development of CNS pathologies including neuronal degeneration and inflammatory demyelination. However, IFN-gamma is considered to be produced exclusively by lymphoid cells. Here, we show that murine microglia, brain macrophages, are able to produce IFN-gamma in response to IL-12, IL-18 and both, by means of RT-PCR method for induction of IFN-gamma mRNA, Western blotting and immunofluorescence for cytoplasmic expression of IFN-gamma in microglia. A murine microglial cell line, 6-3 cells, are also induced to produce IFN-gamma whereas astrocytes did not produce IFN-gamma with above stimuli. Induction of IFN-gamma resulted in the induction of class II MHC antigen expression in microglia. Since IL-12 and IL-18 are produced in the CNS by glial cells, these cytokines may play a critical role in initiation of neural cells-immune cells interaction to induce autoimmune processes in the CNS, in either autocrine or paracrine manner.
Biological roles of two new haemopoietic factors in brain and thymus A. Kikuchi a , H. Tomoyasu b , T. Yamamoto c , M. Kusakabe d , N. Sakuragawa e and I. Kamo e a National Institute of Neuroscience, Tokyo, Japan; b Ohmori Red Cross Hospital, Tokyo, Japan; c Institute of Medical Science, University of Tokyo, Tokyo, Japan; d ANB Tsukuba Institute, Tsukuba, Japan; e School of Medicine, Toho University, Sagamihara, Japan
We found new 80-kDa and 100-kDa haemopoietic factors from coned rat thymic myoid cells. Many cells that produce these factors were detected in myasthenic hyperplastic thymi, and in normal brains, suggesting that they have biological roles other than monocytic cell growth. In the present study, we aimed to understand their potential biological roles in the brain and myasthenic thymus. New born rat brain, and rabbit anti-80-kDa and 100-kDa factor IgG were used for immunohistochemical studies. Neural cells obtained form rat cerebrum were cultured for kinase assay. The 80-kDa and 100-kDa factors were detected in young and aged rat brain tissues. Cultured brain cells and glial cell lines produced these two factors. The two factors purified from these cultures stimulate the growth of microglial cells. The two antibodies react with neural cells. These factors support neural cell growth. In hyperplastic myasthenic thymi, these factors were consistently detected. The two factors also support the growth of B cells. These results suggest that the two new haemopoietic factors play important physiological roles in brain and regulatory roles in autoimmune disease.
Chemokine secretion and chemokine receptor expression in brain cell aggregate cultures treated with interferon-gamma F.A. Marques, I.A. Romero, D.K. Male and A.J. Loughlin The Open University, Milton Keynes, UK A 3-dimensional brain cell aggregate culture system, which approximates to the brain in vivo in terms of glial and neuronal development and cell-cell interactions, was used as a model of the CNS parenchyma in vitro. Cultures were established from brain tissue of 16-day gestation Sprague-Dawley rat embryos. In untreated aggregates, the chemokine receptors CCR1, CCR5 and CX3CR1 were detectable at the mRNA level. Treatment with IFNgamma resulted in an increase in expression of CCR5 and CX3CR1 but not CCR1. There was no detectable expression of CCR2 or CXCR3 in unstimulated aggregates, nor was the expression of these receptors affected by IFN-gamma treatment. At the protein level, CCR5 and CX3CR1 were detected by immunocytochemistry, though IFN-gamma had no discernable effect. IFN-gamma induced secretion of the ligands for these receptors (CCL5 and CX3CL1 respectively) in a time-and dose-dependent manner. The responses to IFN-gamma observed in this study are modest compared to those observed in isolated glial cell populations. These results suggest that the nature and extent of the inflammatory response in the CNS cannot be predicted from studies of isolated cell populations, but critically depends on interactions between cells and a complex milieu of secreted mediators. Our approach complements existing in vivo and in vitro studies and allows us to assess the effect of inflammatory mediators on cells within a complex CNS environment.
Capsaicin up-regulates the production of cytokines by dorsal root ganglion (DRG) neurons in culture N.E. Saade, H. Al Tannoury, M. El-Sabban, B. Safieh-Garabedian and S.J. Jabbur American University of Beirut, Beirut, Lebanon Background and Aims: Previous work from our laboratory has characterized the effects of endotoxin (ET) treatment on the secretion of proinflammatory cytokines by cultured DRG neurons. The aim of this study was to investigate whether the challenge of DRG cells by capsaicin can alter their secretion of tumor necrosis factor (TNF) alpha, interleukins (IL) 1, 6 and 10 and prostaglandin E2 (PGE2). Methods: Primary cultures of adult rat DRG neurons were treated at day 4, with either ET (5 microg in 1 ml) or with capsaicin (0.1, 1 or 10 microg in 1 ml) for 30 min. After treatment, cells were washed and re-supplied with fresh media and the supernatant was sampled at 1.5, 3 and 4.5 h . The concentrations of all the mediators were determined by ELISA. Results: Capsaicin produced a significant increase of the levels of IL6, IL10 and PGE2, reaching more than 10 folds their initial concentrations. This increase was comparable to (albeit slightly less than that observed with ET challenge. The optimal effects were obtained with the concentration of 1microg of capsaicin. Conclusion: (1) Capsaicin challenge to DRG culture upregulates the level of pro and anti-inflammatory mediators. (2) Our results provide a molecular background for the concept of neurogenic inflammation. (Supported by grants from CDRE and the LNCSR).
Effects of chronic expression of IL-1beta in the brain C.C. Ferrari a , A.M. Depino a , F. Prada a , O. Podhajcer a , H. Perry b , D. Anthony c and F. Pitossi a a Gene Therapy Laboratory, Institute for Biochemical Research, Buenos Aires, Argentina; b CNS Inflammation Group, University of Southamptom, Southampton, UK; c Molecular Neuropathology, University of Southampton, Southampton, UK Interleukin-1beta (IL-1) has been implicated in the pathology of several chronic neurodegenerative diseases including multiple sclerosis, Alzheimer's disease and prion disease. However, the effects of long-term IL-1 expression in the brain parenchyma on CNS integrity have not been investigated. We used a recombinant adenovirus producing IL-1 to examine the effects of chronic IL-1 expression in the CNS parenchyma. IL-1 expression was detectable until 30 days post-injection. Between days 8 and 14, there was marked and restricted recruitment of neutrophils, vasodilatation and breakdown of the blood-brain barrier (BBB) ipsilateral to the injection side. Microglia and astrocyte activation was evident through days 2 to 14. Disorganization of the nervous tissue and vast demyelination were observed at 8 and 14 days. No signs of neurodegeneration were observed. Oligodendrocytes and neurons have no evident ultrastructural alterations. Interestingly, at 30 days, the nervous tissue has completely recovered, appearing normal with no signs of demyelination, infiltration or BBB breakdown. In summary, our data show that chronic expression of IL-1, in contrast to its acute delivery, can reversibly damage CNS integrity and implicates this cytokine or downstream components as major mediators of demyelination in chronic inflammatory and demyelinating diseases.
Hepatic CC chemokines control the magnitude of the inflammatory response within the injured rodent brain S.J. Campbell a , V.H. Perry a , F. Pitossi b , A.G. Butchart a , M. Chertoff b , S. Waters a and D.C. Anthony a a University of Southampton, Southampton, UK; b University of Buenos Aires-CONICET, Buenos Aires, Argentina
Hepatic CXC chemokines, behaving as acute phase proteins, regulate neutrophil mobilisation and recruitment following focal IL-1h-mediated inflammation to the rat brain. To determine whether this response was specific to CXC chemokines or whether it represented a more generalised response to acute brain inflammation, we examined brain and liver production of MCP-1, a CC chemokine, when rats were microinjected with TNF-a into the brain. As early as 2h after the TNF-a challenge, MCP-1 mRNA and protein were observed in the liver by Taqman RT-PCR and ELISA. The serum MCP-1 level was also elevated between 2 and 4 h, which was consistent with maximal mobilisation of leukocytes into the blood. Monocyte recruitment was most marked in the liver after 6 h, but was delayed in the brain until 24 h. Elevated hepatic and serum chemokines are implicated in the control of leukocytosis and leukocyte recruitment to the brain and liver, since dexamethasone pretreatment attenuated the hepatic MCP-1 response, modulated leukocyte mobilisation and reduced monocyte entry not only to the brain but also to the liver. Thus hepatic chemokine production controls and amplifies the CNS response to inflammation by controlling the rate, timing, magnitude and composition of leukocyte recruitment to the damaged brain.
Interleukin-1 beta contributes to the early regulation of inflammation after spinal cord injury T.K. Rice, J.E.A. Wells and V.W. Yong University of Calgary, Calgary, Canada
After spinal cord injury (SCI), a number of inflammatory molecules become rapidly elevated. The role of neuroinflammation in SCI is unclear. The objective of this study is to examine neuroinflammation after SCI and to determine whether a particular molecule regulates this process. SCI is induced in CD-1 mice using a clip compression model and the temporal expression patterns of several cytokines, chemokines and matrix metalloproteinases (MMPs) are determined using RNase Protection Assays and real-time PCR. Interleukin (IL)-1 beta is one of the early inflammatory molecules upregulated after SCI. IL-1 beta transcripts increase within 30 min of SCI. Transcript levels of several chemokines (MIPs, MCP-1 and IP-10) are also elevated by 30 min after SCI, but their expression level is low. The expression of several MMPs becomes prominent later. To determine if IL-1 beta is involved in regulating the expression of other inflammatory molecules, wild-type mice and mice genetically deficient for IL-1 beta are compared for differences in inflammatory molecule expression following SCI. IL-1 beta deficient mice show reduced levels of several cytokines (IL-1 alpha, IL-6), chemokines (RANTES, IP-10, MCP-1, MIPs) and MMP-3, 9 and 12. We conclude that IL-1 beta is upregulated early after SCI and that it may be a key regulator of neuroinflammation in this condition.
Granulocyte macrophage-colony stimulating factor (GM-CSF) and triidothyronine (T3) increase oligodendrocyte production in the corpus callosum (CC) following intracerebroventricular (ICV) infusion T. Dubois and S. Weiss University of Calgary, Calgary, Canada
We have found that both GM-CSF and T3 produce more oligodendrocytes from embryonic neural stem cells (NSCs). Thus, we hypothesized that these two factors would also increase oligodendrocyte production from adult NSCs. Both GM-CSF and T3 increased the percent of terminally differentiated oligodendrocytes in neurospheres derived from adult subventricular zone NSCs. To ask whether these factors might regulate NSC production of oligodendrocytes in vivo, we infused (ICV) solutions of either a vehicle control, GM-CSF or T3 into the lateral ventricles of adult mice and evaluated oligodendoglial development through immunocytochemical methods. Both GM-CSF and T3 increased the numbers of immature and maturing oligodendrocyte progenitors as well as terminally differentiated oligodendrocytes in the CC, compared to control infusions, suggesting that they increase oligodendroglial production through all stages of development. Both GM-CSF and T3 also decreased the number of TUNEL-positive cells in the CC suggesting that they reduce cell death. However, only GM-CSF increased the percent of BrdU+ cells within the population of mature oligodendrocytes, suggesting that GM-CSF is enhancing oligodendrocyte survival whereas T3 principally regulates differentiation. Further studies are underway to investigate the actions of these two factors following demyelinating lesions with the aim of further understanding whether enhancing the survival and differentiation of newly generated oligodendrocytes might improve remyelination. Supported by the MS Scientific Research Foundation of Canada.
108 Alphaviral expression of transforming growth factor beta 1 inhibits autoimmune encephalomyelitis in mice M.J.V. Vähä-Koskela, J.C. Holmlund and A.E. Hinkkanen Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland Introduction: Current treatment strategies for chronic, inflammatory diseases of the central nervous system (CNS) are almost solely based on down-regulation of the immune system by systemically administered drugs. Although some drugs may show efficacy in experimental settings, successful treatment of CNS disorders is greatly impeded by the limited access or insufficient quantities of these compounds at the site of inflammation. Aims: To assess the feasibility of live, neurotropic virus to interfere with CNS inflammation by expression of immunomodulatory cytokines. Methods: Genes for both latent and constitutively active transforming growth factor (TGF) beta 1 were cloned into replicationcompetent Semliki Forest virus vector VA7, described elsewhere (V7h7-Koskela et al., 2003) . To study their function in vivo, vectors were administered intraperitoneally to groups of Balb/c mice induced with EAE. Results: The TGF beta vectors were shown to produce biologically active TGF beta-1 in cell culture. When administered to mice with EAE, the vectors were able to significantly inhibit both the development and the mean severity of the disorder, compared to mice receiving only saline. Conclusions: Replication-competent, avirulent alphaviral vectors may serve as vehicles for non-invasive gene delivery into CNS. Anti-inflammatory immunomodulation by virally mediated cytokine expression offers a formidable treatment strategy for inflammatory CNS disorders, where therapy by conventional drug administration may be limited.
Potential role of interleukin-15 (IL-15) in autoimmunity and impact of IGIV on IL-15 expression N. Eller, S. Inoue and D. Scott Food and Drug Administration, Bethesda, USA Interleukin-15 (IL-15) is a four-alpha helix cytokine that shares some receptors subunit usage and biological functions with IL-2. It is involved in the generation and regulation of a normal immune response from monocyte activation to memory CD8+ T cell development. Immune cells such as monocytes/macrophages, microglia, and dendritic cells, are among the few cell types that produce IL-15. Il-15 may be membranetethered or associated with the IL-15Ralpha subunit and very little is secreted from cells, making protein detection difficult. IL-15 has been associated with autoimmune diseases such as multiple sclerosis (MS), and rheumatoid arthritis. It is unclear whether IL-15 has a role in disease initiation or if it is a marker of disease and disease pathogenesis. We developed a method for inducing, detecting, and modulating IL-15 on the surface of human and mouse cells or cell lines using FACs analysis. This method can be used to correlate IL-15 levels with clinical disease. Immune Globulin, Intravenous (IGIV) has been shown to have immunomodulatory effects and has been used in the treatment of MS with varying results. Using a mouse macrophage cell line, RAW 264.7, we have shown that IGIV reduces expression of IL-15 and to a lesser extent, TNF-alpha. Current studies are assessing the role of IL-15 in induction and persistence of experimental autoimmune encephalomyelitis (EAE) in an adoptive transfer model.
Differential macrophage expression of IL-12 and IL-23 upon innate immune activation defines rat experimental autoimmune encephalomyelitis Å . Andersson, R. Kokkola, J. Wefer, H. Erlandsson-Harris and R.A. Harris Karolinska Institutet, Stockholm, Sweden Aim: we compared analysis of the activation phenotypes of MQ derived from autoimmune-susceptible and autoimmune-resistant rat strains. We included investigation of function of expressed receptors, intracellular signaling pathways, cytokines and other soluble mediators released after activation of cells using a panel of stimuli embracing many activation routes. Methods: Real-Time PCR, ELISA, Western blot and Immunohistochemistry. Results: The major differences recorded between DA, BN and PVG rat MQ following activation were in cytokine production and in the balance of NOS/arginase activity. The resultant phenotype representing autoimmune-susceptible DA rat MQ is lower TNF, IL-6, IL-1beta, p35 and NO production with an associated high arginase activity and high expression of p19. Conversely, autoimmune-resistant BN MQ had a phenotype with higher TNF, IL-6, IL-1beta, p35 and NO production with an associated low arginase activity and low p19 expression. Conclusion: Activation of MQ from the autoimmunesusceptible strain was associated with alternative macrophage activation indicated by induction of arginase activity, a lower production of classical proinflammatory mediators and a high expression of IL-23, while MQ from the autoimmune-resistant strains was associated with a higher production of proinflammatory mediators, a classical activation phenotype and preferential induction of IL-12. These MQ phenotypes thus reflect disparate genetic cellular programmes that define autoimmune susceptibility.
The cytokine expression after peripheral nerve injury L.M. Kylliäinen a , K. Kimppa a,b , S. Ruohonen a , M. Tenhami a and M. Rfytt7 a a University of Turku, Turku, Finland; b Åbo Akademi University, Turku, Finland
The aim of the study was to enhance the knowledge of the gene expression during the inflammatory response after peripheral nerve transection by microarrays. The left sciatic nerve of male SD rats was transected. The nerves were allowed to regenerate freely and the first distal section beside the point of injury were taken 12 h, 24 h, 3, 7, 14, 21, 35 and 49 days after the transection. Control samples were collected from non-operated animals. Samples from four rats were pooled in each time point and total RNA was extracted. The samples were hybridized to Affymetrix Rat Genome U34 oligonucleotide microarrays. Sample preparations and hybridizations were performed according to Affymetrix's recommendations. Each sample was compared to control and Signal Log Ratio (SLR) was counted. We decided that values SLRN1.5 or SLRbÀ1.5 are relevant and the expression can then be considered either increased or decreased. Expression of many cytokines and cytokine-related genes were detected and the results correlated well to previous findings. An interesting finding was the significant expression of IL-6 mRNA from 12 h up to 49 days after transection. Also increased expression of inflammatory cytokines IL-1 beta, IL-1 alpha, IL-10 and TGF-beta was noted during the first days after transection. Chemokines MIP-1 alpha, MIP-1 beta and MCP-1 were found to be upregulated.
Interleukin-23 in acute inflammatory demyelination of the peripheral nervous system W. Hu, H.-P. Hartung and B. Kieseier Heinrich-Heine University, Düsseldorf, Germany Interleukin (IL)-23 is a newly identified heterodimeric cytokine comprising the p40 subunit of IL-12 and a private p19 subunit. Mounting evidence suggests that IL-23 rather than IL-12 is critically involved in the pathogenesis of various immune-mediated disorders. In the present study, we analyzed the sequential mRNA expression pattern of IL-23 in sciatic nerves from Lewis rats with myelin-induced experimental autoimmune neuritis (EAN), a classical animal model of the Guillain-Barré syndrome (GBS), using a semiquantitative reverse transcriptase-PCR. IL-23 mRNA was found to be upregulated just prior to the onset of clinical symptoms at day 11 post-immunization (p.i.), with peak levels at day 13 p.i., just preceding maximum clinical disease severity, and declined thereafter. In sural nerves from patients with GBS, IL-23 was found to be expressed, but not in non-inflammatory controls. Macrophages were identified as the cellular source of IL-23 in the inflamed PNS by immunohistochemistry. Thus, our present data suggest that IL-23, primarily expressed by macrophages, plays an important role during the early phase of EAN and therefore appears to be critically involved in the initiation rather than perpetuation of an immune response in immune-mediated inflammation of the peripheral nerve.
The expression of cytokines in silicone tube-treated peripheral nerve injury M. Röytt7 a , S. Ruohonen a , L.M. Kylli7inen a , K. Rfytt7 a , I. Kivi b , M. H7m7l7inen a , A. Pertovaara a and H.S. Taskinen a a University of Turku, Turku, Finland; b Leiras Medical Company, Turku, Finland In order to help axonal reinnervation when the loss of structural continuity occurs, silicone tubes have been used. After peripheral nerve injury, an activation of different cytokines occurs to induce inflammation and/or to help axonal reinnervation. We investigated the expression of cytokines as well as the morphological changes after implantation of silicone tube after removal of a piece of sciatic nerve. The clinical improvement was studied by pinprick and toe-spread tests. The samples from the tube, distal and proximal stumps were taken 1, 3, 5, 7, 14, and 42 days postoperatively. The following cytokines were studied: IL-1h, TNF-a, IFN-g, IL-10 and TGF-h. Neurofilament antibody was used for axonal reinnervation. The sutures used to fix the silicone tube induced foreign body reaction. There was a high initial expression of the studied cytokines but then the expression somewhat decreased. The exception was IFN-g, which showed an increased expression up to five days. After day three the expressions in the tube increased up to day 42, except IFN-g, which declined. The contralateral, uninjured nerve showed some expression at early time points.The present study shows that the insertion of silicone tube induces changes in the cytokine expression. This suggests that these are involved in the building up the missing part of peripheral nerve. Objective: To characterize the production of monocyte chemotactic protein (MCP)-1 and interleukin-1 receptor antagonist (IL-1ra) in the cerebrospinal fluid (CSF) of the patients with Guillain-Barré syndrome (GBS) and Fisher syndrome (FS). Methods: CSF samples from GBS patients (n=8, M/F=3:5, mean age=61:50) and FS patients (n=8, M:F=4:4, mean age=39:38) were collected by lumbar puncture (LP) within 14 days after disease onset. In some cases repeated LP were permitted. CSF samples from eight patients with other non-inflammatory neurological disorders (OND: ALS=4, SCD=4) served as control. Quantitative sandwich ELISA assay for IL-1ra and MCP-1 (R&D) was employed. Results: CSF MCP-1 levels in both GBS and FS patients were significantly increased compared to OND, however, CSF IL-1ra levels significantly increased only in GBS. Strong correlation between CSF MCP-1 and IL-1ra levels was found in FS (R 2 =0.78), not in GBS (R 2 =0.11). Both MCP-1 and IL-1ra levels increased at clinical peak. In GBS, MCP-1 levels decreased prior to IL-1ra during convalescence. Conclusions: MCP-1 may play an important role in both GBS and FS. Contrary the up-regulation of IL-1ra in FS was not clear. This difference may reflect the diversity of PNS involvement area or different cytokine milieu in GBS and FS. In GBS, IL-1ra may indicate recovery from disease although further investigation is needed.
Decreased synthesis of IL-10 during the acute phase of ischemic stroke G. Gromadzka a,b , B. Tarnacka a , I. Sarzynska-Dlugosz a and A. Czlonkowska a,b a Institute of Psychiatry and Neurology, Warsaw, Poland; b Medical University, Warsaw, Poland
During the acute phase of ischemic stroke (IS) local and systemic inflammatory reaction develops. Inflammation leads to increased brain damage and worsen IS outcome. Important mediators inhibiting inflammatory processes are anti-inflammatory cytokines. The aim of study was to evaluate changes in mRNA expression in peripheral blood cells (PBC), and in serum concentration of anti-inflammatory cytokine: interleukin-10 (IL-10) during the acute phase (first 7 days) of IS. Material and methods: 108 patients with IS and 68 healthy control subjects were investigated. Material for investigations was: EDTA-blood and serum. IL-10 mRNA expression in PBC was investigated by semi'quantitative RT-PCR method; serum concentration of IL-10 was investigated by immunoenzymatic test. Results: Decreased IL-10 mRNA expression in PBC and serum concentration of IL-10 was noticed in patients with IS. The lowest synthesis of IL-10 was noticed in patients above 70 years of age, with superficial infarction, with multiple lesions (above 2), and with large lesion area (above 1 lobe) as noticed in CT scan of the brain performed on admission, and also in patients who died during hospitalization. Conclusions: During the acute phase of ischemic stroke the synthesis of anti-inflammatory cytokine IL-10 is diminished. Decreased synthesis of IL-10 is associated with worse prognosis.
Interleukin 18 is increased in sera of acute ischaemic stroke patients and correlates with the size of early brain damage J. Losy a,b and J. Zaremba a a University School of Medicine, Poznan, Poland; b Institute of Experimental and Clinical Medicine, Poznan, Poland Proinflammatory cytokines released during ischaemia by activated brain resident and peripheral blood cells are important mediators of inflammatory response,the process which exacerbates cerebral damage. Interleukin 18(IL-18) is a proinflammatory cytokine able to induce gene expression and synthesis of other proinflammatory cytokines,chemokines and adhesion molecules. Our hypothesis is that IL-18 participates in the inflammatory response during ischaemic stroke. The objective of the study was to determine IL-18 levels in sera of 23 ischaemic stroke patients,compare results with the group of 15 controls and study the relation between IL-18 levels and the volume of hemispheric computer tomography hypodense areas representing early ischaemic changes. Blood sampling and cranial CT were performed within 24 h of stroke. Fifteen tension headache patients served as a control group. IL-18 levels were significantly higher in stroke patients in comparison with controls. Moreover IL-18 levels correlated positively with the volume of early CT hypodense areas (r=0.82; pb0.0001). The results suggest involvement of IL-18 in early inflammatory response during stroke.
Insulin down regulates the overproduction of pro-inflammatory cytokines in peripheral immune cells in Alzheimer's disease S.B. Solerte, D. Taccani, E. Locatelli, E. Ferrari and M. Fioravanti Department Internal Medicine and Geriatrics, University of Pavia, Pavia, Italy Neuroinflammation and immune dysregulation have been implicated in the neurodegenerative processess linked to Alzheimer's disease (AD). The present study was undertaken to evaluate IL-1-beta and TNF-alpha production and seceretion in the supernatants of lymphomononuclear natural killer (NK) cells (7.75Â10 6 cells/ml) of 23 subjects with mild to moderate AD. Cytokines were measured after incubation of NK cells with LPS (1 Ag/ml), beta-amyloid 1-42 fragment (A-beta 1-42; 5 and 10 Ag/ml), IL-2 (100 U/ml) and Insulin (1 and 10 AM/ml) co-incubated with LPS, Abeta 1-42 and IL-2. A significant increase of spontaneous and modulated (by LPS, A-beta 1-42, IL-2) release of IL-1-beta and TNF-alpha by NK cells was demonstrated in AD subjects compared to 23 matched healthy subjects ( pb0.001). The incubation of NK cells with insulin alone significantly reduces the spontaneous secretion of both cytokines in AD subjects ( pb0.001). The co-incubation of insulin with A-beta 1-42 and IL-2 has determined a dose-dependent normalization of IL-1-beta and TNFalpha release from NK cells in AD subjects. However, Insulin action was completely blocked during exposure with 10 AM of p-38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. The present study confirms that NK immune cells are up regulated for the secretion of inflammatory neurotoxic cytokines in AD. The normalizing effects of insulin could depend on MAPK modulation, so involving the insulin intracellular signaling pathways in immune-mediated AD neuropathology.
The gender influence of the IL6 gene expression in a model of Parkinson's disease (PD) A. Ciesielska a , I. Joniec b , A. Przybylkowski b , I. Kurkowska-Jastrzebska a , G. Gromadzka a , A. Czlonkowska a and A. Czlonkowski b a Institute of Psychiatry and Neurology, Warsaw, Poland; b Medical University, Warsaw, Poland
The incidence of PD changes with gender. The higher prevalence of PD in men may suggest a link between estrogens levels and PD. The inflammation is involved in etiopathogenesis of PD. IL6 has been suggested to play a role in regulating neuronal survival in the injured nigrostriatal system. Estrogens could modulate the immune reaction by regulating of this cytokine expression. We examined the contribution of IL6 in the gender difference existing in PD. The levels of IL6 mRNA (RT-PCR) and dopamine (DA) (HPLC) were assessed in the striatum of male and female C57BL/6 mice (12 months old) after 6 h; 1, 3, 7, 14, 21 days post 1-methyl-4-phenyl-1,2,3,6 tetrahydropiridine (MPTP) intoxication. In male and female, the decrease in the level of DA was observed within 1-14 days following MPTP injection, achieving minimal level at 7 day. The male showed significantly greater reductions in striatal DA. MPTP caused an increase of IL6 mRNA in striatum of male and female, but the increase in IL6 was significantly higher in female. In female and male, IL6 expression moderately increased at 1 day and peaked at 7 days post MPTP intoxication. MPTP induced smaller neurotoxicity and greater IL6 gene expression in female than male mice. It couldn't be excluded that, this sexually dimorphic in IL6 mRNA expression may be, in part, responsible for some protection against MPTP intoxication in female mice.
Multiple IL-6 anomalies in patients with major depression S. Alesci a , P. Martinez a , S. Kelkar a , I. Ilias a , A.R. Ayala a , J. Licinio b , G.P. Chrousos a and P.W. Gold a a National Institutes of Health, Bethesda, USA; b University of California, Los Angeles, USA In addition to affective, cognitive, and neuroendocrine abnormalities, depressed patients have increased risk of cardiovascular disease and bone loss. Interleukin-6 (IL-6) is a major inducer of sickness behavior, a potent proinflammatory and prothrombotic agent, and an activator of osteoclastogenesis. In healthy individuals, IL-6 secretion increases with age, correlates with adiposity, and shows a characteristic circadian pattern. The purpose of this study was to compare the 24-h secretion of IL-6 in nine depressed patients and nine gender-, age-and BMI-matched controls, in an attempt to link changes of this cytokine with the manifestations of major depression (MD). Diagnosis of MD was based on the DSM-IV. Plasma IL-6 was measured by high-sensitivity ELISA. The rhythmicity and complexity of IL-6 secretion were assessed by cosinor analyses and approximate entropy (ApEn) algorithms, respectively. Compared to healthy subjects, depressed patients showed highly significant elevations in mean 24 h plasma IL-6 levels (4.7F0.3 vs. 3.4F0.09 pg/ml, meanFSEM, Pb0.001), with 2-to 3fold higher morning values, and a reversal of IL-6 circadian rhythm. The physiologic complexity of IL-6 secretion was reduced in the depressed, as shown by their lower approximate entropy values (0.828F0.029 vs. 0.963F0.024, p=0.035). We conclude that MD is associated with multiple IL-6 abnormalities, which may contribute to the neuropsychological impairment and comorbidity of this psychiatric illness with cardiovascular disease and osteoporosis.
Cytokines and chemokines in subacute sclerosing panencephalitis (SSPE) G. Saruhan-Direskeneli a , S.P. Yentur a , V. Demirbilek b , G. Yilmaz b , E. Onal a , O. Cokar c , Z. Yapici a , G. Akman-Demir a , A. Gokyigit a and C. Gurses a a Istanbul University, Istanbul, Turkey; b Cerrahpasa Medical Faculties, Istanbul, Turkey; c Haseki Hospital, Istanbul, Turkey
To assess the role of cytokines and chemokines in SSPE, cerebrospinal fluid (CSF) and serum samples from 60 SSPE patients, 38 patients with neurological infectious or inflammatory diseases (IN) and 27 patients with noninflammatory neurological diseases (NIN) were examined by ELISA for IFN-gamma, IL-10, IL-4 and IL-12 as well as for CXCL8 (IL-8), CXCL10 (IP-10) and CCL2 (MCP-1) levels. An increased IL-12 response was detected in SSPE: Both median IL-12 CSF and serum levels (19.6 and 128.4 pg/ml) were higher in SSPE than in IN (6.5 and 68.5 pg/ml, pb0.001) and in NIN (1.8 and 70.3 pg/ml, pb0.001 and p=0.05) groups. IL-10 CSF levels were also higher in SSPE than in NIN group (2.6 vs. 0.1 pg/ml, pb0.001), whereas IL-4 and IFN-gamma levels did not differ between groups. CXCL10 level was increased in CSF of SSPE compared to NIN (332.2 vs. 16.2 pg/ml, pb0.001). However, higher CXCL8 was detected in the serum of SSPE (11.5 pg/ml) compared to IN and NIN groups (3.1 and 2.2 pg/ml, p=0.03 and p=0.01, respectively). The increased systemic and intrathecal IL-12 secretion without accompanying IFN-gamma induction may implicate an initial immune activation without an effective cellular response in the pathogenesis of SSPE. Selective intrathecal increase of CXCL10 points to the differential attraction of inflammatory cells to nervous system accompanied by local regulatory effects of IL10. This study is supported by the Istanbul University Research Fund (1530). Numerous soluble mediators have been implicated but not yet convincingly documented in multiple sclerosis (MS). Subjects from Sardinia are relatively homogeneous and ethnically distant compared to Swedes. Both populations have a high MS incidence. We studied cytokine expression patterns in mononuclear cells (MNC) of female untreated MS patients from Sassari, Sardinia, and Stockholm, Sweden, compared to sex-and age-matched healthy controls from both areas. mRNA expression was measured by real-time PCR. Among 20 molecules examined, only IL-12p40 was significantly increased ( pb0.05) in MS compared to controls. Higher expression of IL-12p40 mRNA was confined to MS from both Sardinia and Stockholm, compared to corresponding controls. MS patients from Sardinia also expressed higher ( pb0.05) TNF-alpha compared to corresponding controls. No difference for TNF-alpha was observed between MS vs. controls from Stockholm. IL-6 was remarkably higher in MS compared to controls ( pb0.001) from Stockholm, and compared to MS ( pb0.05) and controls ( pb0.01) from Sardinia. No differences were observed for other members of the IL-12 family (IL-12p35, IL-12Rbeta1 and 2, IL-23p19, IL-23R, IL-27p28); IFN-gamma and IFN-gammaR; IL-4; IL-6R; IL-10 and IL-10R1; IL-18 and IL-18R1; TGF-beta1; the signalling molecules STAT4 and 6. The results corroborate a pro-inflammatory state in MS in general as reflected by high expression of IL-12, i.e., an inducer of pro-inflammatory cytokines, and of the inflammatory cytokines TNF-alpha and IL-6. The extent of expression of TNF-alpha and IL-6 differs between MS patients from different areas, probably reflecting influence of genetic and environmental factors. The object of the study was to identify changes in complement factor C3 concentrations and the degree of C3 activation in serum and cerebrospinal fluid (CSF) in early multiple sclerosis (MS), i.e., at time for diagnosis of disease. The MS-patient group was compared to a control group with no neurological disease. The method for determination of the protein concentration of albumin was immunoturbidimetric assay, for total C3 in serum was radial immunodiffusion used, for C3 in CSF was ELISA performed with polyclonal antibodies, and finally for activated C3 in serum and CSF were ELISA performed by use of a specific monoclonal antibody. A significant increase of the mean value of activated C3 concentration in CSF of MS patients to that of the control group was observed. The significant increase for MS patients was still valid upon determination of the fraction of activated to total C3 in CSF. The activated C3 level in serum was not significantly different from that of the control group, and no significant change in concentration of total C3 was obtained in neither CSF nor serum. The conclusion is that the complement system is activated in the CSF of a group of multiple sclerosis patients at the early stage of diagnosis. The aim of our study was to evaluate whether high dose of methylprednisolone (1000 mg) given for five consecutive days during the disease exacerbations have any impact on immune parameters detected in peripheral blood of MS patients. The following cells phenotypes were determined using two-colour flow cytometry before steroids and after 7 days from starting therapy: CD3+CD19-(T cells),CD3-CD19+ (B cells), CD3+CD4+ (T h cells), CD3+CD8+ (Ts/c cells), CD3-CD56+16+ (NK cells) and T cells, B cells, monocytes, granulocytes producing IL-6 and IL-8 as well. We have studied 20 patients (12 women and 8 men) in mean age of 34.75F10.27. Improvement of disability was found: before therapy mean EDSS was 4.1F1.48, after therapy 2.97F1.34. Significant increase of B cells, decrease of T cells and NK cells was observed after treatment. Trend to decrease of T cells secreting IL-8 and significant decrease of monocytes secreting IL-8 was also noted. It is known that IL-8 might be one of the cytokines responsible for blood-brain barrier disruption and migration of immune cells to the central nervous system. In post mortem studies of MS, patients' brains increased expresion of IL-8 receptors was detected. In this aspect, our results might be important for explaining GS effects during the MS exacerbations.
Upregulation of STATs and interferon-stimulated genes in simian immunodeficiency virus encephalitis via IL6 and interferon-gamma E. Roberts, M. Zandonatti, C. Flynn, K. Roinick and H. Fox The Scripps Research Institute, La Jolla, CA, USA
We previously reported upregulation of STATs 1 and 3, along with interferon (IFN)-stimulated genes (ISGs) and NF-IL6, in frontal lobes of simian immunodeficiency virus-infected monkeys with encephalitis (SIVE). Immunohistochemistry (IHC) revealed STAT1 in astrocytes, endothelial cells, some microglia/macrophages and large neurons. STAT1/ISG upregulation may confer anti-viral and anti-apoptotic properties in astrocytes and microglia, however, STAT1 increase in neurons is linked to dendritic retraction. As these factors may impact on the neuropathology of human (H)IVE and HIV-associated dementia, we examined the pathways involved in STAT and ISG expression. Using frontal lobe tissue, we found increases in IFN-gamma and IL6 mRNA, but not in IFN-alpha or -beta. Interestingly mRNA of STAT1 and ISGs was also upregulated in acutely infected (2 weeks post inoculation) monkeys, however, IHC only showed STAT1 in perivascular cells, endothelium and a very limited number of astrocytes. Although IL6 was upregulated in the CNS in acute infection, the IFNs were not. These results suggests that IL6 alone is not able to induce STAT/ISGs in astrocytes and neurons, thus increases in STAT/ISG expression in encephalitis could be due to the action of both IFN-gamma and IL6. Interestingly, many of the ISGs found in the SIVE cases have previously been described as only being upregulated by increases in IFN-alpha/beta, which may not be the scenario in these cases.
Pivotal role of IL-12 and IFN-gamma axis in controlling tissue parasitism and inflammation in the central nervous system during Trypanosoma cruzi infection J. Lannes-Vieira a , V. Michailowsky a,b and R. Gazzinelli a,b a Department of Immunology-IOC, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; b Laboratory of Immunopatology, CPqR, Oswaldo Cruz Foundation, Belo Horizonte, Brazil
Cytokines are proposed to play a pivotal role controlling tissue parasitism and pathogenesis of experimental Chagas' disease, an infection caused by the intracellular protozoan Trypanosoma cruzi. Symptomatic meningoencephalitis rarely occurs, being more frequently found in children before 2 years of age and in immunosuppressed transplanted or HIV-infected patients. Central nervous system (CNS) alterations as enlargement of perivascular spaces and inflammatory infiltrates are observed during the acute infection of C3H/He mice with T. cruzi, while C57BL/6 mice are less susceptible (Silva et al., 1999; Roffê et al., 2003) . Herein, we investigated the role of the Th1 cytokines IL-12 and IFN-gamma in the establishment of T. cruzi-elicited meningoencephalitis. IL-12 and IFNgamma knockout (KO) mice in C57BL/6 background were highly susceptible to T. cruzi-induced meningoencephalitis as characterized by increased numbers of parasite nests, inflammatory infiltrates, neurological signals and mortality. Interestingly, the inflammatory infiltrates detected in the CNS of T. cruzi-infected IL-12KO mice were mostly devoid of CD8+ T cells. Further, when infected animals were treated with suboptimal doses of Benznidazole (an anti-parasitic drug), the CNS and the heart were the main sites of reactivation of parasitism and inflammation in IL-12KO and IFN-gammaKO mice, respectively. Thus, our results suggest that the level of IFN-gamma deficiency is the major determinant of the site of reactivation of T. cruzi infection in immunocompromised hosts. Human adult microglia express functional Toll-like receptors C.S. Jack, V.C. Montgrain, N. Arbour and J.P. Antel Montreal Neurological Institute, McGill University, Montreal, Canada Classical Toll-like receptor (TLR) signaling activates antigen presenting cells (APCs) and induces pro-inflammatory cytokine secretion. An alternate pathway induced by TLR3 and TLR4 ligands leads to the production of interferon (IFN)-beta, a current therapy in MS. The aims of our study were to assess the expression of TLRs on adult human microglia, the resident APCs of the CNS, and to determine whether signaling through TLR3 and TLR4 can stimulate production of IFN-beta and interferon-inducible protein 10 (IP-10). TLR expression in microglia isolated from surgical specimens was assessed by RT-and QRT-PCR and by flow cytometry. Functional signaling was assayed by ELISA (IP-10 and cytokines) and by QRT-PCR (IFN-beta). In microglia maintained under basal culture conditions, TLR4 mRNA is consistently detected, while its surface expression is low to undetectable. In contrast, although there is marked variation amongst individuals, TLR3 and TLR2 protein is more strongly expressed at the cell surface. In vitro ligation of TLR3 and TLR4 by their respective ligands, double stranded (ds)RNA and LPS, consistently induces the expression of IFN-beta and the secretion of IP-10 in microglia. Notably, ligation of TLR3 results in a significantly stronger IFN-beta and IP-10 response than TLR4 ligation. Signaling via the above TLRs also induces TNF-alpha, IL-1beta, and IL-6. Local production of IFN-beta within the CNS may modulate the complex pro-inflammatory responses characteristic of MS. Microglia are resident semi-professional phagocytes of the CNS. Microglial activation is common to many neurological conditions including virus infection, prion disease and Alzheimer's disease. The innate immune system recognises conserved molecular patterns on microorganisms by a set of pattern recognition receptors of which the Toll-like Receptors (TLR) are members. A multitude of TLRs have now been cloned, though their exact functions and tissue distributions are yet to be characterised. Microglia have been shown to express a few of these receptors. To investigate the expression profile of TLRs on microglia and other CNS cell types during viral encephalitis, groups of Balb/c, nu/nu and IFNalpha/betaRÀ/À mice were infected with SFV and tissues sampled at multiple time points. Gene expression analysis was undertaken using quantitative RT-PCR and tissue sections were studied by immunocytochemistry. In the resting mouse brain, there was extensive expression of TLRs in microglia. Levels of TLRs were differentially modulated during SFV infection and in nu/nu mice demonstrated a positive correlation to levels of viral and IFNalpha transcripts. Stimulation of cultured microglia with lipopolysaccharide resulted in a different pattern of TLR expression to that observed with virus infection.
Potential dual role of astrocytes in innate and autoimmune CNS responses induced by Theiler's virus infection P. Carpentier and S. Miller Northwestern University Feinberg School of Medicine, Chicago, IL, USA Theiler's murine encephalomyelitis virus (TMEV) establishes a persistent central nervous system (CNS) infection in glial cells of susceptible mice, leading to the development of an autoimmune demyelinating disease. Because of its autoimmune nature and the prominent infiltration of CD4+ T cells and phagocytes into the central nervous system, TMEV-induced demyelinating disease (TMEV-IDD) is considered a highly relevant animal model for multiple sclerosis. We have previously shown that innate immune stimulation of astrocytes with Toll-like receptor ligands results in the production of innate immune effector molecules, but not the induction of antigen presenting cell (APC) functions. Here, we have infected astrocytes with TMEV and induce the expression of proinflammatory cytokines, chemokines, adhesion molecules, and type I IFNs. The mechanisms by which astrocytes recognize viral infection is being investigated and preliminary data indicates it is dependent on replicating virus. Later in the course of TMEV-IDD, CD4+ T cells and macrophages/microglia produce large amounts of IFN-gamma and TNF-alpha. In contrast to viral infection, these cytokines do stimulate APC functions of astrocytes. We therefore propose a model in which astrocytes are important for early CNS inflammation and recruitment of the peripheral immune cells to the CNS during TMEV infection, but are only capable of contributing to CD4+ T cell activation late in the autoimmune phase of disease. Acute CNS infection by the neurotropic coronavirus JHMV is controlled by CD8+ T lymphocytes. However, virus persistence in glia is associated with sustained inflammation and ongoing myelin loss. During acute infection perforin mediated cytolysis controls virus replication in macrophages, microglia and astrocytes. By contrast, infection of IFN-gamma deficient mice suggested that IFN-gamma but not perforin controlled virus replication in oligodendrocytes. To specifically implicate an antiviral role of IFN-gamma-mediated signaling in oligodendrocytes, novel transgenic mice expressing a dominant negative IFN-gamma receptor 1 chain (dnIFN-gammaR1) under control of the proteolipid protein promoter were derived. Expression of the transgene was readily detectable by elevated levels of the R1 chain on the surface of oligodendroglia from transgenic compared to wild type (wt) mice. Increased R1 expression was associated with enhanced R2 chain expression. Peripheral immune responses were not affected. Following CNS infection, clinical scores, inflammation and demyelination were similar in both groups. However, virus was still recovered from the CNS of transgenic mice at day 21, while wt mice controlled infection by day 14. Histological analysis revealed an inability to clear virus from oligodendrocytes in transgenic mice. These finding indicate that IFNgamma mediated anti-viral mechanisms are dependent on signaling in oligodendrocytes, but that demyelination is not dependent upon viral persistence in oligodendrocyte infection.
Interferon-gamma is not necessary for Borna disease virus-induced immunopathology but required for antiviral defence by CD8 T cells J. Hausmann a , K.R. Engelhardt a , K. Baur a , H.J. Rziha b and P. Staeheli a a University of Freiburg, Freiburg, Germany; b Federal Research Centre for Virus Diseases of Animals, Tübingen, Germany
Mouse-adapted variants of the highly neurotropic, non-cytolytic Borna disease virus (BDV) cause meningoencephalitis and fatal neurological disease in MRL mice. Disease is mediated by CD8 T cells recognizing the immunodominant epitope TELEISSI in the N protein of BDV. N-specific immune priming of wild-type mice by a prime-boost protocol conferred CD8 T cell-dependent protection against BDV infection and disease. Interestingly, immune priming efficiently protected perforin-deficient mice against infection with BDV, whereas no protection was seen in interferongamma-deficient (GKO) mice. Moreover, adult GKO mice were highly susceptible to BDV infection, whereas adult wild-type mice were resistant. Kinetic analysis of adult mice showed that BDV was able to establish an initial infection, which was subsequently cleared in wild-type but not GKO mice. We further observed that more than 70% of brains from infected GKO but not from wild-type mice showed T cell-dependent severe neuronal damage in the hippocampus, suggesting a neuroprotective role of interferon gamma in BDV-induced meningoencephalitis. In conclusion, BDV-specific CD8 T cells require interferon-gamma for mediating antiviral defence but not for inducing neurological disease, suggesting that two distinct effector mechanisms are at work. Moreover, interferon gamma appears to be neuroprotective in BDV-induced central nervous system immunopathology.
Tumor necrosis factor-alpha and interleukin-1 are required for host defense in a mouse model of Staphylococcus aureus-induced experimental brain abscess T. Kielian, E.D. Bearden, A.C. Baldwin, N. Esen University of Arkansas for Medical Sciences, Little Rock, AR, USA Brain abscesses represent a significant medical problem despite recent advances made in detection and therapy. Using the Staphylococcus aureus-induced brain abscess model established in our laboratory, we have sought to define the functional importance of IL-1, TNF-alpha, and IL-6 in the host anti-bacterial immune response using cytokine gene knockout (KO) mice. The results demonstrate that although they share many redundant activities, IL-1 and TNF-alpha are important for containing bacterial infection in evolving brain abscesses as evident by increased mortality and bacterial burdens in IL-1 and TNF-alpha KO mice compared to wild type (WT) animals. In contrast, IL-6 was not found to be a major contributor to the host anti-bacterial immune response. Microarray analysis was used to evaluate the downstream consequences originating from the lack of IL-1 on subsequent proinflammatory mediator expression in brain abscesses from IL-1 KO and WT animals. Although numerous genes were significantly induced following S. aureus infection, only IL-1 beta and two chemokines, CCL9 (macrophage inflammatory protein-1 gamma) and CXCL13 (B lymphocyte chemoattractant/BLC), were differentially regulated in IL-1 KO versus WT animals. These results indicate that IL-1 and TNF-alpha play a pivotal role during the acute stage of brain abscess development through regulating the ensuing antibacterial inflammatory response.
The intermediate filament GFAP is important for the control of experimental murine Staphylococcus aureus-induced brain abscess and Toxoplasma encephalitis W. Stenzel a , M. Deckert a , S. Soltek b and D. Schlqter b a University of Cologne, Cologne, Germany; b University of Heidelberg, Heidelberg, Germany CNS infections can be controlled effectively by cells of the immune system, but organ specific cells such as astrocytes play an important part in the host response as well. The functional role of astrocytes exerted via their intermediate protein glial fibrillary acidic protein (GFAP) in CNS infections was studied in murine Staphylococcus aureus-induced brain abscess. Compared to wild type (WT) mice, GFAP0/0 mice developed larger and poorly demarcated inflammatory lesions, paralleled by a significantly increased intracerebral bacterial load, a diffuse leukocytic infiltration even of the contralateral hemisphere, purulent ventriculitis, vasculitis, and severe brain edema. These observations were correlated with the lack of a bordering function of activated astrocytes, which strongly upregulated their GFAP expression in the abscess surrounding of WT mice. Clinically important, this lack of restriction of inflammation markedly aggravated the course of disease with manifestation of seizures and a severe weight loss in GFAP0/0 mice. These data were paralleled by observations in the model of Toxoplasma encephalitis (TE), during which the intracerebral parasitic load was significantly increased with tachyzoite-induced tissue necrosis in the brains of GFAP0/0 mice in chronic TE. Collectively, these findings delineate a host defense function of astrocytes via restricting pathogenic spread and multiplication within the CNS, thereby contributing to protection of the highly vulnerable brain parenchyma. Neurotropic coronavirus infection of mice results in acute demyelinating encephalomyelitis followed by non-productive viral persistence. Acute replication is resolved by CD8 T cells within 2 weeks post infection (p.i.), whereas viral persistence is controlled by neutralizing antibody. The kinetics of CNS B cell recruitment and differentiation were investigated. Virus specific antibody secreting cells (ASC) within the CNS increased prominently between 15 and 21 days p.i. and were maintained at high frequencies during persistence; by contrast, only low frequencies were detectable in bone marrow. Alterations in antibody specificities within CNS ASC during persistence provided evidence for ongoing dynamic regulation. Whereas spike and nucleocapsid protein specific ASC accounted for most virus specific ASC between days 10 to 14 p.i., they gradually declined thereafter. Furthermore, many ASC of unknown specificity were associated with persistence. Flow cytometric analysis of infiltrates isolated from the infected CNS indicated a gradual increase in CD19+ B cells by day 15 p.i. Delayed plasmablast recruitment was reflected by emergence of syndecan positive cells only between days 10 and 14 p.i. While the relative percentage of syndecan positive cells remained constant after day 21, ongoing differentiation was suggested by an increasing loss of class II surface expression. Thus, both altered specificities and phenotypes support a dynamic process within the B cell compartment during CNS viral persistence. This contrasts the apparent senescence of T cell reactivity during persistence.
COX-2 inhibition increases CNS immune activation and SIV replication during acute SIV infection J. Mankowski, S. Queen, P. Tarwater, R.J. Adams, M.C. Zink and J.E. Clements Johns Hopkins University, Baltimore, MD, USA During acute infection, productive viral replication is present in the brain of SIV (simian immunodeficiency virus)-infected macaques although encephalitis does not develop at this time. Early viral replication in the brain is accompanied by CNS immune activation including endothelial cell ICAM-1 expression and elevated microglial expression of CD68 and MHC class II. Upregulation of COX (cyclooxygenase)-2 expression and subsequent production of arachadonic metabolites in the brain may be a key pathway regulating CNS immune responses during acute infection. To determine whether COX-2 inhibition during acute SIV infection alters CNS immune activation, thereby modulating viral replication in the CNS, six pig-tailed macaques were treated with rofecoxib, a selective COX-2 inhibitor, during the initial 10 days of SIV infection. CNS immune activation and CNS viral burden were measured and compared with a group of SIV-infected, untreated animals. Both endothelial ICAM-1 expression and parenchymal microglial activation (measured by image analysis of immunostained tissue sections) were elevated in rofecoxib-treated, SIV-infected animals at 10 days post-inoculation as compared to untreated, infected animals, demonstrating that arachadonic acid metabolites such as prostaglandin E2 may down-regulate CNS immune responses. In parallel with increases in CNS immune activation, CNS viral replication measured by real time RT-PCR was significantly elevated in the basal ganglia of rofecoxib-treated animals versus untreated, infected animals. These findings demonstrate the complexity of immunomodulation in the CNS with COX-2 pathway products playing diverse roles. Psychological stress has profound effects on the pathogenesis of multiple sclerosis (MS). These effects are mediated by interactions between the endocrine, immune and nervous systems. Our laboratory has been investigating the impact of stress on the susceptibility to Theiler's virusinduced demyelination (TVID) (a mouse model of multiple sclerosis) in genetically susceptible strains of mice. In TVID, restraint stress activates the hypothalamic pituitary-adrenal axis and the autonomic nervous system, which results in immunosuppression mainly through the increased production of glucocorticoids. Stressed mice have decreased NK cell activity and decreased chemokine expression (Ltn, IP-10 and RANTES) in the brain and spleen. Reduced chemokine expression may account for the decreased inflammation in the CNS resulting in increased viral replication and dissemination. The higher viral load subsequently leads to an earlier disease onset and more severe clinical and histological signs of demyelinating disease. Our results have important implications for understanding the pathogenesis of MS and suggest that stressful events during early infection with an agent capable of inducing demyelination, may result in immunosuppression and failure to eliminate the pathogen which in turn may lead to the development of MS. Funded by grants to C.J.R.W. and M.W.M. from National Multiple Sclerosis Society RG 3128 and NIH/ NINDS R01 39569.
Microglial dynamics in WNV encephalitis D. Getts, I. Matsumoto and N.J.C. King University of Sydney, Sydney, Australia Fatal cases of West Nile Virus (WNV) encephalitis show microglial activation, nodule formation and leukocyte infiltration. Because of the pivotal importance of Interferon-gamma (IFN-gamma) in both microglial activation and virus eradication, we investigated the role of IFN-gamma in microglial responses and leukocyte infiltration in WNV encephalitis. Eight-week-old C57BL/6 (WT) and IFN-gamma knockout (IFNGKO) mice were inoculated intranasally with WNV. Flow cytometric and immunohistochemical analysis was performed to characterise infiltrating and constitutive cell populations. Microglial activation was clear in both strains from day 3, concurrent with detectable neuronal WNV infection. Nodule formation, around infected neurones was evident by day 5, concurrent with leukocyte infiltration. Nodules were significantly greater in the WT ( Pb0.01) after day 5, correlating with increased macrophage numbers. Regions of high nodule formation did not correlate with those of high neuronal infection. Nodule formation in both strains was maximal on day 6, with a reduction on day 7, when maximal viral and leukocyte loads were seen. Numbers of MHC-II+ microglia remained similar in the WT, but significantly reduced in IFNGKO by day 7, compared to MHC-II+ macrophages, which increased 4-fold in the WT by day 7. Microglial activation is IFN-gamma independent, possibly induced by signals from infected neurones. In contrast, microglial nodule formation, correlating temporally and numerically with differential leukocyte infiltration in both strains, is enhanced by IFN-gamma. Differential MHC-II+ cell numbers in each strain, correlating with the presence of IFN-gamma may have functional implications.
West Nile virus encephalitis: T cell function in IFN-gamma-deficient mice S. Rana and N. King University of Sydney, Sydney, Australia West Nile virus (WNV) is an emerging neurotropic flavivirus. In a murine model of WNV encephalitis, C57BL/6 wildtypes (WT) administered a sublethal dose (10^5 pfu intraperitoneal) of WNV (Sarafend) showed a 60% mortality. However, only 20% mortality was observed in IFN-gamma knockout mice (KO). Since IFN-gamma is an important antiviral cytokine that can modulate lymphocyte function and activation, we examined the brains, spleens and CNS-draining cervical lymph nodes (CLN) of infected mice, examining chemokine mRNA, cellular populations and effector CTL activity. A differential upregulation of chemokine (IP-10, RANTES, MIP-1, MCP-1) mRNA was found, with surprisingly greater expression in KO compared to WT brains. Infiltration of inflammatory leukocytes was evident from day 5 post-infection (p.i.), and was still maintained at day 30 p.i., beyond the period of detectable virus. No difference was found between the two strains. However, by using a sensitive fluorolysis CTL assay, we found that at day 8 p.i., the peak of CNS infection, deep CLN and CNS derived-leukocytes from KO mice have a reduced capacity to specifically lyse WNV-infected target cells. The finding that WT and KO splenocytes are equally able to lyse targets throughout the course of infection, suggests that fewer WNV-specific CD8 T cells, possibly from the deep CLN, are trafficking into the KO CNS. Alternatively, there may be a direct role played by IFN-gamma in mediating cytotoxic activity against WNV-infected cells.
Herpes simplex virus 1 (HSV1)-induced central nervous system (CNS) demyelination L.F. Kastrukoff a , C.M. Jones b and F.R. Carbone b a University of British Columbia, Vancouver, Canada; b University of Melbourne, Melbourne, Australia HSV1 induces recurrent multifocal CNS demyelination in specific strains of mice. Following mucosal inoculation with virus, three stages of demyelination occur with the first the result of an immune response directed at infectious virus. To further investigate the immune mechanisms mediating the development of this stage of demyelination, 10-to 12-week-old male C57BL/6 (BL/6) nu/nu and BL/6 bg/bg mice were lip inoculated with HSV1 lab strain 2. BL/6 mice, pretreated with anti-CD4 or anti-CD8 mAb, along with gamma-delta T-cell-deficient (B6.129P2-tcrdtm1mom) and Bcell-deficient mice (B6.129S2-Igh6tm/cgn) were also studied. The spread of virus in the CNS along with the development of CNS demyelinaton were determined. Virus spreads through the CNS in BL/6 nu/nu and BL/6 bg/bg mice but not in BL/6 mice treated with either anti-CD4 or anti-CD8 mAb, gamma-delta T-cell-deficient, or B-cell-deficient mice. CNS demyelination develops only in BL/6 bg/bg mice. Taken together, the results suggest that the local replication of virus and its spread in the peripheral nervous system (PNS) are affected by both the innate and adaptive immune systems. In contrast, natural killer (NK) cells and T-cells play a role in the spread of virus in the CNS and in the development of the first stage of demyelination. Herpes simplex virus (HSV) is a common neurotropic virus, which infects epithelial cells and subsequently the trigeminal ganglia (TG) and brain tissue. In this study,d we focused on how immunomodulation with roquinimex (Linomide) affects the course of corneal HSV infection in BALB/c mice. We addressed the questions how immunomodulation affects the local as well as the systemic immune response and whether roquinimex could facilitate the spread of HSV to central nervous system (CNS). Viral culture was used to detect replicating HSV. The cytokine response in brain and TG was studied with quantitative rapid real-time RT-PCR method and in stimulated splenocytes with EIA. No increase in mortality to HSVinfection was seen, nor was the acute infection enhanced. The expression of IL-23p19 mRNA was decreased in brains of roquinimex-treated infected mice. Also, the expression of IL-12p35 and IFN-gamma mRNAs decreased. At the same time the expression of IL-12p40 was increased. No significant changes were seen in IL-4 and IL-10 mRNA expression. In systemic immune response, Roquinimex treatment suppressed the production of IFN-gamma but also the production of IL-10. In conclusion, downregulation of Th1-immune response in Th2-prone mice did not lead to increased mortality of corneally infected mice. These results are important regarding the safety of immunomodulative treatment during viral infection.
Diffusion-tensor imaging of the temporal lobe in acute herpes-simplexvirus encephalitis J. Sellner, C. Herweh, S. Heiland, M. Hartmann and U. Meyding-Lamadé Ruprecht-Karls-University Heidelberg, Heidelberg, Germany
Herpes-simplex-virus encephalitis (HSVE) still carries significant morbidity and serious sequelae in survivors despite advances in treatment and critical care. Magnetic resonance imaging (MRI) and especially diffusionweighted MRI can improve diagnostic sensitivity and contains information of prognostic value. Furthermore, diffusion-tensor imaging (DTI) enables the in vivo characterization of the structural and functional integrity of the brain and could be of further diagnostic and therapeutic value in HSVE. Clinical impairment and cranial MRI changes were studied in five patients with acute, PCR-proven HSVE. We studied the fractional anisotropy (FA) and the mean diffusability (MD) in the primarily affected temporal lobe in comparison with values from five healthy controls. Data were aquired by placing the region of interest over the whole temporal lobe, and performed with: DSE, 6 non-colinear gradients with b=0 and 1000. The FA was 0.204884F0.0362 in patients with HSVE and 0.208863F0.0158 in healthy controls. Furthermore, the MD was 1097F150Â10 -6 s/mm 2 in HSVE and 1053F68Â10 -6 s/mm 2 in healthy controls. Despite severe clinical impairment, we could not find significant differences of the FA and the MD studying the temporal lobe in a series of five patients with HSVE. Diffusion changes in small regions might not be displayed by our data aquisition from the whole temporal lobe. A ROI analysis in correlation with MR-changes and clinical impairment may provide more accurate information. We investigated the cytokine response to intranasal herpes simplex virus type 1 (HSV-1) infection in trigeminal ganglia (TG) and in brains of BALB/c mice. Four-to 6-week-old mice were infected with HSV-1 (F) and samples were taken at five time-points: day 0 (uninfected), day 5, day 7, day 10 and day 31 post infection. Virus was detected with rapid culture and HSV-PCR. Samples were analyzed for the cytokines interleukin (IL)-4, IL-10, IL-23, IL-12p35, IL-12p40 and IFN-gamma with the real-time PCR method, using LightCycler. Alfa-TIF (VP16) expression was also analyzed with the LightCycler system. Replicating virus was observed in TG after day 5 whereafter the amount decreased. HSV-DNA was detected in brains from day 5 on. In TG, we found a significantly elevated expression of mRNA for IFN-gamma during acute HSV infection (day 5 and day 7) when compared to uninfected mice ( pb0.05). The IL-23 and IL-4 expressions were significantly downregulated in TG from day 7 and day 10, respectively. The IL-4 expression decreased in brain at day 5, day 7 and day 31 in brain. The cytokine response to intranasal infection differs in some extent from that to ocular infection (Broberg et. al 2002, Journal of Interferon and cytokine research, vol 22: 641-651) .
Dynamics of neurotrophic factor expression in experimental herpes-simplex virus encephalitis J. Sellner a , T. Lenhard a , J. Haas a , R. von Einsiedel b and U. Meyding-Lamadé a a Ruprecht-Karls-University Heidelberg, Heidelberg, Germany; b St7dtisches Klinikum Magdeburg, Magdeburg, Germany Herpes-simplex virus 1 is the most common cause of non-epidemic focal encephalitis (HSVE) and still associated with high morbidity and mortality. Neurotrophic factors play an important role in neuronal survival and recovery after acute injury to the central nervous system (CNS). We studied the mRNA-expression of glial cell line-derived neurotrophic factor (GDNF), brain-derived neuroptrophic factor (BDNF) and neurotrphin-3 (NT-3) in an established model of murine Herpes-simplex-Virus encephalitis. The regulation of these neurotrophic factors was determined during the acute phase of disease (e.g., 3 and 7 days post inoculation) and recovery (2 months post inoculation). Our results demonstrate a sequential modulation of mRNA expression during the acute phase of GNDF ( Pb0.05) and NT-3 ( Pb0.01), and a persisting overexpression of NT-3 mRNA even 2 months after acute encephalitis ( Pb0.001). Furthermore, the administration of acyclovir as well as adjunctive methylprednisolone to acyclovir promoted a significantly increased expression of GDNF during acute encephalitis and a downregulation of BDNF and NT-3. Our results indicate a differential expression pattern of neurotrophic factors during acute viral encephalitis, which may have impact on neuronal survival and regeneration. The promotion of neurotrophic factors in acute viral encephalitis may represent an additional therapeutic target for treatment of this highly disabling disease in humans.
Effects of antigen-specific tolerance of CD8+ T-cells on a viral infection of the central nervous system M. Teague and S. Miller Northwestern University, Chicago, Illinois, USA Antigen-specific tolerance of CD4+ T-cells induced by the injection of antigen coupled, ECDI-fixed cells into recipient mice is a procedure that has been pursued as a possible therapy for autoimmune diseases, as it prevents and/or ameliorates several CD4+ T-cell mediated diseases in mice. We have begun to analyze the use of this method for tolerance of CD8+ T-cells. We have found that effector function of specific CD8+ Tcells can in fact be effectively inhibited by encounter with ECDI-fixed cells bearing specific antigen. Infection of SJL/J mice with Theiler's murine encephalomyelitis virus (TMEV) leads to a demyelinating disease similar to multiple sclerosis in humans. C57BL/6 mice, however, clear the virus and are thus resistant to disease. The role of CD8+ T-cells in this disease has been controversial thus far. We have shown that in C57BL/6 mice, induction of tolerance in CD8+ T-cells specific for the immunodominant TMEV CD8+ T-cell epitope, followed by TMEV infection, renders these mice susceptible to viral persistence and motor dysfunction. We have also shown a modest effect on viral persistence and disease in susceptible SJL/J mice. Thus, this method can be used to assess the roles of specific CD8+ T-cells in a disease scenario. In addition, coupled cell tolerance of CD8+ T-cells is an attractive potential therapy for diseases in which CD8+ T-cells are pathogenic.
Role of NF-kappaB in Theiler's virus-induced cytokine/chemokine gene activation and demyelinating disease B.S. Kim, J.P. Palma, A.C. Fuller and H. Kang Min Northwestern University Medical School, Chicago, USA Theiler's murine encephalomyelitis virus (TMEV) infection in the CNS induces a demyelinating disease similar to human multiple sclerosis. To delineate the early events in the virally induced demyelinating disease, chemokine and cytokine gene activation upon viral infection in glial cells was examined. Infection of SJL/J primary astrocyte cultures induces select proinflammatory cytokine and chemokine genes. In addition, some of the proinflammatory cytokines function synergistically for virus-induced chemokine gene expression. The cellular gene activation by TMEV is dependent on the NF-kappaB pathway. These results demonstrate that infection of astrocytes and other glial cells by TMEV induces early NF-kappaB-mediated cytokine/chemokine gene activation involved in CNS inflammatory responses leading to demyelination. Interestingly, C57BL/6 mice deficient in the NF-kappaB p50 subunit permit viral persistence in the CNS and clinical manifestation. A significant decrease in CD8+ T cell infiltration to the CNS and an increase in CD4+ T cell and macrophage infiltration are found in these knock out mice as compared to control C57BL/6 mice. However, fewer CNS-infiltrating T cells, both CD4+ and CD8+, are specific for TMEV. It is clear from these studies that the NF-kappaB p50 subunit is important in the resolution of CNS infection in resistant mice. Further studies are being undertaken to address the role of NF-kappaB subunits and upstream signals involved in the cellular activation and pathogenesis of TMEV-induced demyelinating disease.
Zinc-binding motif in leader protein of Theiler's murine encephalomyelitis virus is critical for viral persistence and demyelination K. Asakura a , H. Murayama b , T. Himeda a and Y. Ohara a a Kanazawa Medical University, Ishikawa, Japan; b Sendai City Hospital, Sendai, Japan DA strain and other members of the TO subgroup strains of Theiler's murine encephalomyelitis virus cause a biphasic disease characterized by acute self-limiting gray matter inflammation followed by chronic inflammatory demyelination in the spinal cord in susceptible strains of mice. The second phase serves as an experimental model of multiple sclerosis. DA subgroup synthesizes L* protein from an alternative initiation codon. The function of L* is not fully understood. L* is considered to play a key role in viral persistence though it is controversial for demyelination. We previously demonstrated that L* is expressed exclusively in neurons in vivo in the acute phase of infection in the central nervous system (CNS) by generating a mutant virus which expresses epitope-tagged L* (K. Asakura et al., J. Virol. 76:13049-13054, 2002) . However, in this study, the mutant virus failed to grow in L929 though it grew in BHK-21 and failed to persist in the CNS. Here, we explored the basis of this failure by generating a panel of new mutant viruses, which express epitope-tagged L*. Two of the new mutant viruses successfully caused persistent infection and demyelination in the spinal cords and enabled us to identify L* immunohistochemically in the demyelinated lesions. These mutant virus studies revealed that sparing zinc-binding motif in the leader protein of TMEV has crucial effect on its persistent infection in the CNS. It is well established that early life stress can induce long-lasting changes in behavioral and neuroendocrine reactivity to stress. However, little is known about the long-term consequences of neonatal stress on susceptibility to infectious and autoimmune diseases. The present study investigated the effects of neonatal stress on susceptibility to a virally mediated animal model of multiple sclerosis, Theiler's murine encephalomyelitis virus (TMEV) infection. TMEV induces a biphasic infection of the CNS in which polio-like symptoms are observed during acute infection followed by autoimmune-mediated demyelination during late disease. In these experiments, BALB/cJ pups were exposed daily to one of three conditions during their first 2 weeks of life: (1) 180-min of neonatal maternal separation (NMS), (2) 15-min of NMS, or (3) undisturbed. During adulthood, mice were inoculated intracranially with Theiler's virus and sacrificed at days 14, 21, or 35 post-infection. Prolonged 180-min NMS decreased viral clearance in the spinal cords of male and female mice at days 21 and 35 pi, whereas brief 15-min NMS increased viral clearance. Both the 15-and 180-min NMS mice exhibited blunted corticosterone responses to TMEV infection, and increased adrenal and lymph node weights. These changes were accompanied by alterations in histological signs of meningitis and microgliosis in spinal cord, and alterations in motor function. These findings suggest that maternal separation has long-term effects on neuroimmune, neuroendocrine, and behavioral responses to TMEV infection. The present study examines the effects of social stress on an animal model of multiple sclerosis, Theiler's murine encephalomyelitis infection (TMEV). TMEV induces a biphasic infection of the CNS in which polio-like symptoms are observed during acute infection followed by neuroinflammatory demyelination during late disease. We have previously shown that the effects of social disruption (SDR) on acute Theiler's virus infection are dependent upon the timing of SDR application in relation to infection. When SDR was applied prior to infection (PRE-SDR), it led to glucocorticoid (GC) resistance, and these mice developed more severe disease course, with increased inflammation. In contrast, when SDR was applied concurrent with infection (CON-SDR), GC resistance failed to develop, disease course was less severe and inflammation was moderate. The current study extends this line of work by examining the impact of SDR on the course chronic phase of disease. Three groups of infected Balb/cJ mice received SDR either immediately prior to or concurrent with infection, or were nonstressed. In addition to replicating the pattern of acute behavioral impairments, we found that the PRE-SDR group developed earlier and more severe hind limb impairment, impaired rotarod and inclined plane performance, reduced sensitivity to von Frey stimulation, and reduced spontaneous locomotor activity. These converging lines of evidence demonstrate that social stress exacerbates both the acute and chronic phase of Theiler's virus infection.
Treatment options in SSPE: experience with beta interferon B. Anlar a , A. Guven b and O.F. Aydin c a Hacettepe University, Ankara, Turkey; b Social Securitý Hospital, Ankara, Turkey; c SUCH Hospital, Ankara, Turkey Aim: Efficient treatment methods are lacking for subacute sclerosing panencephalitis (SSPE). We compared the results obtained with two forms of h-IFNs in SSPE. Method: h-IFN1a in two forms: 60 Ag/week IM, named h-IFN1a 1/wkIM, or 22 Ag 3/week SC, named h-IFN1a 3/wkSC, were given in a non-randomized fashion to 48 patients diagnosed with SSPE. All received oral inosiplex 50-100 mg/kg. Those who received the treatment for N3 months and had at least 1 year's follow-up were evaluated. Results: Treatment groups were similar in terms of age, sex, and duration of disease while stage at diagnosis differed: no Stage 1 patients were in the h-IFN1a 3/ wkSC group ( pb0.02). Mean follow-up was 15.4F9.8 months. More patients in the h-IFN1a 3/wkSC group stabilized or improved ( pb0.05). Survival and duration of treatment were significantly shorter in the h-IFN1a 1/wk IM group. Fatality rate at 6 and 12 months, and frequency of side effects were not different between groups. Conclusion: Despite the difference in the stages of the patients, the proportion tients with satisfactory outcome was higher with h-IFN1a 3/wkSC. Overall progression was similar in all groups. Randomized studies are difficult to realize in SSPE because of the clinical variability of the disease. Open studies give some, although limited, evidence about treatment, and suggest h-IFN at high doses combined with inosiplex might constitute an applicable option. To investigate antigen-specific T cell responses in SSPE, we analyzed proliferation and cytokine secretion from 37 SSPE patients and 45 healthy controls (HC) to specific antigens and to measles vaccine (MV). PBMCs of SSPE and HC groups were stimulated with MBP, MOG, alpha-B-crystallin, MV and PPD. Proliferation was measured as stimulation indices (SI). IFNgamma, IL-12 and IL-10 production were determined in culture supernatants by ELISA. The median SI values to MBP, MOG and alpha-Bcrystallin did not differ between groups. There was a trend towards a decrease in IL-12 production in response to MBP in SSPE patients than in HCs (0.0 vs. 4.4 pg/ml, p=0.051, respectively). Proliferation (1.8 vs. 25, pb0.001), as well as IFN-gamma, IL-12 and IL-10 production in response to PPD were impaired in SSPE patients (0.1 vs. 681.0, 0 vs. 14 pg/dml, both pb0.001, and 0 vs. 2.6 pg/ml, p=0.02, respectively). Proliferative responses to MV were not different among the groups. However, in vitro IL-12 secretion of SSPE patients to MV was lower than HCs (0.0 vs. 3.6 pg/ml, p=0.001). The results did not demonstrate any by-stander cellular responses against myelin antigens, implicating that the central nervous system is not primary target of an autoimmune response in SSPE. The previously reported lower IL-12 response to measles virus seem to persist in SSPE.
Role of neuroimmunophilin ligands in lipopolysaccharide (LPS) induced peripheral and central hyperalgesia A. Singh and S.K. Kulkarni Institution UIPS, Panjab University, Chandigarh, India
Exposure of tissues to endotoxin lipopolysaccharide (LPS) leads to induction of inducible nitric oxide synthase (iNOS). FK506 an immunosuppressant is reported to suppress LPS-induced (iNOS) and nitrite levels in macrophages and other tissues. The present study was aimed to assess the modulatory effect of FK506 on lipopolysaccharide induced central as well as peripheral hyperalgesia possibly through iNOS inhibition. LPS induced a significant hyperalgesia when measured by both central as well as peripheral methods. Dose of FK506 (0.1-0.75 mg/kg i.p.) having no analgesic activity, dose dependently and significantly reversed peripheral as well as central hyperalgesia. Aminoguanidine, a specific iNOS inhibitor not only dose dependently (10-50 mg/kg i.p.) and significantly reversed LPS-induced hyperalgesia, but potentatiated the lower subeffective dose of FK506. FK506 dose dependently and significantly reversed elevated nitrite levels in brain and paw of the LPS-treated mice and rats, respectively. Elevated MPO (Myeloperoxide) levels in LPS treated paw in rats were also significantly reversed by FK506 treatment. Again aminoguanidine not only reversed LPS-induced elevation of myloperoxide (MPO), nitrite levels but also potentaited the effects of lower dose of FK506. FK506 possibly through iNOS inhibition reversed LPS-mediated alteration in pain (hyperalgesia) and restored elevated MPO and nitrite levels in LPS treated animals, thus may be screened as a possible adjuvant in the treatment of systemic hyperalgesia.
A novel role for olfactory ensheathing cells in neuroimmunological defence in the rodent olfactory system A. Vincent, A. West and M.I. Chuah University of Tasmania, Hobart, Australia
The dendrites of olfactory receptor cells are exposed to the external environment whilst their axons project directly into the olfactory bulb in the CNS. This provides a route for pathogens into the brain that bypasses the blood-brain barrier, yet infection via this route appears to be limited by local defences within the olfactory system. Given the close apposition of olfactory ensheathing cells (OECs) to olfactory receptor axons along their entire projection, these glial cells could constitute an important component of the olfactory immune defence. We recently detected enriched transcripts for several immune factors in cultured neonatal OECs by microarray analysis, including lysozyme (Lyz), the chemokines growth-regulated oncogene (Gro, CXCL1) and monocyte chemoattractant protein 1 (MCP1, CCL2), and the interleukin 6-responsive transcription factor CCAAT/enhancer binding protein beta (C/EBPb). Lyz, MCP1 and Gro expression were confirmed by real-time RT-PCR. We detected all gene products by immunostaining in cultured OECs, but did not detect significant levels of either chemokine in OECs in the normal adult olfactory system. Lyz mRNA is inducible in cultured OECs in the presence of Staphylococcus aureus but not Escherichea coli (E. coli) or lipopolysaccharide (LPS, 1-25 Ag/ml). Conversely, Gro and MCP1 are strongly induced by E. coli and LPS. We are currently testing the function of these immune factors in OEC in culture and in the olfactory system.
Mengo virus encephalomyelitis is prevented by the polyanion COAM through interferon-independent chemokine induction and enhanced phagocytosis S. Starckx, A. Billiau, J. Van Damme and G. Opdenakker University of Leuven, Leuven, Belgium
The polyanionic compound COAM (chlorite-oxidized oxyamylose) protects mice from lethal encephalomyelitis caused by intraperitoneal (IP) Mengo virus infection. The in vivo protective effect is associated with multiple biological activities of COAM, such as a reduction of plasma virus titers. We found that IP injection of 2 mg COAM induces a dramatic increase in peritoneal leukocyte counts which is explained by a selective infiltration of polymorphonuclear leukocytes (PMNs). PMNs migrate into the peritoneum in response to a gradient of granulocyte chemotactic protein-2 (GCP-2). The presence of GCP-2 and the subsequent migration of PMNs coincide with the release of MMP-9/ gelatinase B in the peritoneal cavity. Although the phagocytic activity of peritoneal macrophages or the two murine macrophage cell lines was not significantly affected by COAM, the increased number of PMNs in the peritoneum was responsible for the enhanced phagocytic efficiency and the accumulation of proteolytic enzymes. The antiviral activity of COAM was not mediated by the induction of interferons since COAM, in contrast to polyI:C, does not protect mouse fibroblasts against the cytopathogenic effect of Mengo virus in vitro. In addition, the observed effects do not rely on TLR3-or TLR4-dependent mechanisms. Altogether, the experimental results indicate that the in vivo antiviral activity of COAM originates in the retention of the virus within the peritoneal cavity due to increased phagocytosis and killing of the virus. The immune system plays a key role in the dissemination of prion infections from the periphery to the central nervous system (CNS). Follicular dendritic cells are critical for prion replication in lymphoid tissue and subsequent neuroinvasion, whereas myeloid dendritic cells (DC) have been indicated as candidate vectors for prion propagation to the CNS. Since nothing is known on the ability of DC to migrate to the CNS during prion diseases, we investigated the immunohistochemical localization of CD205+ DC in the brain of C57BL/6 mice intraperitoneally infected with the mouse-adapted KFu strain of Gerstmann-Str7ussler-Scheinker syndrome, a human genetic prion disorder. In normal brain, CD205+ cells were present in the meninges and choroid plexus whereas in the majority of mice sacrificed from 120 to 300 days post-infection (dpi), scattered CD205+ dendritiform cells were also detected in the cerebral cortex, subcortical white matter, thalamus and cerebellum, sometimes in perivascular location. Brains from mice in the late stage of infection showed severe spongiform degeneration in the medulla oblongata, thalamic nuclei and several white matter areas. Accumulation of PrPSc was observed predominantly at the thalamic level at 240-260 dpi and became more diffuse during the terminal stage of the disease. These findings demonstrate that myeloid DC can enter the CNS of prion infected mice, suggesting a possible role for these cells in the pathogenesis of prion disorders. Cerebral malaria (CM) is a serious complication due to infection with Plasmodium falciparum. Patients often present with neurological symptoms that can progress to death. The pathogenesis of CM is not well understood, although it is believed to be largely a host-mediated event, with cytokines playing a prominent role. The role of chemokines is less clear, although recent work suggests that the expression of some chemokine receptors, such as CCR5, on leukocytes sequestered within the brain may be crucial for the development of murine CM. The expression of known chemokines and chemokine receptors was examined and compared between mouse models of malaria. Mice infected with Plasmodium berghei ANKA develop neurological symptoms similar to human CM, and die 6-7 days postinoculation (p.i.), whereas mice infected with P. berghei K173 do not present with cerebral manifestations, but die 12-14 days p.i. with overwhelming parasitaemia and anaemia. RT-PCR was used to determine the relative expression of chemokine genes in the brains of these mice. Several chemokines and receptors, including CCL2 and CCR1, were found to be up-regulated in mice that presented with CM, compared to mice without cerebral symptoms. Laser capture microdissection techniques were also employed in order to determine the cellular source of chemokines. Vessels were separated from parenchyma cells and the mRNA levels of candidate chemokine and chemokine receptor genes were assessed using RT-PCR. Changes in the expression of chemokines may play an important role in the pathogenesis of CM.
Parasitic glycoconjugates-role in neurocysticercosis pathogenesis J.I. Alvarez and J.M. Teale University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Neurocysticercosis (NCC) is an infection of the central nervous system by the larvae of the helminth Taenia solium. Symptom severity is associated with the intensity of the immune response. First, there is a long asymptomatic period where the host immunity seems to be incapable of resolving the infection. Helminth teguments are dynamically responsive to adverse environments and can be rapidly shed. Because more than 95% of NCC patients exhibit humoral response against at least 2 carbohydrate antigens (glycoconjugates-GCs), we analyzed the expression of different GCs in NCC infected specimens. To determine the GCs present in the parasite's tegument, we used fluorochrome labeled lectins with specificity to different carbohydrates. All the lectins utilized labeled the tegument, but interestingly the GCs bound by isolectinB4, wheat germ agglutinin and concavalinA were also found to be taken up by macrophages/microglia. Peanut lectin binding GCs, on the other hand, remained on the parasite throughout the infection and were not detected in host cells. To confirm that the lectin-binding GCs staining cells were of parasitic origin, we developed an antibody against the 12-kDa glycoprotein of T. solium. The antibody and lectins colocalized with macrophages/microglia indicating parasitic origin. Thus, constant release of GCs may help the parasite to persist in the adverse host environment because massive antigen uptake by immune cells could interfere with normal immune function. Additionally, these molecules likely provide a source of persistent antigen responsible for life-long sequelae in many NCC patients.
Gram negative lipopolysaccharides indirectly induce baxonal stunningQ in rats G. Ifergane, M. Bersudsky, A. Dori and I. Wirguin Soroka University Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, Israel
While tissue dysfunction during sepsis is common, cell death in the heart, kidney, liver, and lung is relatively minor. It was suggested that sepsis activates defense mechanisms that cause cellular processes to be reduced to basic bhousekeepingQ roles. We examined whether similar processes are induced in the peripheral nerve when the blood-nerve barrier (BNB) is breached. we systemically injected Lewis rats (presensitized by KLH) with Campylobacter Jejuni (Cj) or E. coli lipopolysaccharide (LPS). The exposure caused transient conduction blocks within 24 h, but only if accompanied by a simultaneous minor neural insult (intraneural (IN) injection of saline) and was not associated with morphological abnormalities. IN injection of the LPS itself did not result in conduction abnormalities. In a second set of experiments, we incubated Lewis rats splenocytes with Cj or E. coli-LPS for 48 h. The splenocytes LPS reactive medium (SLRM) was injected into Lewis rats sciatic nerves, and caused a transient conduction block appearing 24-72 h after the IN injection. Our results suggest that gram-negative LPS induces an acute immunological response mediated by soluble factors, which are capable, when penetrating the BNB to cause transient baxonal stunningQ and functionally affect neural conduction. This pathophysiological process could be involved in the pathogenesis of critical illness neuropathy in septic patients with multisystem failure. BBB dysfunction in HIVE is accompanied disruption of tight junctions (TJ) of brain microvascular endothelial cell (BMVEC). TJ proteins, claudin-5 and occludin are down-regulated paralleling Mo egress into the CNS. As small G-proteins (such as Rho) play a role in BMVEC TJ assembly, we hypothesized that loss of TJ integrity is associated with Rho activation triggered by Mo brain migration. To investigate this, we utilized artificial BBB system to explore the relationship between TJ, Rho/Rho kinase (RhoK) activation and trans-endothelial Mo migration. Mo passage across the BBB in response to macrophage chemotactic protein (MCP-1) (increased in HIVE) was evaluated with inhibitors of Rho (C. botulinum C3 transferase) and RhoK (Y-27632). HIV-1 infected Mo migrated more efficiently than uninfected Mo in response to MCP-1. Mo/BMVEC coculture led to Rho activation and occludin phosphorylation. C3 and Y-27632 blocked passage of infected and uninfected Mo. RhoK inhibitor prevented BBB leakage and occludin phosphorylation associated with Mo migration. BMVEC transfection with dominant negative mutant resulted in occludin up-regulation at TJ, while dominant active (DA-RhoK) transfection led to dislocation of occludin from the membrane and loss of BMVEC contacts. When DN-RhoK transfected BMVEC were used in BBB constructs, Mo passage across the BBB was reduced by 84%. These observations suggest that pharmacologic manipulation of Rho/RhoK may prevent Mo egress across the BBB during HIVE. Although HIV is present in the brains of all infected individuals, only 30% of these individuals develop neurological disease, indicating that viral and host characteristics act together in the development of neurological disease. To identify and characterize the salient genomic and functional features that allow some viral strains to induce neuroAIDS, we used the rhesus macaque model of neuroAIDS. We serially passaged microglia-associated SIV through the brains of rhesus macaques and isolated virus after the final passage to generate nine unique, brainadapted SIV clones from the brains of two macaques with neurological disease. Sequence analyses of these brain-adapted clones show a remarkably high level of similarity in the gp120, gp41, nef and LTR regions of their genomes, with sequences that are distinct from other neurovirulent strains of SIV. Functional characterization of representative brain-adapted clones shows a robust in vitro infection of CEMx174 cells, primary macrophages and primary microglia. They also demonstrate an infectious phenotype distinct from other clones, characterized by a very early peak p27Gag levels followed by a slow decline. This unique infection phenotype may be a result of their ability to initially infect a much larger percentage of cultured cells. Positive in vivo selection of brain-adapted strains of SIV resulted in a near-homogeneous strain of virus with distinct properties that may give clues to the viral basis of neuroAIDS. The cognitive and motor disorders occurring in HIV-infected individuals result in significant morbidity and mortality, and remain a serious problem even in the era of highly active antiviral therapy. Although numerous potential neurotoxic molecules have been identified, the nature of the CNS disorder has remained elusive. Using SIV-infected rhesus monkeys, we have carried out functional, pathological, and molecular analyses of different stages of the neurological disease. Distinct as well as overlapping sets of genes are differentially regulated in acute infection, chronic asymptomatic infection, and during AIDS with CNS disease (neuroAIDS). The greatest number of differences is found in neuroAIDS, where transcriptional profiling has revealed 188 known genes to be significantly upregulated. The function of only approximately half of these genes have been characterized; another 28 upregulated transcripts were also identified which have yet to be genetically classified. These proteins encoded by novel genes have intriguing predicted properties, including transcriptional regulation, alteration of RNA secondary structure, and control of protein subcellular localization. The viral-host interaction occurring in the CNS environment can have profound effects on cellular processes. The elucidation of these effects will enable the understanding of how HIV infection leads to CNS dysfunction. Schwann cells (SC), primarily responsible for the formation of myelin within the PNS, might be involved in the local immune response in the PNS. Several lines of evidence indicate that SCs display immune-related functions, such as the expression of MHC class II antigens, suggesting that SCs can acquire the role of a facultative antigen presenting cell. Toll-like receptors (TLRs) have emerged as key receptors responsible for recognizing specific conserved components of microbes, such as lipopolysaccharide (LPS), and have been implicated to play a critical role in various inflammatory disorders. LPS may be a relevant antigen causing immunemediated demyelination of the PNS. We studied the expression of TLRs in these PNS resident cells. In SCs from rat various TLRs were detectable in vitro, out of which TLR-2 and -4 were found to be regulated by substrates, such as interferon-alpha, or phorbol 12-myristate 13-acetate (PMA) in a time-and dose-dependent fashion, as measured on the RNA-level by RT-PCR as well as on the protein level by immunofluorescence. After stimulation with LPS, the secretion of various inflammatory mediators was induced, as determined by a qualitative protein array and quantitated by ELISA. These mediators include chemokines, growth factors, and protease inhibitors. This induction was effectively blocked by specific anti-TLR-2 and anti-TLR-4 antibodies as well as inhibitors of nuclear factor-kappa B. Thus, our data indicate that SCs might act as a link between innate and acquired immunity via TLRs in the inflamed PNS. Skeletal muscle is an immunological microenvironment and the site of many desirable and undesirable immune reactions. Toll-like receptors (TLR) are crucial players of the innate immune system sensing the invasion of microorganisms and inducing antimicrobial responses. To elucidate mechanisms of innate immune responses in human muscle, we investigated the expression of TLR1-9 mRNA in cultured human myoblasts and TE671 rhabdomyosarcoma cells using QRT-PCR. Muscle cells showed constitutive expression of TLR3 mRNA. TLR3 was up-regulated by its ligand Poly(I:C), a synthetic analog of dsRNA, and by IFN-gamma, but not by the TLR4 ligand lipopolysaccharide (LPS) or TNF-alpha. TLR3 protein was located mainly intracellular assessed by flow cytometry and western blot analysis. Poly(I:C) induced NF-kappaB activation and IL-8 secretion in muscle cells. In vivo, muscle fibers of non-inflamed muscle and of inflammatory myopathies expressed TLR3, analysed by immunhistochemistry and QRT-PCR. Importantly, TLR-3 mRNA was highly up-regulated in HIV-myopathies corroborating our in vitro observations. Taken together, our findings may contribute to the understanding of muscular responses to microbial stimuli during infections and the pathogenesis of autoimmune inflammatory disorders. Guillain-Barré syndrome (GBS) is an acute autoimmune polyradiculoneuropathy with a clinical presentation of flaccid paralysis with areflexia, variable sensory disturbances, and elevated cerebrospinal fluid protein without pleocytosis. Although GBS previously had been viewed as a unitary disorder with variations, it currently is viewed as a group of syndromes with several distinctive subtypes. Fifteeen to 40% of the patients with GBS develop the syndrome after being infected by the Gram-negative bacterium Campylobacter jejuni (C. jejuni), a leading cause of acute gastroenteritis in humans. There is a strong tendency for the appearance of antibodies that bind to gangliosides, particularly GM1, to be measurable in the serum of patients with GBS, particularly those with axonal variants and types associated with C. jejuni infection. We have investigated the localization of GM1 in C. jejuni and nervous tissues by using immunoelectron microsopy and con-focal laser microscopy. GM1 was exclusively localized in both central and peripheral nerve myelin. GM1 was not observed in central nerve axons or peripheral nerve axons. We could not find GM1 in C. jejuni by immunoelectron microscopy. Further studies should be taken to clarify the role of antecedent infections of C. jejuni and the role antiganglioside antibody responses.
162a A lentiviral expression system demonstrates that L* protein of Theiler's virus has an anti-apoptotic effect in a macrophage cell line Y. Ohara, T. Himeda, K. Asakura, Y. Kontani Kanazawa Medical University, Kahoku-gun, Ishikawa, Japan DA subgroup strains of Theiler's virus (TV) persist in the CNS of infected mice and induce demyelination. This demyelinating disease serves as an experimental model of multiple sclerosis. The mechanism(s) by which the virus persists and demyelinates remains unknown. DA subgroup strains synthesize a 17-kDa protein, called L*, from an initiation site out-of-frame with the polyprotein. Previous studies using a mutant virus, DAL*-1 (in which the L* AUG is substituted by an ACG) showed that L* has an anti-apoptotic effect in a macrophage cell line, P388D1. In order to confirm and better characterize the anti-apoptotic effect of L*, we used a lentiviral vector to generate P388D1 that constitutively expresses L* (L*/P388D1 cells). The expression of L* was confirmed by Western blotting with anti-L* antibody. DNA laddering was seen in control/ P388D1 cells from 10.5 through 12 hours p.i.. In contrast, no laddering was detected following the infection of DAL*-1 in L*/P388D1 cells. Caspase-3 was activated in control/P388D1 cells, not in L*/P388D1 cells, the same time course as in the case of DNA laddering. The above data suggest that L* has an anti-apoptotic activity in macrophages that can function in cis or trans. The anti-apoptotic activity of L* may play an important role in TV pathogenesis. Identification of novel markers for encephalitogenicity of effector T cells in EAE U. Boehlmann and B. Becher University Hospital of Zurich, Neuroimmunology Center, Zurich, Switzerland TH1 lymphocytes were thought to play a central role in the initiation and potentially in the propagation of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). We have recently shown that the TH1/TH2 paradigm cannot be used as a reliable marker for encephalitogenicity. We identified two mutant mouse strains (IL-12/ 23p40KO and IL-12p35KO) in which auto-aggressive T cells are virtually indistinguishable by virtue of expression of known suspect genes (TH1/ TH2), yet one strain is EAE hyper-susceptible, the other is resistant. Since the activation status of the T cells is correlated with the expression of specific genes, we used high-density gene expression arrays to discover potential encephalitogenicity genes induced by IL-23. We have identified several genes which could have a potential pathogenic function. Currently, the involvement in encephalitogenicity of target genes is being verified in vivo using RNAi knock-down or transgenic over-expression of the target gene. For gene delivery in vivo, we use recombinant lentiviral vectors infecting mouse embryos to generate transgenic mice constitutively expressing the siRNA of the target gene or to infect MOG-reactive T cell lines prior to adoptive transfer into naive recipient mice. Preliminary data clearly show that several of the newly identified genes confer encephalitogenic potential and are critical for the development of EAE. Taken together, our approach will allow us to reliably unmask potential encephalitogenicity genes in vivo.
Silencing T-bet prevents autoimmune demyelinating disease A. E. Lovett-Racke, A.E. Rocchini, J. Choy, S.C. Northrop, R.Z. Hussain and M.K. Racke UT Southwestern Medical Center, Dallas, TX, USA T-bet is a transcription factor that regulates IFN-gamma production in Th1 cells and NK cells. We developed an anti-sense oligonucleotide (AS-Tbet) and small interfering RNA (siRNA-Tbet) specific for T-bet as a tool to determine the role of T-bet in the differentiation of encephalitogenic T cells. Transfection of naive MBP-specific T cells with AS-Tbet or siRNA-Tbet prior to differentiation resulted in decreased IFN-gamma production and a dramatic reduction in the ability of these cells to transfer experimental autoimmune encephalomyelitis (EAE) to naive recipient mice. In addition, in vivo administration of AS-Tbet or siRNA-Tbet to mice at the time of immunization with myelin antigen prevented EAE.T-bet and IFN-gamma expression was suppressed in the lymph nodes and spleens of these mice. This study suggests that T-bet may be a viable target for therapy in Th1mediated diseases, such as multiple sclerosis.
Role of CD200 in immune-modulation and neural protection in EAE T. Chitnis, J. Imitola, W. Elyaman, P. Chawla, M. Sharuk and S.J. Khoury Brigham and Women's Hospital, Harvard Medical School, Boston, USA Background: The CD200-CD200R pathway may play an important role in the regulation of immune responses in the periphery as well as the CNS. Design/Methods: EAE was induced in C57BL/6 mice (WT) by immunization with peptide 35-55 of myelin oligodendrocyte glycoprotein. CD200Fc molecule (Trillium Therapeutics) was administered iv from d0-10 or d10-20 postimmunization. Neuronal cortical cultures were prepared from E18 Wlds and WT embryos and exposed to LPS-activated microglia in vitro, with or without an anti-CD200 blocking antibody (Trillium Therapeutics). Results: CD200Fc (that signals the CD200R) suppressed clinical disease when administered during both the priming and effector stages of EAE. Axonal loss and demyelination were reduced in the CNS of mice that received CD200Fc. In treated animals, there was increased production of IL-4 by primed splenocytes in vitro after re-stimulation with MOG peptide. We have previously found significantly higher expression of CD200 by Wlds neurons than wild-type neurons. Neuronal cultures from Wlds mice were protected after addition of LPS-activated microglia, in contrast to WT neurons. Addition of anti-CD200 Ab to Wlds cultures abrogated protection, suggesting that neuronal protection in Wlds is related to expression of CD200. Conclusions: Signaling through CD200R is associated with protection in EAE and increased Th2 cytokine production. Neuronal overexpression of CD200 confers protection from microglial-induced injury. Macrophage stimulating protein (MSP) modulates innate immunity through its tyrosine kinase receptor, RON. The extent to which RON and MSP are expressed in the central nervous system (CNS) and influence neuroinflammation is unknown. Here, we investigated the effects of RON and MSP in multiple sclerosis (MS) and an established mouse model of MS, experimental allergic encephalomyelitis (EAE). RON mRNA and protein abundance in the CNS was diminished in both EAE ( pb0.05) and MS ( pb0.01) relative to controls while MSP levels were unchanged. MSP inhibited TNF-alpha ( pb0.05), IL-1beta and MMP-9 ( pb0.001) expression in monocytoid cells. During EAE, RON heterozygote and null animals showed augmented IL-1beta ( pb0.05), IL-12 ( pb0.01), MMP-12 ( pb0.05) and TNF-alpha ( pb0.001) with diminished IL-4 levels ( pb0.01) in spinal cord compared to wild type littermate controls. Moreover, neurological disease in heterozygote and null animals exhibited a more rapid onset and overall worsened severity ( pb0.01) in terms of neurobehavioral deficits together with accentuated demyelination, axonal injury and neuroinflammation, relative to wild type controls. Thus, the MSP-RON axis represents a distinct pathway in the CNS by which neuroinflammation and its pathogenic effects are regulated.
Experimental autoimmune encephalomyelitis in histidine decarboxylase-deficient mice R. Pedotti a , S. Musio a , S. Scabeni a , B. Gallo a , L. Poliani a , H. Ohtsu b and R. Mantegazza a a National Neurological Institute C. Besta, Milan, Italy; b Tohoku University, Sendai, Japan
Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are generally perceived as T helper 1 (Th1) mediated diseases. However, recent observations suggest that both allergic and autoimmune mechanisms are involved in the development of EAE, and pharmacological blockade of histamine, one the main mediator of allergic responses in mice and in humans, might prevent EAE. In order to study the contribution of endogenous histamine to the expression of EAE, we induced EAE in histidine decarboxylase (HDC) deficient mice, which express no histamine-producing activity and lack histamine. Upon immunization with myelin oligodendrocyte glycoprotein (MOG) 35-55, HDCÀ/À mice developed EAE, with an incidence somehow lower compared to that of the wild type mice (HDCÀ/À). The onset of EAE was delayed in HDCÀ/À compared to HDC+/+ mice, but those HDCÀ/À mice that developed the disease presented a more severe course compared to wild type mice, with higher EAE-related mortality and hyper acute course in some cases. T cells proliferation to MOG 35-55 was similar in HDCÀ/À and +/+ mice, but T cells from HDCÀ/À mice producegreater amount of interleukin-10 and interferon-gamma. Furthermore, HDCÀ/À mice produce higher levels of IgG antibodies against MOG 35-55 compared to +/+ mice. These results suggest that endogenous histamine importantly modulates EAE. Histamine is a ubiquitous regulator of diverse physiologic processes including inflammation and neurotransmission. Four subtypes of histamine receptors are currently recognized. Genetic and pharmacological studies have shown that histamine H1 and H2 receptors expressed on T-cells and antigens presenting cells play an important role in regulating T-effector cell functions leading to autoimmune inflammatory responses within the CNS. The histamine H3 receptor, which is not expressed by hematopoietic cells, is predominantly presynaptic and serves an important autoregulatory function associated with the release of histamine and other neurotransmitters. Here, we show that despite having identical T-cell effector responses, C57BL/6J mice with a disrupted histamine H3 receptor (H3RKO) immunized with MOG35-55 for the induction of experimental allergic encephalomyelitis develop earlier, more severe clinical signs during the acute phase of the disease compared to wild-type controls. Enhanced clinical disease in H3RKO mice is also associated with earlier disruption of the blood-brain barrier and significantly greater histopathologic disease. Failure of H3RKO mice to limit the acute phase of the autoimmune inflammatory response and clinical disease is consistent with inhibition of negative regulation of presynaptic autoregulatory function in maintaining tissue barrier permeability. Additionally, genetic complementation studies indicate that a structural polymorphism within the third intracellular loop of the H3R may underlie eae8, a previously identified QTL controlling the severity of clinical signs and weight loss, a phenotype known to be regulated by central H3R activity. Macrophage migration inhibitory factor (MIF) has recently been shown to regulate T cell activation, cell division and macrophage cytokine production. Importantly, increased levels of MIF were found in the cerebrospinal fluid of multiple sclerosis (MS) patients. Antibody blocking of MIF was shown to blunt acute EAE in mice by interfering with homing of T cells to the CNS. We explored the role of MIF in relapsing-remitting EAE in wild type (WT) C57BL/6Â129 mice immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 and in MIFÀ/À C57BL/6Â129 mice. Acute EAE was similar between WT and MIFÀ/À mice, but MIFÀ/À mice showed a dramatic decrease in the number and severity of disease relapses compared to WT mice. Pro-inflammatory cytokine levels (TNF-alpha, IFN-gamma and IL-6) were sharply decreased in MIFÀ/À mice relative to WT controls at all time points, while IL-4 and IL-10 secreting cells were significantly increased in MIFÀ/À mice. Corticosterone levels were markedly elevated in MIFÀ/À mice during the acute disease and recovery periods. Our results show that MIF exerts a profound effect on disease relapses, mediated in part by the regulatory effects of glucocorticoid hormones on cytokine production. (Supported by NIH grants AI43376, NS48316 and National MS Society grants RG3091 and RG3272).
Autoreactive T cells persist in rats protected against EAE and can be activated through innate immunity S.B. Conant, N.A. Wolf and R.H. Swanborg Wayne State University, Detroit, USA Lewis rats are rendered unresponsive to experimental autoimmune encephalomyelitis (EAE) by immunization with myelin basic protein (MBP), or MBP68-86, the dominant encephalitogenic epitope, in incomplete Freund's adjuvant. However, protected rats harbor inactive encephalitogenic T cells. We investigated whether these cells could be activated in vitro. Although they respond poorly to MBP68-86, they proliferate vigorously, and secrete IFN-gamma if cocultured with MBP68-86 plus IL-2 or IL-12, suggesting that they are anergic. We could also activate these cells to proliferate and secrete IL-6 with peptide plus CpG oligonucleotide, but not control oligonucleotide; thus, products of innate immunity can activate anergic autoreactive T cells. Anti-TGF-beta-1 Ab released the unresponsive T cells from the anergic state in the presence of MBP68-86 alone (i.e., without CpG), whereas TGF-beta-1 inhibited proliferation of MBP68-86 plus CpG-activated T cells. CD4+CD25À T cells isolated using magnetic beads proliferated vigorously to CpG plus MBP68-86, whereas CD4+CD25+ cells proliferated poorly. TGF-beta has been implicated in T regulatory cell (Treg) activity, and since Treg cells frequently express the CD4+CD25+ phenotype, our results are consistent with the hypothesis that Treg cells maintain autoreactive T cells in the anergic state, but these can be released from anergy by products of the innate immune system (e.g., CpG) which override regulatory mechanisms that maintain immune homeostasis. (Supported by NIH and National MS Society).
The failure of interferon gamma receptor null mice to recover from EAE is NOT due to the absence of nitric oxide S.A. Fordham, M.A. Staykova and D.O. Willenborg Australian National University Medical School, Canberra, Australia IFN-gamma receptorÀ/À (gamma RÀ/À) mice develop severe, usually fatal EAE when immunised with MOG peptide 35-55 whereas wild type mice develop milder disease. Furthermore, passive transfer of disease with MOG35-55 specific lymphoid cells from IFN-gamma RÀ/À mice produces, in knockout (IFN-gamma RÀ/À) mice, severe EAE from which recipients fail to recover. Identical cells produce equally severe disease in IFN gamma R+/+ mice but recipients recover. These results provided evidence that IFN-gamma is not essential for EAE development but plays an important role in down-regulating disease. We hypothesised that IFN-gamma stimulates NO production which limits disease. This hypothesis is incorrect. Transfer of encephalitogenic T cells from immunised gamma RÀ/À mice into inducible nitric oxide synthase knockout (iNOSÀ/À) mice, which can respond to IFN-gamma but cannot make NO, results in severe EAE yet the animals recover. We also show that EAE in 3 different strains of mice each lacking the IFN-gamma R and immunised with three different encephalitogens all develop severe, usually fatal EAE with identical pathology-extensive neutrophilic infiltration. We conclude that the inability of IFN-gamma RÀ/À mice to recover from EAE is not due to their inability to shut off effector T cells but to the fact that the absence of IFN-gamma responsiveness results in a malignant neutrophilic inflammatory response rather than a relatively benign mononuclear response. , while in its presence, inhibition by NO is reduced. The interaction between these two free radicals may regulate immune response intensity and autoimmunity, as studied in EAE. The identity of the source(s) of NO and O 2 À in the spleen is unknown. Using cell separation, we show that Gr-1 spleen cells from primed mice are responsible for regulation of T cell proliferation by NO and O 2 À in an MHC-unrestricted manner. This was demonstrated in the presence of activated spleen cells from naRve DO11.10 mice, which do not produce NO or O 2 À . Furthermore, non-adherent Gr-1 cells are a major source of O 2 À production. FACS analysis indicated that these Gr-1 cells are CD11b hi , CD11c neg , B220 neg , I-A/I-E neg and F4/80 lo , possibly representing immature myeloid Gr-1 cells. Purified Gr-1 + cells contain different stages of granulocyte maturation. M. tuberculosis in CFA is responsible for the Gr-1 cell increase in the spleen following immunization, which starts as early as 4 days p.i. and continues until at least 4 weeks p.i. These studies provide a novel way for innate immune cells to regulate adaptive immunity.
Antibody responses to the glial progenitor cell proteoglycan NG2/AN2 exacerbate experimental autoimmune encephalomyelitis (EAE) E. Mathey a , A. Schubart b , K. Williams a , J. Trotter c and C. Linington a a University of Aberdeen, Aberdeen, Scotland, UK; b Harvard Medical School, Boston, USA; c Johannes Gutenberg-Universität, Mainz, Germany
Remyelination in the adult central nervous system is carried out by a population of glial progenitor cells (NG2/AN2 positive) that migrate to the lesion site and repair injury. However, in multiple sclerosis (MS), this endogenous repair mechanism is inefficient and ultimately fails as the disease progresses. The reasons for failure of remyelination in chronic MS lesions are poorly understood; however, the presence of autoantibodies to NG2 in the cerebrospinal fluid of MS patients may be indicative of an autoimmune response against the cells responsible for remyelination. We have used a DNA vaccination strategy in rats to generate a specific autoantibody response to the first two LNS domains of the NG2 protein.
The antibody response can be assayed by Western blotting, ELISA and flow cytometry. This DNA vaccination strategy has allowed us to assess the effects of an NG2-specific antibody response on the modulation of disease severity in adoptive transfer EAE and on remyelination in MOG induced EAE. Our findings indicate that self-tolerance to the NG2 protein can be disrupted in the rat and that the consequent autoantibody response enhances the clinical and histological severity of EAE. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) receptors expressed in the nervous system include S1P1, S1P3 and LPA2 (in neuronal cells), LPA1 and S1P5 (in white matter tracts) and S1P2 (in both neuronal cells and white matter tracts). In the immune system lymphocytes express S1P4, unstimulated T helper cells express LPA2 and S1P4, whilst macrophages express LPA1 and S1P3. Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease with pathological features equivalent to multiple sclerosis (MS). RNA prepared from spinal cords of control and EAE rats was profiled for S1P and LPA receptor expression using Taqman analysis. Average (n=5) mRNA levels for S1P1, S1P5 and LPA1 receptors were each decreased 0.4-, 0.6-and 0.5-fold respectively, in EAE samples in comparison to control. However, mRNA for S1P2, S1P4, LPA2 and LPA3 receptors were increased 3.3-, 12.5-, 2.7-and 1.4-fold in comparison to controls. S1P3 receptor mRNA expression was not detected in control group but was induced in the EAE group. The significant increase of S1P4 receptors reflects the huge infiltrating lymphocytes in the EAE model and suggests that targeting this receptor may influence the progression of EAE and potentially MS. The multifunctional cytokine Oncostatin M (OSM) is involved in the progression and resolution of the inflammatory process (Hurst et al., 2002) . While the exact functional role of this cytokine in neuroinflammation is currently unknown, several studies have documented elevated levels of OSM in the brains of patients with the progressive relapsing-remitting neurological inflammatory disease multiple sclerosis (MS). OSM may play an important role in the progression of neuroinflammation by virtue of its spatial and temporal expression and its role in leukocyte recruitment. In this study, we have evaluated the spatial and temporal expression of OSM in neuro-inflammation using the chronic relapsing Biozzi mouse model of MS. Brain and spinal cord tissues were collected from mice at different stages of the disease process and semi-quantitative PCR used to determine the presence of OSM mRNA. Evidence will be presented to indicate that tissue levels of OSM mRNA are associated with the severity of symptoms and phases of disease. Cell adhesion molecules are of fundamental importance in autoimmune diseases such as multiple sclerosis (MS) and an animal model for MS, the experimental autoimmune encephalomyelitis (EAE), by affecting the interaction between target and effector cells. Here, we define the role of a cell adhesion molecule, termed ninjurin-1, in the interaction between neurons and activated microglia cells. In peripheral nerve regeneration, this molecule is upregulated and promotes nerve outgrowth; here, we show that ninjurin-1 expression is regulated by cytokines and located at the interface between the injured axons and activated microglia. The homophilic binding of ninjurin-1 between neurons and microglia was efficiently blocked by a oligopeptide. Moreover, we identified ninjurin-1 in injured axons and glial cells in EAE. Therefore, we suggest that ninjurin-1 is implicated during the pathology of EAE whereby cytokines regulate the adhesion of glial cells to injured axons.
The role of the central dopamine depletion in regulation of experimental autoimmune encephalomyelitis in C57BL mice E. Balkowiec-Iskra a , I. Kurkowska-Jastrzebska b , I. Joniec a , A. Czlonkowska b and A. Czlonkowski a a Medical University, Warsaw, Poland; b Institute of Psychiatry and Neurology, Warsaw, Poland
Circumstantial evidence suggests the involvement of central dopaminergic system in the regulation of immune response. The aim of our study was to investigate the influence of central dopamine depletion on experimental autoimmune encephalomyelitis (EAE) progression. Dopamine depletion was obtained by treatment with 1-methyl-4phenyl-1,2,3,6-tetrahydropiridine (MPTP) (40 mg/kg). EAE was elicited 7 days after MPTP intoxication by immunization with MOG 35-55 (150 Ag). In mice with injured dopaminergic system, onset of the symptoms was observed 15 days earlier (3 day vs. 18 day) than in mice not treated with MPTP. In MPTP+EAE group, two attacks were noted with a longer total time comparing to EAE group, in which only one short attack was observed.
Similarly, remission of symptoms in EAE group appeared earlier than in MPTP+EAE group. However, the disease index did not differ between the groups. Histological analysis revealed stronger infiltration by inflammatory cells in the meninges and in perivascular space of the white matter of the lumbar spinal cord of MPTP+EAE mice. In our previous studies, MPTP has been shown to have no direct action on immune cells. Herein, we demonstrate, that EAE induction and progression may be dependent on central dopamine action. We suggest indirect mechanisms to be involved in reported results. Murine EAE is an established multiple sclerosis (MS) model generated in our laboratory by inducing autoimmunity to a peptide (amino acids 35-55) from myelin oligodendrocyte glycoprotein (MOG). Use of a single injection protocol, and two different mouse strains, generates diseases resembling relapsing-remitting (RR) MS (NOD/Lt mice), or primary progressive (PP) MS (C57Bl/6 mice). Current evidence of axonglia and glia-glia communication lead us to undertake a time course study of astrocyte responses in both EAE types. We report the significant finding that the two MS types have different astrocyte response patterns: (1) in the pre-clinical period, considerable astrocyte hypertrophy occurs in RR-EAE whereas PP-EAE shows a much smaller increase;
(2) with progression, astrocyte hypertrophy occurs throughout white matter and grey matter with or without parenchymal inflammation;
(3) during remission (RR-EAE), astrocyte hypertrophy remains; (4) astrocyte processes in RR-EAE intercalate between and separate inflammatory cells into small groups whereas inflammatory foci in the PP-EAE are free of astrocyte processes; (5) in RR-EAE astrocyte response is related to disease stage; but to clinical score in PP-EAE. These observations suggest that the astrocyte response is considerably less active in PP-EAE, giving rise to the hypothesis that the genetic context of this response may influence the particular form of MS expression. Introduction: The signalling molecule sonic hedgehog (SHH) is involved in several processes of CNS development. Recently, the protein concentration of SHH has been shown to be upregulated during active disease in a spontaneously demyelinating mouse model and to be decreased in multiple sclerosis (MS) white matter. Specific object of our study was to investigate the expression pattern of SHHduring lesion evolution in myelin-oligodendrocyte-glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a model strongly mimicking MS. Methods: MOG-EAE was actively induced in DA rats. Histological evaluation was performed on paraffin-embedded CNS sections on days 20 to 120 after active immunization. Results: In actively demyelinating and inactive lesions immunoreactivity for SHH was present in macrophages, astrocytes and endothelial cells. In addition, astrocytes in the lesion surrounding showed staining for SHH. In early remyelinating and remyelinated lesions, expression of SHH was found in astrocytes, macrophages, endothelial cells and axons, which showed signs of acute injury. The highest density of SHH expression on axons was found in early remyelinating lesions. Conclusion: We provide the first immunohistochemical analysis of the expression pattern of SHH in autoimmune encephalomyelitis. Our study demonstrates that SHH plays a role in repair mechanisms of the adult CNS. The localization of SHH on injured axons in early remyelination might indicate a role of SHH as axonal survival factor.
Alterations of neurotrophic factors during the course of experimental autoimmune encephalomyelitis J. Ah-Sue and V.W. Yong University of Calgary, Calgary, Canada
Neurotrophic factors can confer neuroprotection, regeneration and immunomodulation in diseases of the central nervous system, including multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Elevation of certain neurotrophic factors have been observed in EAE and MS. In this study, we sought to determine the expression profile of neurotrophic factors in relation to the stage of disease during progression and remission of EAE in order to gain insights into their possible functions. Relapsing-remitting EAE was induced in female, 129/SvEv mice using myelin oligodendrocyte glycoprotein as the immunogen. Mice were sacrificed 6 days postimmunization, at first sign of disease, during peak disease, and in remission. The lumbar/sacral spinal cords were extracted and analysed using real time-polymerase chain reaction. Multiple neurotrophic factors were elevated in EAE, including ciliary neurotrophic factor and insulinlike growth factor-1. A prominent juncture for the upregulation of several neurotrophic factors was at peak disease and at resolution (remission). Current experiments seek to evaluate the cellular sources and the function of neurotrophic factors in EAE, particularly with regards to immunomodulation. Multiple sclerosis (MS) is caused by a combination of genetic predisposition and environmental factors, resulting in T cell infiltration into the CNS. Many environmental factors have been implicated in disease although results of studies performed to date remain inconclusive. A possible genetic/biochemical factor implicated in MS is the dysregulation of matrix metalloproteinases (MMPs), a family of zinc containing proteases with many physiological functions. In many diseases, these proteases are upregulated and acquire harmful functions. Specifically, MMP-9 has been implicated as detrimental in experimental autoimmune encephalitis (EAE), an animal model of MS and in MS. In this study, we have examined the activation of MMP-9 by heavy metals, and have found that Hg exerts a biphasic response on MMP-9. Importantly, at 100 nM, Hg increases MMP-9 activity two-fold ( pb0.001). This combination of Hg and MMP-9 increases the levels of interferon-gamma by human and mouse T cells in culture comparably ( pb0.001). The requirement of MMP-9 was demonstrated by the inability of Hg to increase interferon-gamma levels in T cells from MMP-9 null mice. Recovery of cytokine levels followed exogenous addition of MMP-9 to Hg-treated null cells. Ongoing experiments address the facilitation of EAE in Hg-treated MMP-9 and wildtype mice. We postulate that MMP-9 dysregulation and Hg exposure, leading to altered T cell reactivity, may underlie the progression of some cases of MS. Murine EAE is a well established model that recapitulates many clinical and physiopathological aspects of MS. A close examination of the earliest changes in gene expression, even those preceding inflammation, has not been reported yet. We collected serum and CNS tissue from NOD mice both before immunization with MOG 35-55 and at 13 timepoints encompassing the first clinical attack (days 3 to 19 postimmunization). Microarray-based gene expression profiling was performed in spinal cords from four mice at each timepoint (82 arrays in total), while other three animals were used for histopathological staining at the same time intervals. Supervised and unsupervised machine learning algorithms were used to identify expression signatures that correlated with disease stage and histological profiles.
Remarkably, the earliest expression changes in the CNS were observed before any histological evidence of inflammation. In addition, a mass spectrometry-based proteomic profile was obtained from the sera of each animal and was correlated with gene expression patterns, in the search for early markers of neuroinflammation. Such high frequency expression data will enable the formulation of more precise models of disease pathogenesis. Our study demonstrates the utility of combined genomics and proteomics approaches towards unraveling the molecular pathways leading to clinical disease and recovery in EAE, and eventually MS.
Chemokine receptors CCR7, CCR8 and CX3CR1 in chronic relapsing experimental autoimmune encephalomyelitis A.R. Glabinski, B. Bielecki and K. Selmaj Medical University, Lodz, Poland
Chemokines are small molecules which direct migration of inflammatory cells towards the tissue sites of inflammation. Because of that ability, chemokines are involved in pathogenesis of inflammatory diseases of the central nervous system (CNS). In the present study, we analyzed expression of three lately described chemokine receptors CCR7, CCR8 and CX3CR1 in the CNS and in peripheral tissues during chronic-relapsing experimental autoimmune encephalomyelitis (ChREAE), an animal model of multiple sclerosis (MS). Methods: Female (SWRxSJL/J)F1 mice were immunized with an encephalitogenic PLP139-151 peptide. Chemokine receptors expression was analyzed using quantitative Rnase Protection Assay (RPA). Analysis was performed during first attack, remission and the second attack of ChREAE. Animals without ChREAE were used as controls. Results: We observed upregulation of all three analyzed chemokine receptors (CCR7, CCR8 and CX3CR1) in the spinal cord during the first and the second ChREAE attacks. In the brains, this upregulation was less prominent. Expression of CCR7 and CX3CR1 in the spinal cord correlated well with the disease severity. In spleens, expression of all three chemokine receptors was comparable in all phases of ChREAE. In the blood, expression of CCR7 was lower during ChREAE than in control normal animals. Conclusion: Our results suggest that chemokine receptors CCR7, CCR8 and CX3CR1 are involved in pathogenesis of ChREAE and that they may be potential target for treatment of the CNS autoimmune disorders.
188 TWEAK, the TNF-related weak inducer of apoptosis, and its receptor Fn14 in experimental autoimmune encephalomyelitis (EAE) A. Müller, X. Pedré, I. Kleiter, G. Giegerich and A. Steinbrecher University of Regensburg, Regensburg, Germany TWEAK, a member of the TNF superfamily, mediates prominflammatory effects by its receptor Fn14. We examined the consequences of TWEAKand Fn14-blockade in chronic relapsing experimental autoimmune encephalomyelitis (EAE). Inducing a neutralizing antibody response TWEAK or Fn14, respectively, we found a significant amelioration of the clinical course of MOG-induced EAE in rats and of PLP(139-151)induced EAE in SJL mice accompanied by a significant decrease of spinal cord infiltrates. In addition, we performed DNA-vaccination with an eukaryotic expression plasmid encoding for TWEAK resulting in a more severe disease course compared to control vaccinated animals. Further in vitro and ex vivo studies suggest that blockade of Fn14-mediated signalling interferes with the recruitment of immune effector cells into the CNS.
Expression of adrenomedullin (ADM) receptors in the central nervous system of Lewis rats during acute experimental allergic encephalomyelitis (EAE) C. Paul a and C. Bolton b a University of the West of England, Bristol, UK; b William Harvey Research Institute, London, UK
Loss of normal blood-brain barrier (BBB) function is an integral feature of the neuroinflammatory process in multiple sclerosis (MS) . Previously, we demonstrated that restoration of BBB function is associated with an improved clinical outcome in models of MS. In vitro studies have shown ADM capable of regulating BBB characteristics including transendothelial electrical resistance, permeability and p-glycoprotein pump activation. Therefore, ADM-mediated control of BBB function may be a viable therapeutic target for controlling the course of MS. Clearly, it is essential to determine whether the receptor pathway is intact during a neuroimmune challenge such as occurs in EAE the model of MS. Using immunohistochemistry, we have assessed the expression of ADM receptors and receptor-activity-modifying proteins (RAMP) 2 and 3, which confer calcitonin receptor-like receptor specificity for ADM, in the CNS of animals with acute EAE. Preliminary studies indicate that normal spinal cord RAMP2 expression is strongly associated with morphologically identified capillary structures while RAMP3 exhibits a very low vascular presence. In EAE CNS tissues, RAMP2 expression is maintained both immediately before and after the height-of-disease. RAMP3 expression is similarly preserved. Hence, the receptor pathway with respect to RAMP2 and 3 expression appears intact at the cerebrovasculature during EAE. Therefore, the potential exists to employ ADM and related agonists to restore BBB function during neuroinflammation. Experimental autoimmune encephalomyelitis (EAE) is a well-characterised autoimmune disorder of the central nervous system (CNS). Investigations into the enzymes controlling the complex alterations in the extracellular matrix (ECM) environment may lead to a greater understanding of their role in CNS inflammation. ADAMTS enzymes are metalloproteinases that degrade versican, aggrecan, brevican, phosphacan and neurocan. These are found within the CNS and promote cell adhesion, migration and influence neurite outgrowth. To investigate the potential role of ADAMTSs, white matter from the spinal cord of rats at pre-disease, peak and recovery stages of acute EAE and naive controls were assessed for the expression of ADAMTS-1, -4 and -5, their natural inhibitor, TIMP-3 as well as the ECM components, phosphacan and neurocan, at the mRNA and protein level. ADAMTS-4, -5, TIMP-3 and phosphacan were all downregulated in the CNS at peak disease compared to naRve controls, whereas ADAMTS-1 and neurocan were upregulated in pre-diseased animals compared to naRve controls. This provides the first report of ADAMTS expression in EAE. This work was funded by The Wellcome Trust. Previous work found that MMP-12 is highly expressed in the spinal cord of mice afflicted with EAE, an animal model of MS. Currently, we are investigating whether there is an interaction between MMP-12 and OPN, and their function(s) in EAE progression. In vitro, we found that MMP-12 time-dependently cleaved OPN into 2 major products, which was inhibited by BB-94, a metalloproteinase inhibitor. EAE was produced in 8-9 weeks old female C57BL/6 mice via immunization with myelin oligodendrocyte glycoprotein and animals were sacrificed either at the initiation of disease, at the ascending phase of disease, at peak disease, and at score stabilization. The spinal cords were examined for mRNA and protein expression levels of OPN and MMP-12. Western blot analyses showed that at disease onset, OPN cleavage products first appeared, reaching a maximum expression during the ascending phase of disease. The mRNA expression of OPN and MMP-12 showed a similar trend. Current experiments seek to identify the MMP-12 cleavage products of OPN, and their role in disease. We postulate that the interaction between MMP-12 and OPN may have important roles in EAE onset and progression. Experimental autoimmune encephalomyelitis (EAE) induced with recombinant myelin oligodendrocyte glycoprotein (rMOG) in rat is an accurate model of multiple sclerosis (MS) immunopathology. A growing body of experimental evidence points to a critical involvement of matrix metalloproteinases (MMPs) at critical checkpoints in the immunopathogenesis of the disease. However, our knowledge on the new MMP family members expression profile in inflammatory demyelination is limited. In the present study, we analyzed the expression pattern of all 22 known rat MMPs in a rMOG-EAE model in DA rats during the clinical course using real time RT-PCR. Central nervous system infiltrating T-cells were detected by immunohistochemistry on cryostat sections, and RNA was extracted from these areas out of consecutive sections. MMP mRNA levels were measured and their variations related to early stage non-symptomatic animals. The mRNA of MMP-12 was found to be most strikingly up-regulated, remaining highly expressed even during the recovery phase of the disease. Membrane-bound MMP-14 and recently described MMP-23 were among other up-regulated enzymes. On the other hand, MMP-13 and MMP-27 revealed a persistent down-regulation during the course of the disease. Our results demonstrate a highly differential regulation of MMP mRNA expression in inflammatory infiltrates in the rMOG-EAE rat model. Further studies on specific expression patterns of selected MMPs might help to broaden our understanding on the specific role of MMPs in inflammatory demyelination.
Inducible nitric oxide synthase (iNOS)-mediated axonal injury in experimental autoimmune encephalomyelitis F. Aboul-Enein, P. Weiser, M. Bradl and H. Lassmann Brain Research Institute, Vienna University, Vienna, Austria
Multiple sclerosis (MS) is a T-cell mediated inflammatory disease of the central nervous system (CNS) of supposed autoimmune nature. Since the hallmark of MS pathology is the demyelinated plaque associated with relative preservation of axons despite complete destruction of their myelin sheaths, axonal injury has been interpreted only as a secondary consequence of primary demyelination. Axonal degeneration is recognized to be an important cause of neurological deficit. However, pathogenetic mechanisms underlying axonal injury within inflammatory diseases of the CNS remain still largely undefined. As there is only little evidence for direct immune attack, free radicals as oxygen and/or nitrogen species may be crucial for axonal pathology. For this purpose, we induced acute monophasic EAE in the Lewis rat by passive transfer of encephalitogenic MBPspecific T-cells in wild type and in PLP overexpressing transgenic Lewis rats in presence or absence of demyelinating anti-MOG antibodies.
Performing immunohistochemistry, we found in both experiments expression of inducible Nitric Oxide Synthase (iNOS), a key enzyme of free radical production, exclusively confined to perivascular macrophages and/ or microglia. Acute axonal injury, identified by the accumulation of amyloid precursor protein, correlated strictly with numbers of iNOSpositive macrophages/microglia. Anti-MOG antibody mediated demyelination resulted in an augmentation of axonal injury and iNOS expression. Our findings may have major implications for therapy of inflammatory CNS diseases. The BN rat is reported to be resistant to induction of Th1 type experimental autoimmune encephalomyelitis (EAE) and a number of mechanisms have been suggested. We provide evidence that the high production of nitric oxide (NO) in the periphery results in this resistance. Spleen cells from BN rats make 3-6 times more NO when stimulated with IFN gamma than do cells from PVG or Lewis rat and the BN rat makes substantially more NO than either EAE susceptible strain following immunisation with CFA. If carbonyl iron is used as adjuvant, there is no increase in NO levels in the BN rats and they are rendered highly susceptible to EAE. CFA given simultaneously with neuroantigen and carbonyl iron drives up NO levels and the resistance is restored. EAE produced using carbonyl iron is characterised by extensive presence of macrophage/microglia in the central nervous system (CNS) lesions and the cytokine profiles in the lymph nodes and CNS do not differ from the ones in the EAE Lewis rats.
Expression of iNOS and COX-2 in TMEV-IDD N.G. Carlson, K.E. Hill, H.E. Watt and J.W. Rose VA-SLCHCS, University of Utah, Salt Lake City, UT, USA Objective: We recently reported that COX-2 and iNOS are expressed in chronic-active plaques from MS patients. We proposed that these enzymes contribute to enhanced glutamate-mediated excitotoxicity of oligodendrocytes and neurons. Confocal microscopy of COX-2 and iNOS expression in the Theiler's murine encephalomyelitis virus (TMEV) induced demyelinating disease (TMEV-IDD) was examined to assess whether their expression precedes the onset of neuropathology and clinical disease. Methods. Mice were given IC injections of TMEV and were assessed for clinical symptoms, demyelination and inflammation and intervals of 1, 2, 3, 4, 5 and 9 weeks post injection. Expression of iNOS and COX-2 was examined using immunofluorescence confocal microscopy in combination with the cell-specific markers CNPase (for oligodendrocytes) and GFAP (for astrocytes). Results: COX-2 and iNOS were detected in isolated regions of the brain as early as 2 weeks post-injection. Purkinje cells of the cerebellum expressed iNOS.iNOS and COX-2 was also observed in regions of the hippocampus. After 4 weeks, just prior to the onset of symptoms, moderate amounts of iNOS were observed in spinal cord regions near oligodendrocytes. COX-2 was also observed in association with iNOS both nearby and within oligodendrocytes. Conclusion: The expression of COX-2 that we observed in oligodendrocytes may have important consequences with respect to vulnerability of these cells to glutamate-mediated excitotoxicity analogous with neuronal excitotoxicity. Expression of COX-2 may then render oligodendrocytes more vulnerable to glutamatemediated excitotoxic death.
Oxidative modification alters the autoantigenicity of myelin oligodendrocyte glycoprotein-implications for induction of disease and tolerance in mice and men M. W3llberg a , J. Bergquist b and R. Harris a a Karolinska Institutet, Stockholm, Sweden; b Uppsala University, Uppsala, Sweden
Myelin antigens may become autoantigenic through modification of their structure, but this concept has received little or no attention to date. Understanding the effects of chemical modification of myelin antigens by mimicking in vitro processes which are likely to occur during inflammatory reactions in vivo may provide clues as to the antigenic nature of the proteins underlying MS development. We studied modification of myelin oligodendrocyte glycoprotein (MOG), with malondialdehyde (MDA), which is physiologically generated through lipid-peroxidation. Using IEF, FPLC and FTICR mass spectrometric analyses, we demonstrate that MDA modification leads to changes in protein weight and charge. DBA/1 mice immunised with MDA-MOG developed a more severe form of EAE, and antigen-specific recall responses were elevated to MDA-MOG compared to normal MOG. Formulation of a MOG-alum vaccine with MDA-MOG improved its protective efficacy. Finally, CSF from MS patients had higher titres of anti-MOG IgG to MDA-MOG than to control MOG. We believe that oxidative modification of MOG leads to enhanced autoantigenicity that promotes induction of disease induction, induction of protection and diagnosis of human MS. To develop a therapeutic strategy using single amino acid substitutions of MOG 8-21 and PLP 56-70 we first selected those alanine and lysine amino acids substitutions that did not induce EAE in the mice but in vitro induced strong T cell responses. While many substituted peptides were encephalitogenic suggesting those residues were not critical for ether binding to MHC or activating T cells, others did not induce either disease or proliferation. In addition several peptides were non-encephalitogenic but were immunogenic in vitro suggesting that these peptides may induce regulatory responses. Two such alanine substitutions of MOG 8-21 (MOG P-A10 and MOG I-A11) selected on basis of not inducing EAE and with a high T-cell response in vitro were administered in a tolerance regimen during remission in an attempt to inhibit chronic relapsing EAE. Conclusion: While nonencephalitogenic single amino acid substitutions of MOG 8-21 and PLP 56-70 inhibit relapsing disease in mice immunised with MOG 8-21, the therapy was significantly more effective when the wild-type peptide is used. Objective: Intravenous injection of pertussis toxin (PT) greatly facilitates the induction of several experimental autoimmune diseases {Mochizuki, 1983 #68} including experimental autoimmune encephalomyelitis (EAE). We investigated whether the enhancement of autoimmunity by PT is related to differences in expression of the apoptotic protein caspase 3 by autoreactive CD4 T cells. Methods: SJL mice were immunized with proteolipid protein (PLP) 139-151 in CFA. One experimental group of mice received in addition intravenous injections of 300 ng pertussis toxin. Mice were examined for EAE disease score and histological signs of EAE. Single cell suspensions were prepared from draining lymph nodes and analyzed by flow cytometry after staining with MHC class II tetramers specific for PLP139-151 (I-AsPLP139-151) and antibodies against CD4 and caspase 3. Results: PT was required for PLP139-151/CFA immunized mice to develop clinical and histological signs of EAE. Administration of PT increased the absolute number of autoreactive T cells in the draining lymph nodes 10 days after immunization. The expression of caspase 3 in autoreactive T helper cells was reduced in mice that receive PT. Conclusions: We demonstrate that the induction of autoimmunity by PT is associated with inhibition of apoptosis in autoreactive T helper cells.
High threshold for autoantigen-specific stimulation and induction of experimental autoimmune encephalomyelitis in T cell-specific IKK2deficient mice B. The nuclear factor (NF)-kB family of transcription factors plays a pivotal role in T cell activation and survival during adaptive immune responses. IkB kinase 2 (IKK2 or IKK2beta) is part of the IKK complex, a central component of the intracellular signalling pathway leading to canonical NF-kB activation. We used mice that lack IKK2 specifically in T cells to investigate the role of IKK2 in autoantigen-specific T cell activation and induction of autoimmune disease. We found highly impaired myelinoligodendrocyte-glycoprotein (MOG)35-55-specific T cell activation in vitro and complete resistance to MOG 35-55-induced EAE in T cellspecific IKK2-deficient C57BL/6 mice. In contrast, T cell-specific IKK2deficient mice transgenic for a pathogenic MOG35-55-specific T cell receptor (2D2 TCR) were susceptible to MOG35-55-induced EAE. Therefore, inefficient priming and/or expansion of IKK2-deficient T cells seems to be responsible for the resistance of T cell-specific IKK2deficient mice to induction of EAE. Pharmacological inhibition of IKK in vitro reduced MOG35-55 specific proliferation and cytokine production of 2D2 transgenic T cells. Our data suggest an IKK2-dependent threshold for autoantigen-specific T cell activation and underscores the potential value of IKK inhibition for the therapy of autoimmune diseases. Immunization of SJL mice with encephalitogenic peptide PLP139-151 induces EAE when the antigen is resuspended in IFA supplemented with 4 mg/ml of MT. We have studied the effect of presence of MT during immunization on two TCR repertoires specific for PLP139-151. Frequency of usage and polarization of these repertoires is not affected by the presence of MT. The Th1 promoting effect of adjuvant appears to be due to recruitment towards Th1 of a wider spectrum of TCR repertoires, rather than to a more robust expansion of the same repertoires. Th1 cells appear in the LN by day 4 after immunization, by day 10 in the spleen and in the CNS at the onset of the first episode of EAE. They disappear from the CNS during remission and are again detectable during the plateau phase of the first relapse of disease. To investigate the influence of MHC genes on manifestation of central nervous system (CNS) autoimmune disease, we studied myelin oligodendrocyte glycoprotein (MOG)-induced EAE in H-2 congenic C3H mice. We found that clinical signs and pathology differ dramatically according to the MHC haplotype. MOG-induced EAE in C3H.Fej mice (H-2k) is severe and clinically characterized by proprioception defects and spastic reflexes. Pronounced parenchymal inflammatory infiltrates are observed in the brain, comprising both CD4+ and CD8+ T cells, B cells, macrophages and neutrophils. CD8+ T cells preferentially localize in the brain while CD4+ T cell subsets predominate in the spinal cord. In contrast, C3H.SW mice (H-2b) develop a mild, chronic EAE characterized by ascending paralysis. Little inflammation is observed in the brain of C3H.SW mice.
In both strains, the spinal cord is severely targeted with large lesions composed predominantly of neutrophils, the extent of the lesions correlating with antibody deposition. MOG-specific T cell responses in C3H.Fej mice encompass several MOG epitopes that differ from the single MOG epitope (MOG35-55) targeted in C3H.SW mice. The role of immune cell subsets, T cell specificities and antibody responses in determining the pathogenesis of disease will be discussed. This model will elucidate immunological parameters that influence clinical disease, and may provide insight into the complex and heterogeneous pathogenesis of multiple sclerosis. The hypothesis that astrocytes participate in maintaining the immune privilege of the central nervous system (CNS) is mainly based on in vitro data showing that they inhibit mitogen and antigen-driven lymphocyte proliferation. We have previously shown that rat T cells pre-incubated with astrocytes completely inhibited proliferative response of naRve lymphocytes to mitogenic stimuli when co-cultivated at 1:1000 ratio, respectively. In this study, we demonstrated that isolated mononuclear cells from spinal cords (SCMNC) of DA rats in different phases of experimental autoimmune encephalomyelitis (EAE) are unresponsive to mitogenic stimuli during acute phase but not during relapse of EAE. Furthermore, co-cultivation of SCMNC obtained from rats at the peak of EAE with naRve lymphocytes, inhibited proliferation to ConA of latter cell population. This inhibitory effect was restricted to SCMNC, as cells obtained from the lymph nodes of the same animals did not influence the mitogen-induced proliferation of responder cells. Interestingly, SCMNC isolated from animals in the recovery phase of EAE were unable to inhibit proliferation of naRve cells. Further studies of the observed phenomena would contribute to the understanding of mechanisms involved in spontaneous recovery during inflammatory diseases of CNS. Although there are many ways of inhibiting autoimmune demyelinating disease in rodent models, there has been poor translation of these in multiple sclerosis (MS) and progressive disease continues despite immunotherapy that inhibits blood-brain barrier dysfunction and relapse rate. Prophylactic autoimmune tolerance can be induced by a variety of different means, yet therapeutic tolerance in established disease provides a more difficult goal. In the robust, relapsing Biozzi ABH mouse model of MS, using a combination of a transient-deletion of primed, auto-reactive T cells (by CD4-specific antibodies) followed (1 week later) by intravenous myelin-antigen administration (antigen-coupledsplenocytes), established relapsing disease in experimental autoimmune encephalomyelitis can be effectively silenced following disease induction. Unfortunately, when treatment was initiated late during chronicrelapsing disease (after three paralytic attacks), despite inhibition of further relapses; mice demonstrated evidence of secondary progression, including increased spasticity and deterioration in mobility. This suggests that autoimmune therapy in MS should begin as early as possible to have an optimum chance for success and indicates that once sufficient deficit is accumulated, targeting relapsing, immunological components of disease alone is unlikely to be sufficient to control progression based on a clinical outcome. Therefore, there appears to be correlation between that observed in humans and that found in rodent models.
The Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system. Experimental Autoimmune Encephalomyelitis (EAE) is an animal model used to study MS and is mediated by encephalitogenic CD4+ Th1 cells. T-box expressed in T cells, or T-bet, is a transcription factor that is believed to play a key role in regulation of the IFN-gamma gene and in the differentiation of pathogenic autoimmune Th1 cells. To determine the role that T-bet plays in the differentiation of encephalitogenic Th1 cells, we investigated the effects of silencing T-bet expression on both the IL-12/STAT4 and the IFNgamma/STAT1 pathways. To achieve T-bet suppression, we designed anti-sense oligonucleotides and small interfering RNAs specific for T-bet. T-bet suppression resulted in reduced STAT1 protein levels but did not affect the IL-12/STAT4 pathway. In addition, T-bet was found to bind the IFN-gamma and STAT1 promoters via chromatin immunoprecipitation assay. Therefore, T-bet appears to regulate the differentiation of encephalitogenic Th1 cells through the IFN-gamma/STAT1 pathway and may be a potential target for therapeutics for Th1-mediated diseases such as MS. Motor-evoked potentials (MEP) by transcranial electrical stimulation were studied as an in vivo method for assessing corticospinal system changes in chronic experimental autoimmune encephalomyelitis (EAE), obtained by immunizing C57Bl/6 mice with MOG35-55, in parallel with neuropathology. Cortical MEP were recorded in 95% of EAE mice 20 days post-immunisation (20 dpi), were absent in 41.1% and 47.3% of mice at 40 and 80 dpi, respectively. At 120 dpi, we did not record any MEP in the majority of the EAE mice. In mice with preserved MEP, latencies were significantly increased, with a peak at 20-40 dpi and a slight revovery afterwards. At the latest time-points, MEP amplitudes significantly decreased, suggesting chronic axonal loss. When considering overall MEP issues, the method sensitivity almost approached 100% in EAE. Neuropathology demonstrated inflammatory infiltrates since 10 dpi, a peak of either inflammation or demyelination at 20 dpi, then both of them tended to fade out later on. Axon loss was firstly detectable between 20 and 40 dpi, reaching its maximum at 80-120 dpi. Thus, the small-sized, slowed or even absent MEP observed in the latest EAE stages, in keeping with neuropathological findings, suggests that acute axonal damage and chronic axonal loss are characterizing feature of EAE in C57Bl/6 mice, perhaps as a consequence of early inflammatory/demyelinating events occurring in the first 20 dpi. Therefore, MEP can contribute not only to gain insight on EAE pathophysiology, but also to quantitatively assess experimental neuroprotective therapies in CNS demyelinating diseases.
Estimation of EAE severity using video-based motion analysis of tail motility D. Schmitz, T. Tomov, W. Neiss and D. Angelov Institute of Anatomy, University of Cologne, Cologne, Germany
To avoid biased clinical scoring of EAE in rats, we developed a method to objectively evaluate onset and severity of disease. Since tail atony is the earliest and most consistent symptom of EAE, we video-taped tail movements before the appearance of paraparesis [at 9 days postinduction (dpi)] and at the peak of paraparesis (13 dpi). Using Motus 2000 (PEAK Performance Technologies), we analysed the common motor performance of tail muscles evaluating the angle between tail and body. Under normal conditions, the tail lies loose on the inspection table. Upon lifting the animal, the tail acquires horizontal position due to reflex contraction of the musculature. We measured the deviation of the tail from the longitudinal axis of the animal's body in the sagittal plane. Thereby, the dorsal flexion was estimated in negative and the ventral flexion-in positive degrees. In intact animals the dorsal flexion was -4.608F3.108 and the ventral flexion-2.65F1.608 (n=8), showing that tail movements in intact rats occur generally in the negative region. At 9 dpi, the dorsal flexion was À14.70F9.208 and the ventral flexion +13.60F11.208 (n=8). Atd 13 dpi, the dorsal flexion increased dramatically to +32.42F13.88 and the ventral flexion to +35.04F14.18(n=8). Video-based motion analysis of tail motiliy is a valuable tool to estimate onset, clinical progress and results after EAE-treatment. Although multiple sclerosis (MS) is considered to be an autoimmune disease in the central nervous system (CNS), the immune responses that take place in the CNS and lymphoid organs remain to be elucidated. Here, we have successfully induced various subtypes of EAE in LEW.1AV1 rats carrying RT1av1 on the Lewis background genes by immunization with recombinant rat MOG in various solutions with adjuvants. The purpose of the present study was to analyze in more detail the clinical and immunopathological features of MOG-induced EAE in LEW.1AV1 rats. Immunization with high doses of soluble MOG with pertussis toxin (PT) induced acute, frequently fatal, EAE, whereas medium doses of partially aggregated MOG without PT produced relapsing and remitting (RR)-EAE. Secondary progressive (SP)-EAE was induced in some rats by immunization with the immunization protocol having the intermediate nature between the above two. The optic nerve (approximately 60% of the immunized rats) and spinal cord (100%) were frequently involved and detectable both clinically and pathologically. Histological examination revealed that despite of the variety in the clinical subtypes, progression of the pathological processes was strikingly uniform, i.e., initial inflammation with minimal demyelination followed by predominant demyelination with minimal lymphocyte infiltration. These findings suggest that the lesion during the later stage is maintained by humoral factors. Taken together, this experimental system can serve as a model of neuromyelitis optica (NMO). Further analysis will provide useful information to elucidate the pathogenesis and to develop the immunotherapy of NMO and other MS. Adult mouse neural precursor cells (aNPCs) were in vitro labelled with increasing concentrations of Fe using three different super paramagnetic iron oxide (SPIO) contrast media (EndoremR; ResovistR; SineremR). Iron uptake, cell viability, proliferation and differentiation were in vitro tested 72 h and 8 days after in vitro labelling. We prepared agarose phantoms containing dispersed unlabelled and labelled cells or free SPIO and measured at 3T (with a 23-mm surface coil) signal change with routinely used sequences and R1 and R2 relaxivities. Increasing doses of SPIOs were used (from 0.01 to 0.8 mg/ml Fe). Uptake of all SPIOs was much higher when aNPCs were incubated in vitro with both polylisine-L and iron nanoparticles. Mild cell toxicity was measured when cells were incubated with 0.8 mg/ml Fe. All relaxation parameters paralleled increased cell concentration of the iron particles. Between contrast media tested, EndoremR showed the highest signal decrease. aNPCs can be successfully labelled in vitro with novel SPIOs contrast media. This method might allow in vivo aNPC tracking. Expression of endothelial selectins was documented during MS (multiple sclerosis) and EAE. Alpha(1,3) fucosyltrasferases (FucT) catalyze glycosylation of P-selectin glycoprotein ligand-1 (PSGL-1), a ligand for endothelial E-and P-selectin. The GOAL of this study was to determine the role of FucTs in the induction of EAE. METHODS FucT-deficient mice for FucT-IV, FucT-VII and double deficient mice for FucT-IV and VII were generated on C57Bl/6 genetic background. Intravital microscopy studies were performed in inflamed brain vessels. EAE was induced by using MOG35-55 peptide. RESULTS: Th1, but not Th2, cells efficiently roll and arrest in inflamed brain venules. Anti-E-and P-selectin and anti-PSGL-1 antibodies blocked tethering and rolling of Th1 lymphocytes in brain venules. Th1 lymphocytes produced from FucT-IVÀ/À mice efficiently tethered and rolled in brain venules when compared with cells from wt mice. In contrast, primary adhesion of Th1 lymphocytes obtained from FucT-VIIÀ/À or FucT-VII and IVÀ/À mice was drastically reduced. The onset of actively-induced EAE in FucT-VIIÀ/À, but not FucT-IVÀ/À, mice was significantly delayed. Disease was significantly inhibited in FucT-VII and IVÀ/À mice when compared with wt and FucT-IVÀ/À mice. MOG35-55-specific T cells obtained from FucT-VIIÀ/À or FucT-VII and IVÀ/À mice displayed no rolling on E-and P-selectin under physiological flow. CONCLUSIONS: Our data unveil a critical role for FucT-VII in the recruitment of activated lymphocytes in inflamed brain venules, and suggest that FucTs play a role in the pathogenesis of autoimmune inflammatory diseases of the brain. Methods: Magnetic column-enriched or -depleted sub-populations of adult rat hematopoietic bone marrow cells (BMC) were transplanted alone or with chicken ovalbumin-specific rat T cells into severe combined immunodeficient (SCID) mice. Recipient SCID mice were assessed for the location and phenotype of transplanted BMC-derived cells and for susceptibility to CNS inflammation induced subsequently by transferred myelin-specific T cells. Results: BMC expressing low or undetectable levels of alpha 4 integrin (VLA-4) were associated with a high potential for T cell-enhanced migration to the CNS, and with the highest diseaseinducing activity, compared to BMC expressing a high level of alpha 4 integrin. BMC expressing low or undetectable alpha M integrin (CD11b) were also associated with enhanced trafficking to the CNS and more severe disease, compared to BMC expressing high alpha M integrin. Conclusions: CNS APC develop from BMC expressing low or undetectable CD49d and CD11b. This developmental pathway is enhanced by non-myelin-specific T cells in an acute, pre-pathogenic time frame, and may be responsible for modulating susceptibility to and severity of CNS inflammation in diseases such as multiple sclerosis. In many TNF-alpha-associated neuropathologies, including MS, cerebral malaria, vascular dementia and HIV encephalitis, the contribution made by high levels of cerebral TNF-alpha expression to the pathogenesis and the outcome of these conditions remain unresolved. We have found that TNFalpha in the brain gives rise to a TNFR2-dependent endothelin-mediated reduction in regional cerebral blood volume (rCBV), which is accompanied by delayed blood-brain barrier breakdown and compromised tissue energy metabolism. We have investigated the role of platelets in these events by microinjecting the pro-inflammatory cytokines IL-1beta or TNF-alpha into the brain parenchyma. We have discovered that TNF-alpha, but not IL-1beta, induces accumulation of GPIIb-IIIa positive platelets within the intact cerebrovasculature and is followed by the adherence of ED-1 positive mononuclear cells in a fashion that is very reminiscent of cerebral malaria pathology. Platelet accumulation was not restricted to the microvasculature and venules, but was also present in arterioles. We next investigated how adenoviral-mediated chronic TNF-alpha expression might affect platelet accumulation. We found that platelets remain within the cerebral vasculature while CBV is chronically reduced. The chronic, adenoviral mediated expression of TNF-alpha is also associated with neuronal cell death and monocyte recruitment. The administration of anti-selectin antibodies successfully reduced platelet and ED-1-postive cell accumulation. These findings indicate that targeting the TNFR2 pathway and TNFinduced platelet accumulation may represent a useful target for the neuropathologies associated with elevated TNF-alpha and reduced CBV. Infiltration of leukocytes into the CNS is a key event in the development of multiple sclerosis (MS). Molecules involved in the interaction between leukocytes and endothelial cells are therefore candidate targets for therapy. CD81 is a member of the tetraspanin familiy of small surface proteins, and it has been implicated in cellular adhesion, motility and proliferation. Also, CD81 regulates the activation of VLA-4 and LFA-1, two major adhesion receptors on leukocytes. We therefore set out to determine the function of CD81 in leukocyte transmigration. A time lapse videomicroscopy assay was used to monitor transmigration of rat NR8383 monocytic cells across confluent monolayers of immortalised rat cerebrovascular endothelial cells. A monoclonal antibody (mAb) against rat CD81 consistently resulted in inhibition of monocyte transmigration by 40-50% as compared to controls receiving no mAb or isotype control. When the endothelial cells were prestimulated with a mixture of IL-1beta and IFN-gamma for 48 h, inhibition by CD81 mAb was further increased to around 60%, similar to the effect of a mAb to VLA-4. We conclude that CD81 is important in the transendothelial migration of monocytes. Furthermore, our results provide strong support for its further exploration as a therapeutic target in MS. Understanding the mechanisms of immune cell migration into MS lesions offers great therapeutic potential. This study focused on CXCL13, a chemokine classified as homeostatic. CXCL13 is essential for B cell migration to lymph follicles, but may also affect T cells, since its receptor CXCR5 is upregulated on T cells during activation. We report that CXCL13 was overexpressed in MS-brain lesions as detected by quantitative PCR. Within MS lesions, CXCL13 was immunolocalized to cells with a macrophage-like appearance in the perivascular area and in the parenchyma. In the CSF of MS patients, CXCL13 was elevated and correlated with intrathecal Ig production, the presence of B cells, plasma cells/plasma blasts, and T cells, but not monocytes. About 20% of the T cells and almost all B cells in the CSF expressed CXCR5. The strong correlation of CXCL13 with both T cell and B cell migration to the brain MS might qualify this chemokine as a future therapeutic target in MS. The mononuclear inflammatory infiltrate, a common characteristic of infective and inflammatory CNS disorders, consists of T-cells, monocytes and activated microglia. Chemokines and chemokine receptors play a key role in the recruitment of inflammatory cells into sites of inflammation. CCR2, a CC receptor, is expressed on T-cells and monocytes in blood and interacts with CCL2. In animal models, the disruption of CCL2/CCR2 interaction using CCL2 neutralising agents or CCL2/CCR2 knockout animals leads to a decrease in inflammatory infiltrate and clinical disease. This study identifies the pattern of CCR2 expression on inflammatory infiltrate in a number of CNS disorders, including multiple sclerosis (MS), West Nile Encephalomyelitis (WNE), using immunohistochemistry and CCL2 binding assays. In the CNS, the majority of T-cells expressed CCR2 whereas activated monocytes sparsely expressed CCR2. Anti-CCR2 antibodies inhibit the binding of CCL2 to CCR2 on frozen MS sections. In the CSF, small percentage of T-cells and the majority of activated monocytes expressed CCR2. CCR2 expressing mononuclear cells were in close contact with activated microglia in WNE. CCR2 expression is differentially regulated on T-cells compared to monocytes in the CNS. CCR2 expression is either upregulated on T-cells upon transmigration across the blood-brain barrier or CCR2 immunoreactive T-cells are preferentially recruited into the CNS. CCR2 may be a potential therapeutic target in CNS inflammatory disorders.
The role of the substance P receptor, NK-1, in CNS inflammatory disease E. Reinke and S. Macvilay, Z. Fabry University of Wisconsin-Madison, Madison, WI, USA Substance P is a neuropeptide shown to affect the immune system, nervous system, and vasculature. It has been shown to have an important role in inflammation and acts to increase activation of pro-inflammatory leukocytes; increase edema, vasodilation, and adhesion molecule expression on vascular endothelial cell; and is involved in the detection of pain by sensory neurons. We have investigated the role of the substance P receptor, neurokinin-1 (NK-1), in central nervous system (CNS) autoimmunity using the model disease experimental autoimmune encephalomyelitis (EAE) in C57Bl/6 and NK-1 receptor knock out mice treated with a subcutaneous injection of the CNS self-peptide, myelin oligodendroglial protein (MOG). Our data show that the NK-1 receptor is expressed on brain microvessel endothelial cells, and that mice lacking the NK-1 receptor experience a less severe disease in the chronic state. This is characterized by lower clinical scores and less demyelination and infiltration in the spinal cord. These results indicate that substance P and its receptor, NK-1, can contribute to CNS inflammatory diseases and opens up a new direction for possible treatments for diseases such as multiple sclerosis. Perivascular accumulation of monocyte-derived macrophages and lymphocytes is a characteristic feature of multiple sclerosis pathology. In order to enter the brain parenchyma, immune cells need to cross the blood-brain barrier (BBB). This barrier is mainly composed of specialized brain endothelial cells, pericytes and perivascular macrophages. Endothelial cells and pericytes are lined by a vascular and astroglial basement membrane (BM), respectively. Here, we studied the molecular composition of both BMs in well-characterized MS lesions using a panel of antibodies directed against several BM proteins. Laminin alpha-4 and -5 chains are present in the endothelial BM, whereas anti-laminin alpha-1 and -2 antibodies specifically decorate the astroglial membrane. Laminin alpha-3, beta-1, beta-2 and gamma-1 antibodies immunostain both BMs. Similarly, fibronectin, heparan sulfate proteoglycans (HSPGs) and glycosaminoglycans (GAGs) localize to both BMs. Interestingly, we observed laminin alpha-4, beta-1 and gamma-1 chain-positive structures within perivascular cuffs. These patterns were also immunopositive for other extracellular matrix (ECM) components including fibronectin, HSPGs and GAGs. It has already been shown that HSPGs and GAGs can bind chemokines, small proteins that control cellular migration. We therefore hypothesize that deposition of ECM/BM components in the perivascular space may act as a conduit network for chemokines and thereby contribute to recruitment of inflammatory cells. Future research is warranted to study the functional role of ECM/BM molecules in multiple sclerosis pathogenesis.
The beta chemokines MCP-1 and MIP-1alpha enhance adhesion of monocytes to human brain microvessel endothelial cells in vitro K. Liu a , R. Prameya b , R. Shahidi b , V. Wu b and K. Dorovini-Zis a a University of British Columbia, Vancouver, Canada; b Vancouver Hospital and Health Sciences Centre, Vancouver, Canada
Chemokines have been implicated in the recruitment of leukocytes to the brain in CNS inflammation. The factors that regulate the trafficking of monocytes across the blood-brain barrier (BBB) remain poorly understood. We investigated the effects of the beta chemokines MCP-1 and MIP-1alpha on monocyte adhesion to human brain microvessel endothelial cells (HBMEC) in an in vitro model of the BBB. Primary cultures were grown to confluence on collagen membranes in a double chamber chemotaxis system. MCP-1 or MIP-1alpha were placed in the lower chamber to establish concentration gradients. HBMEC monolayers were used untreated or treated with TNF-alpha and IFN-gamma for 24 h to mimic inflammatory conditions. Peripheral blood monocytes were placed over HBMEC and incubated for 30 to 60 min at 37 8C. Radiolabeled MCP-1 and MIP-1alpha diffused across the collagen membranes and cytokine-treated HBMEC monolayers in a time-dependent manner and bound to the subendothelial matrix and basal cell surfaces. Chemokine diffusion was minimal across untreated HBMEC. Cytokine treatment increased the adhesion of monocytes to HBMEC by approximately 100%. Concentration gradients of MCP-1 and MIP-1alpha increased the adhesion of monocytes to untreated and cytokine-treated HBMEC. These findings suggest a potential role of MCP-1 and MIP-1alpha in the recruitment of monocytes across the BBB to sites of inflammation in the CNS. Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS) and is widely studied as an animal model of multiple sclerosis (MS). Our EAE model is characterised by disturbances in balance and coordination causing mice to roll in an axial rotatory motion. Inflammatory and demyelinating lesions are found primarily in the brainstem and cerebellum and to a lesser extent in the spinal cord. The inflammatory infiltrate contains lymphocytes, monocytes/ macrophages, and also polymorphonuclear cells (PMNs), which makes this model pathologically similar to acute variants of MS such as Marburg's disease. Cytokines and chemokines play a role in promoting the immune response and determining the lesion composition in EAE and MS. Therefore, we have examined the cytokine, chemokine and chemokine receptor expression in our EAE model to determine the factors involved in leukocyte infiltration into the CNS. Our results indicate that CXCL2/MIP-2 and CXCL1/KC, potent chemotactic agents for PMNs, and their receptor CXCR2 are highly expressed within the CNS. Additionally, CCL5/ RANTES, a chemotactic agent for monocytes/macrophages, eosinophils and basophils, and its receptor CCR5, as well as proinflammatory cytokines IFN-gamma and TNF-alpha, are also expressed within the CNS in this model. The relevance of this data and implications for future research will be discussed.
CXCL10 promotes the secretion of CXCL8 and CCL2 by human astrocytes and brain endothelial cells E.A. Subileau a , H. Davies a , F. Colyer a , J. Greenwood b , D. Male a and I. Romero a a Open University, Milton Keynes, UK; b University College London, London, UK
High levels of the chemokine CXCL10 and its receptor, CXCR3, have been reported in multiple sclerosis (MS) lesions. CXCL10 plays an important role in activated lymphocyte and monocyte diapedesis towards inflamed sites. However, little is known about the effects of CXCL10 on the cells of the blood-brain barrier (BBB). Using transmission electron microscopy and immunogold staining of post-mortem MS brain tissue revealed the presence of CXCR3 in HBEC and surrounding astrocytes both in normal appearing white matter and in white matter lesions. We demonstrated the expression of CXCR3 on primary human brain endothelial cells (HBEC) by immunocytochemistry in vitro. Addition of CXCL10 to the culture medium had no effect on the permeability of HBEC monolayers to dextran-FITC 70 KDa and 4 kDa in an in vitro BBB model, nor was the expression of the adhesion molecules (ICAM-1, VCAM-1 and PECAM-1) modified on HBEC. However, CXCL10 led to an increase in CXCL8 and CCL2 levels in the supernatant of stimulated HBEC and astrocytes. In addition, CXCL10 induced the phosphorylation of ERK1/2 and p38MAPK. In astrocytes, either MEK inhibitor (PD98059) or p38MAKP inhibitor (SB203580) failed to block CXCL10-mediated CXCL8 and CCL2 release suggesting that signalling pathways other than ERK1/2 and p38MAPK may be involved in this process. Our results suggest that CXCL10 could play a role in the infiltration process by amplifying the network of chemokine signals acting on leukocyte migration. Chemokines and their receptors are implicated in leukocyte ingress into the brain during central nervous system (CNS) inflammation, as observed during multiple sclerosis. The expression of several chemokine receptors on T lymphocytes, particularly CCR2 and CXCR3, differs greatly depending on their location in blood, cerebral spinal fluid, or brain tissue, suggesting that these receptors may be modulated during trafficking, or may be required for migration. We addressed receptor modulation on mononuclear cells during diapedesis into CNS parenchyma using a static in-vitro model of the blood-brain barrier (BBB) that recapitulated BBB properties. Using this model, we performed transmigration assays with peripheral blood mononuclear cells and characterized receptor expression profiles on leukocytes. Of T cells, only memory cells transmigrated and CXCR3 was enriched on transmigrated cells compared to input and non-migrated cells. Studies using CXCR3-depleted populations showed that CXCR3 is a surface marker for cells that have the capacity to cross the BBB, but does not play an essential role in extravasation. We have extended this experimental approach to additional chemokine receptors and cell populations. This BBB transmigration assay provides useful information that promotes our understanding of the biological significance of differential chemokine receptor expression in blood compared to parenchymal CNS lesions.
Modeling monocyte/macrophage migration and reverse transmigration in the brain
The Scripps Research Institute, La Jolla, CA, USA Cells of the monocyte/macrophage lineage have been shown to be the principal targets for productive HIV-1 replication within the central nervous system. In addition, HIV-1-associated dementia has been shown to correlate with macrophage abundance in the brain. While accumulation of macrophages in neuroAIDS is likely initiated by increased entry of monocytes into the brain, mechanisms that prevent macrophage egress from the brain and means that prevent macrophage death may also contribute to accumulation. We have developed a novel transendothelial migration model that will allow us to test the ability of chemokines to alter monocyte/macrophage trafficking. The migration and accumulation of peripheral blood mononuclear cells in the ablumenal (i.e., tissue) side of an in vitro endothelial monolayer was investigated. The migrated cells were highly enriched for monocytes, including CD14+CD16+ monocytes. We also investigated the reverse transmigration of monocytes from the ablumenal to lumenal side (i.e., circulation). We hypothesized that within the HIV-infected brain, molecules can be expressed that decrease reverse transmigration of these macrophages, leading to increased number of tissue macrophages. Based on our microarray analyses that showed osteopontin, a potent chemoattractant protein, was upregulated in the brains of simian immunodeficiency virus encephalitis cases, we are currently examining the role of osteopontin in comparison to other chemokines, including fractalkine, in our transendothelial migration model with the hypothesis that osteopontin is involved in the decrease of reverse transmigration.
Lipoic acid prevents monocyte migration across the blood-brain barrier Migration of monocytes across the blood-brain barrier (BBB) is a critical step in the development of inflammatory lesions during multiple sclerosis (MS). Transendothelial migration of monocytes requires the active participation of brain endothelial cells (BEC); they need to rearrange their cytoskeleton and tight junctions. Previously we showed that reactive oxygen species (ROS) are essential for monocyte migration and that superoxide is the pertinent ROS that affects the integrity of the endothelial cell layer. We investigated whether the antioxidant lipoic acid (LA) could inhibit monocyte migration across the BBB. In vitro, LA decreased migration of monocytes across a monolayer of BEC. Using life cell imaging techniques we now have quantitatively assessed that ROS are produced within minutes upon interaction of monocytes with endothelium, inducing changes in the cytoskeleton, which could be prevented by LA.
In vivo, LA dose-dependently prevented the development of clinical signs and monocyte infiltration into the CNS in acute Experimental Allergic Encephalomyelitis (EAE). Together, these results indicate that oxygen radicals are essential for monocyte migration in vitro as well as in vivo. ROS-scavengers like LA may be tools to prevent monocyte migration across the BBB and may therefore be potential candidates for the treatment of MS.
Development of microglia from mouse embryonic stem cells and their migration into the brain parenchyma T. Tsuchiya, K.C. Park, S. Toyonaga, S. Yamada, H. Nakabayashi, E. Nakai, N. Ikawa and K. Shimizu Department of Neurosurgery, Kochi Medical School, Kohasu, Nankoku-shi, Kochi, Japan
Microglia is very similar to peripheral macrophage, and exist at central nervous system (CNS). Microglia function as an antigen presenting cell in several CNS diseases, but the detailed mechanism has remained obscure yet. Microglial progenitor or precursor cells have been not identified. We derived microglia from mouse embryonic stem cells (ES cells) at a high density. Mac1-positive cells were induced by stimulation with GM-CSF following neuronal differentiation of mouse ES cells using the five-step method. CD45low expression was high and CD45high low on induced cells. Microglia expressed MHC class II molecules and expression was enhanced by LPS. On the other hand, detection of MHC class I molecules was negligible on the derived microglia, irrespective of LPS stimulation. Intravenous transfer of GFP+ microglia derived from GFP+ ES cells caused selective accumulation in brain but not in peripheral tissues such as spleen and lymph node. GFP+ cells were detected mainly in corpus callosum and hippocampus but rarely in cerebral cortex, where Iba1, another marker of microglia, is primarily expressed. Furthermore, both GFP+ and Iba1+ cells exhibited a ramified morphology characteristic of mature microglia. Thus, we suggest that mature microglia induced with our protocol may function as brain-specific delivery of medicines, or other bioactive materials, such as proteins or genes. Being an integral part of the blood-brain barrier, astrocytes play an important role in regulating homing of different leukocyte subsets to the inflamed central nervous system (CNS). The aim of this study has been to investigate whether astrocytes produce chemokines which facilitate dendritic cell (DC) migration. Using RNase protection assay and/or ELISA, we found that human astrocytes stimulated in vitro with interleukin-1 beta and tumor necrosis factor produce CXCL12, CCL2, CCL5 and CCL20, that act on immature myeloid DC, but not CCL19 and CCL21, that attract mature myeloid DC. Supernatants of cytokine-stimulated astrocyte cultures were more effective than control cell-free medium and supernatants of unstimulated astrocytes in inducing the migration of monocyte-derived immature DC. The finding that immature DC migrated toward CXCL12 and CCL20 and that desensitization of their corresponding receptors reduced DC migration in response to astrocyte supernatants indicates that astrocytes produce biologically relevant amounts of these chemokines. Immunohistochemical studies showed that in unaffected human brain CXCL12 was expressed in neurons and cerebrovascular endothelia. In brain tissue from multiple sclerosis (MS) patients CXCL12 immunopositivity was also detected in reactive astrocytes within chronic lesions. Expression of CCL20 in MS lesions is under investigation. These findings suggest that astrocytes may actively contribute to DC recruitment in the CNS during autoimmune disease.
Bone-marrow stem cells migrate across the blood-brain barrier and differentiate into microglia in normal and neurodegenerative mouse models A.R. Simard and S. Rivest Laval University, Quebec City, Canada
Stem cells are capable of differentiating into multiple cell types in order to develop or regenerate different tissues. However, it is still unclear whether bone marrow-derived stem cells can migrate across the blood-brain barrier (BBB) in many regions of the central nervous system (CNS), and if these cells can differentiate into microglia. We thus studied the differentiation of bone marrow stem cells upon immigration into the CNS by systemically transplanting stem cells that express green-fluorescent protein (GFP) into lethally irradiated mice. We show that circulating stem cells can migrate across the BBB and differentiate into microglia throughout the CNS of normal mice. These cells were also CD11c-positive, indicating that they may be more potent APCs compared to residential microglia. We also tested whether stem cell immigration into the CNS would be increased in neurodegenerative models. We thus injected the brain with sodium nitroprusside, a chemical that causes extensive neurodegeneration. We found that GFP-positive cell migration and differentiation into microglia were significantly more prevalent in regions afflicted by neuronal death. Taken together, our data show that microglia of blood origin can cross the BBB, are preferentially recruited to damaged areas of the brain and that they could potentially activate cells of the adaptive immune system. These results may have great clinical relevance for both immune-derived neuronal disorders and cancer patients undergoing allogeneic hematopoietic stemcell transplantation.
Characterization of macrophage brain cell populations in experimental cerebral malaria T.F. Pais and S. Chatterjee Instituto Gulbenkian de Ciência, Oiras, Portugal
The brain macrophages comprise a very heterogeneous population of cells, the parenchymal microglial cells, perivascular macrophages, meningeal and choroid plexus macrophages. The relative contribution of each population during infections of the CNS has not always been clearly determined. In this study, we have addressed the activation of brain macrophages in the context of cerebral malaria (CM). Plasmodium falciparum is the infectious agent of CM in humans, a severe cerebral complication characterized by reversible encephalopathy. The kinetic of activation of brain macrophages was investigated on brain slices of infected animals by staining with tomato lectin. Obvious morphologycal changes and increased staining on brain macrophages were observed 2 days before mice start to die. Further characterization of leukocytes was performed by flow-cytometric analysis of several markers of T cells, monocytes, macrophages and activated macrophages. An increased percentage of cells expressing high levels of CD45 but slightly CD11b was observed in moribund mice. This population was mainly derived from donor cells in bone marrow chimeric mice and expressed TCR alpha/beta, CD8 alpha and considerable levels of myeloid markers such as CD11b, Gr-1 and 7/4. A slight increase was also observed in the percentage of CD45 high and F4/80 positive cells. However, we did not detect on brain macrophages increased expression of classical markers of macrophage activation such as MHC class II and CD40 suggesting that during cerebral malaria activated brain macrophages display a different activation phenotype.
Lymphomononuclear infiltration in the CNS of mice with acute GvHD after allogeneic bone marrow transplantation P. Sostak, C. S. Padovan, A. Gerbitz and A. Straube Ludwig-Maximilinans-University, Munich, Germany
Graft-versus-host disease (GvHD), a systemic complication after allogeneic bone marrow transplantation (BMT), most often affects the skin and liver and more seldom may cause peripheral neuromuscular disorders. So far, involvement of the CNS has been denied in autopsy studies, but recent clinical and experimental findings suggest that GvHD might also manifest in the CNS. In an animal model of chronic GvHD, we could already detect cerebral lymphomononuclear infiltrations, microglial activation and upregulation of adhesion molecules. Until now, studies on acute cerebral GvHD have been performed less systematically, therefore, we have investigated the brains of mice with experimental acute GvHD. After conditioning with total body irradiation, the mouse strain CBA received bone marrow from B10.BR mice for induction of acute GvHD. Syngeneic transplanted animals and mice, which were only irradiated but not transplanted, served as controls. The mice brains were investigated 6 weeks after transplantation by immunohistochemistry. In all of the 13 allogenic animals, we found lymphomononuclear infiltrates, most often associated with the leptomeninges and the plexus, more rarely within brain parenchyma. Infiltrations were nearly absent in syngeneic or irradiated controls. Compared to earlier findings, the extend of lymphomononuclear infiltration was lower in acute GvHD than in chronic GvHD.
Our data provide evidence that involvement of the CNS occurs both in acute and chronic GvHD. The different underlying pathophysiological mechanisms need further clarification.
Essential role of VLA-4/VCAM-1 pathway in the establishment of Trypanosoma cruzi-elicited meningoencephalitis J. Lannes-Vieira, E. Roffê, A. Silva and A.P. Marino Department of Immunology-IOC, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil
Central nervous system (CNS) damage can occur during Trypanosoma cruzi infection, especially in immunosuppressed patients. The enhanced susceptibility of C3H/He mice to CD8-mediated acute meningoencephalitis is associated with higher up-regulation of vascular cell adhesion molecule-1 (VCAM-1) on CNS vascular endothelia than in the less susceptible C57BL/6. Further, in vitro adhesion of activated peripheral blood cells to CNS blood vessels was abrogated by anti-VLA-4 antibodies that also inhibited cell migration into the CNS of T. cruzi-infected mice. Lastly, the reactivation of meningoencephalitis in immunosuppressed chronically infected mice was associated with VCAM-1 up-regulation. Therefore, we hypothesize that VLA-4/VCAM-1-pathway plays a pivotal role in the establishment of T. cruzi-elicited encephalitis. Osteopontin (OPN) was found to be one of the major upregulated genes in inflammatory neuropathologies such as MS, Theiler's virus infection, EAE and SIV encephalitis. The role of OPN as chemoattractant to inflammatory cells was studied in vivo using the rodent air pouch model (AP). OPN was injected in subcutaneous APs in Balb/c mice. Cells were harvested from the cavity over a 48-h period. Predominantly mononuclear cells accumulated gradually at early time points, reaching a peak at 5 h, followed by a decrease. Histopathology of the AP adjacent skin showed adherence of OPN-elicited cells. The cells in the exhudate at 5 h after OPN injection into the AP were phenotypically characterized and compared with other chemoattractant, MCP-3. OPN was more powerful than MCP-3, inducing higher number of Mac-1+ cells into the AP. Furthermore, OPN and MCP-3-attracted cells exhibited distinct characteristics: OPN-induced macrophages expressed higher levels of CD44v6 (OPN receptor), CD16 and CD62L (involved in migration of cells to the brain parenchyma), and lower levels of CD86 (related to type-2 response), and CD14 than MCP-3-elicited cells. Differences on expression of CD80, CD11a and CD44pan were not observed. Overall, MCP-3 induced more immature macrophages, while OPN attracted/ stimulate cells with characteristics of migrating and type-1response-inducer macrophages (CD16+CD14low CD62L+CD80+CD86F), similar to what is found in inflamed CNS.
(This work is supported by NIH NS045534-01).
The endoneurial macrophage response during experimental slow progressive neuropathies is mainly of intrinsic origin M. Müller, K. Wacker, E.B. Ringelstein and R. Kiefer University of Münster, Münster, Germany
Macrophages have a major impact on the pathogenesis of peripheral neuropathies and increase during peripheral neuropathies. In addition, we recently demonstrated that an important proportion of the endoneurial macrophage response in traumatic and inflammatory neuropathies is generated by resident macrophages and not only by invading hematogenous macrophages. To prove the hypothesis that resident macrophages are capable of generating the macrophage response during slow progressive neuropathies, we used chimeric mice carrying green fluorescent protein positive bonemarrow. These mice allow differentiation between resident and hematogenous macrophages. After administration of acrylamid the mice developed a progressive neuropathy. Fluorescence microscopy and immunohistochemistry was used to quantify resident GFPÀ and GFP+ hematogenous macrophages. In addition, basic macrophage properties were examined. We found that during acrylamide, induced neuropathy endoreurial macrophages become activated, proliferate, phagocytose and increase in number. The ratio of GFP+ and GFPÀ macrophages changed only slightly towards an increase of GFPÀ macrophages. In addition quantitative analysis of macrophage shape revealed that GFP+ endoneurial macrophages are more activated than GFPÀ macrophages. Taken together, our results point towards an intrinsic generation of the macrophage response in slow progressive length-dependent neuropathies. These macrophages share basic macrophage properties like phagocytosis and MHC-II expression. Differences in the macrophage shape between GFP+ and GFPÀ macrophages could be interpreted as different functional states of long term resident and hematogenous macrophages.
Regulatory substrates of gelatinase B/MMP-9 in neuroinflammation P.E. Van den Steen, I. Nelissen, S. Starckx, P. Proost, J. Van Damme and G. Opdenakker Laboratory of Immunobiology, Rega Institute, University of Leuven, Leuven, Belgium
Gelatinase B/MMP-9 is upregulated in neuroinflammatory diseases and was shown to degrade myelin basic protein (MBP), thereby causing demyelination in, e.g., multiple sclerosis. In addition, gelatinase B participates in the degradation of the blood-brain barrier by cleaving the junctional protein ZO-1. Here, we show that gelatinase B cleaves several immunoregulatory molecules. In particular, gelatinase B cleaves CXC-chemokines. The neutrophil-attracting chemokine IL-8 and its counterpart in the mouse, GCP-2, are cleaved aminoterminally with an increased activity and a positive feedback loop as a consequence. ENA-78 is first potentiated by aminoterminal truncation, and subsequently degraded by additional cleavages. The activity of other neutrophil-attracting CXC-chemokines is either unaffected (human GCP-2) or downregulated (CTAP-III, GROalpha) . In contrast to the neutrophil-attracting chemokines, the CXCR-3 ligands MIG and IP-10, which attract activated T-cells, are cleaved in the carboxyterminal region by gelatinase B. These cleavages are expected to downregulate the activities. Another example of an immunoregulatory substrate for gelatinase B is interferon-beta (IFN-beta), an approved therpeutical agent in multiple sclerosis. IFN-beta is cleaved and inactivated by gelatinase B. In particular, the aglycosyl-form of IFN-beta is more sensitive to cleavage than the glycosylated variant, indicating a role of the sugars in protease resistance. Furthermore, gelatinase B degrades the heatshock protein alphaB-crystallin, resulting in the generation of immunogenic remnant epitopes. These findings have important implications in the understanding and treatment of neuroinflammatory diseases.
Phenotype of infiltrating cells in brains of lupus-prone mice X. Ma and B. Sakic
SLE is an autoimmune disorder that affects morphology and function of the CNS. We study lupus-prone MRL mice to elucidate behavioral dysfunction induced by lupus-like disease. Previous studies have shown that leukocyte infiltration into brains of MRL-lpr mice coincides with neurotoxic CSF. The intrathecal synthesis of IgG is proposed in destruction of periventricular neurons. We predict that antibody-producing cells are present when MRL mice brains become inflamed. Infiltrating leukocytes were characterized by mAbs to CD3, CD4, CD11b, CD19, CD69 and CD138 antigens. The CD3e-and CD4-positive cells were mostly located in the 3rd ventricle. Macrophages (CD11b), B-lymphocytes (CD19) and plasma cells (CD138) were less frequent but detectable in almost all infiltrates (CD11bN CD19NCD138 cells). In comparison to age-matched MRL-lpr mice, the brains of 20-week old MRL+/+ controls did not show cell infiltration. The predominance of CD4 lymphocytes and presence of CD11b cells suggests that T cells and macrophages play a role in cytokine-induced brain damage. The presence of B and plasma cells provides further support for the hypothesis that toxic IgG are synthesized intrathecally. Cytokine expression and cell phenotypes are presently being examined and will be reported at the meeting. This work was supported by funds from the NIH to B. Sakic, who is a recipient of the FSORC career development award.
Signaling delivered through cannabinoid receptor CB1 blocks the recruitment of encephalitogenic lymphocytes in brain venules through a PKA-dependent mechanism B. Rossi a , S. Bach a , M. Fusco b , F. Fachinetti b , A. Leon b and G. Constantin a a University of Verona, Verona, Italy; b Research and Innovation, Padova, Italy
It is known that cannabinoid treatment prevents the development of experimental autoimmune encephalomyelitis, but its mechanisms of action are not well elucidated. The GOAL of this study was to determine a potential role of cannabinoid receptors in the recruitment of lymphocytes in brain venules, a critical event in the initiation of autoimmune inflammation. Methods: PLP 139-151 autoreactive T lymphocytes were produced from SJL immunized mice. Intravital microscopy studies were performed in inflamed brain vessels. Expression of adhesion molecules and apoptosis was studied by flow cytometry. The release of intracellular calcium from stores was determined by using Fura-2AM. Results: SR141716, a selective cannabinoid CB1 receptor antagonist, and SR144528, a cannabinoid CB2 receptor antagonist were used. Both compounds had no effect on intracellular calcium release and did not alter the expression of adhesion molecules and apoptosis of encephalitogenic lymphocytes. Intravital microscopy studies performed in inflamed brain vessels showed that SR141716 has no effect on rolling, but consistently inhibited lymphocyte arrest in brain microcirculation. SR144528 had no significant effect on lymphocyte recruitment in vivo. H89, a PKA (protein kinase A) inhibitor, was able to prevent SR141716-induced inhibition of lymphocyte sticking in brain vessels. Conclusions: Our data help to further understand the antiinflammatory effects exerted by cannabinoids and suggest that signal transduction generated through CB1 interferes with the recruitment of lymphocytes in inflamed brain venules. CD26 is involved in T cell activation and in cell adhesion processes. Furthermore, CD26 exerts unique enzymatic specificity in cleaving dipeptides from chemokines (CXC3R ligands) and neuropeptides, such as neuropeptide Y and substance P. Although all of these functions suggest a functional role of CD26 in autoimmunity, there are conflicting results regarding possible clinical applicability as well as underlying mechanisms. We report here that two substrains of mutant Fischer 344 rats lacking endogenous DPIV enzymatic activity show reduced susceptibility to acute actively-induced EAE (MBP+ CFA+ pertussis). A significant delay of onset was observed in male rats, while in female mutant substrains also the severity of the disease was markedly suppressed. These suppressive like effects in CD26 negative rats were associated with a time dependent and subset-specific modulation of blood leukocyte numbers and spinal chord infiltrating leukocytes. A reduction of circulating B cells at day 7 postimmunization (pi) and the numbers of NKT cells at day 12 pi (peak of clinical score) in the blood but increased numbers of NK and NKT cells as well as reduced number of granulocytes and T cells (CD8+ and CD4+) in the spinal cord at day 12 pi. These results indicate protective-like effects of reduced CD26-like expression/enzymatic activity on EAE and emphasise cell migration/adhesion as a contributing mechanism in rats.
Growing evidence indicates that CD8 T cells may play a major role in the pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The mechanisms controling the recruitment of CD8 T cells in inflamed brain venules is not yet understood. Objective: The study proposes to compare the behavior of autoreactive CD4 versus CD8 T cells in inflamed brain venules. Methods: MOG35-55-specific CD8 and CD4 T cells were produced. Binding of E-and P-selectin chimeras were determined by flow cytomery. Intravital microscopy was performed in inflamed brain venules of C57Bl/6 mice. Results: MOG35-55 stimulated CD8 T cells display increased expression of PSGL-1, VLA-4 and LFA-1 when compared with MOG35-55 stimulated CD4 T. Moreover, CD8 T cells display increased binding to P-selectin-IgG chimera in vitro. In situ analyses revealed that autoreactive CD8 T cells have increased capability to tether and roll in inflamed brain venules when compared with MOG35-55specific CD4 T cells. Importantly, tethering and rolling of CD8 T cells was completely blocked by antibodies to PSGL-1 and P-selectin. Conclusions: Our findings show that inflamed brain endothelium expressing P-selectin preferentially recruit autoreactive CD8 T cells versus CD4 T cells and suggest that CD8 T cells may have a role in early inflammation during the autoimmune process.
Effects of an anti-VLA-4 antibody on chronic experimental allergic encephalomyelitis (EAE) in C57Bl/6 mice J. Rundle, V. Gauden, R. Mead and M. Christie Celltech R&D Ltd, Cambridge, UK
In Lewis rat and guinea pig EAE models treatment with antibodies or small molecule inhibitors of VLA-4 reduces clinical severity (Yednock et al., 1992; Kent et al., 1995; Piraino et al., 2002; Leone et al., 2003) . However, in the chronic relapsing EAE model in the SJL mouse, VLA-4 inhibition may either diminish or exacerbate disease depending on time of treatment (Theien et al. 2001 (Theien et al. , 2003 . The present study investigated the effect of an anti-VLA-4 antibody in a chronic EAE model in the C57Bl/6 mouse. EAE was induced in adult female C57Bl/6 mice by subcutaneous (s.c.) injections of complete Freund's adjuvant containing 0.4 mg/ml Mycobacterium and 0.2 mg of myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 in each flank (0.15 ml; days 0 and 7). Pertussis toxin was administered intraperitoneally (200 ng/mouse; days 0, 1, 7 and 8). Mice were treated with CRL19.11 (mouse chimeric anti-VLA-4 antibody; IgG1) or 101.4 (isotype control; 10 mg/kg s.c. twice weekly) either prophylactically (from day 0) or therapeutically (50% incidence). Prophylactic treatment with CRL19.11 caused a 40% and 62% reduction in maximum and cumulative clinical score, respectively ( pb0.01 Mann-Whitney U), with reduced incidence ( pb0.01 Fischer's exact test). Therapeutic treatment caused a 40% reduction in maximum clinical score ( pb0.01 Mann-Whitney U). In conclusion, the effects of CRL19.11 in this chronic murine EAE model suggest that VLA-4-dependent mechanisms drive both initiation and maintenance of the disease.
Expression of cutaneous lymphocyte antigen (CLA) on activated lymphocytes is critical for efficient tethering and rolling in inflamed brain venules B. Rossi a , L. Piccio b , L. Colantonio c , E. Scarpini b , D. D'Ambrosio c and G. Constantin a a University of Verona, Verona, Italy; b University of Milan, Milan, Italy; c Bioxell, Milan, Italy
We have previously shown that endothelial selectins are expressed by the inflamed brain endothelium during EAE and experimental models of subacute inflammation. CLA represents a fucosyltransferaseVII-induced carbohydrate modification of PSGL-1 (P-selectin glycoprotein ligand-1) responsible for the interaction of PSGL-1 with endothelial selectins.
Objective: The goal of this study was to determine the role of CLA epitope in lymphocyte recruitment into the brain. Methods: Th1 and Th2 lymphocytes CLA+alpha4beta7À or CLA-alpha4beta7+ were generated. Intravital microscopy was performed in subacutely inflamed brain microcirculation. Results: Human and murine Th1 cells efficiently tethered and rolled in brain venules, while in contrast, very few Th2 lymphocytes were able to roll and firmly adhere to inflamed brain endothelium. Anti-PSGL-1 and anti-CLA antibodies almost completely abolished the recruitment of Th1 lymphocytes into the brain. Importantly, we observed a dramatic increase of capture and rolling and subsequent arrest of Th2 lymphocytes expressing CLA when compared with Th2 cells CLAÀ. Moreover, when we compared the adhesive interactions between Th1 cells CLA+ and CLAÀ, we observed that expression of CLA determined a significant increase of tethering and rolling. Conclusion: Our data unveil that CLA epitope of PSGL-1 is critical for the recruitment in inflamed brain venules and may represent an attractive pharmaceutical target to be explored in autoimmune diseases of the brain. Infiltration of the CNS by CD4+ Th1 cells precedes onset and relapses of experimental autoimmune encephalomyelitis (EAE). We reported that (B6ÂSJL) F1 (H-2b/s) mice, with severe relapsing-remitting disease, had extensive infiltration by CD4+ T cells compared to C57BL/6 (B6) (H-2b) mice, which developed mild low-relapsing disease in response to myelin oligodendrocyte peptide 35-55 (MOG35-55). This observation led us to search for mechanisms that specifically regulate trafficking of CD4+ T cells in relapsing H-2b/s mice. We found that the CD4+ cell chemoattractant cytokine IL-16 has an important role in regulation of relapsing EAE. We found that production of IL-16 within the CNS of mice with EAE correlated well with the extent of CD4+ T cell and B cell infiltration during acute and relapsing disease. In the CNS, IL-16 was produced by infiltrating CD4+ T cells, CD8+ T cells and B cells. Treatment with neutralizing anti-IL-16 antibody successfully reversed paralysis and ameliorated relapsing disease. In treated mice, diminished infiltration by CD4+ T cells, lesser demyelination and more sparing of axons were observed. We show an important role of IL-16 in regulation of relapsing EAE and describe a novel therapeutic approach to specifically impede CD4+ T cell chemoattraction in EAE, by neutralization of IL-16. Our findings have high relevance for the development of new therapies for relapsing EAE and potentially MS.
The role of the heme-heme oxygenase system in multiple sclerosis pathology J. van Horssen a , F.A. Wagener b and H.E. de Vries a a VUMC, Amsterdam, The Netherlands; b UMC Nijmegen, Nijmegen, The Netherlands
Multiple sclerosis (MS) is characterized by infiltration of monocytederived macrophages. These activated monocytes secrete inflammatory mediators like reactive oxygen species (ROS) that contribute to axonal damage and demyelination. The inducible heme oxygenase (HO)-1 gene responds to changes in cellular redox potential provoked by ROS. We previously demonstrated an increase of HO-1 and -2 at mRNA level by quantitative PCR and microarray analysis in experimental autoimmune encephalomyelitis (EAE) rat brains, a validated model for MS. HO breaks down heme into free iron, carbon monoxide (CO) and biliverdin, which is converted by biliverdin reductase (BVR) into the antioxidant bilirubin. Iron is directly sequestered and inactivated by co-induced ferritin. Both heme-mediated ROS formation and inflammatory processes may contribute to tissue damage in EAE/MS brains. The aim of our study is to identify the cellular source of HO-1 and -2, BVR and ferritin in EAE rat brains as well as in human MS lesions. Our findings demonstrate that HO-1 and -2 as well as BVR and ferritin are markedly upregulated in infiltrating macrophages, while HO-2 and ferritin are also expressed by astrocytes in active lesions. We therefore hypothesize that the heme-heme oxygenase system may be involved in transendothelial migration of monocytes. In future, we will study the effects of heme, HO activity, CO and bilirubin on monocyte migration. Additional research is needed to further elucidate the role of the heme-HO system in the pathogenesis of MS. CCR6 expression defines the subset of CD25+ regulatory effectormemory T cells M. Kleinewietfeld a , F. Puentes a , G. Borsellino b , L. Battistini b , O. Rötzschke a and K. Falk a a Max-Delbrück-Center for Molecular Medicine (MDC), Berlin, Germany; b Institute of Neuroimmunology, IRCCS Santa Lucia, Rome, Italy
Regulatory CD25+ T cells are the central element of peripheral tolerance. Little is known, however, about phenotypic and functional characteristics of regulatory T cells with regard to memory. In this study, we show that the chemokine receptor CCR6 is expressed on a distinct subset of regulatory CD25+ CD4+ T cells. Similar to their CD25À counterparts, CCR6+ CD25+ T cells exhibit markers of activation, expansion and memory indicative for an effector-memory function. Although regulatory CD25+ T cells comprise only 15% of the CD4+ T cell subset, in naive mice CCR6+CD25+ T cells outnumber the respective population of CD25À T cells by a ratio of 2:1. They are true memory T cells, generated in vivo from naRve CD25+ T cells after the encounter of antigen. In contrast to activation markers, such as CD5 or CD44, induction of CCR6 is not immediate and evident only after several rounds of proliferation. CCR6+CD25+ T cells are enriched in the peripheral blood and accumulate in the CNS after induction of EAE where they counteract infiltrating CCR6+ effector-memory T cells. Thus, CCR6+CD25+ T cells seem to represent a population of bregulatory effector-memoryQ cells, destined to control destructive immune responses directly in inflamed tissues. Here, we report that pharmacologic depletion of fibrin reverses relapsing paralysis in an established polyphasic model of remitting-relapsing experimental autoimmune encephalomyelitis (EAE) induced by proteolipid protein (PLP139-151) in SJL/J mice. To investigate the therapeutic effects of fibrin depletion, we pharmacologically depleted mice from fibrin after the development of the first paralytic incidence. Untreated mice relapsed on days 35 and 50 after immunization. By contrast, fibrin-depleted mice recovered faster than untreated mice and never relapsed. Rotarod behavioral tests demonstrated that fibrin-depleted mice had increased motor skills when compared to the untreated group. Our current research on the cellular and molecular mechanisms of fibrin in EAE will be presented. Overall, these experiments identify fibrin as a potential target for therapeutic intervention in MS. Supported by the NMSS RG-3370 and the Wadsworth Foundation grants to KA. Our previous studies demonstrated that oligomeric recombinant TCR ligands (RTL) can treat clinical signs of experimental autoimmune encephalomyelitis (EAE) and induce long-term T cell tolerance against encephalitogenic peptides. We produced a monomeric I-AS/PLP-139-151 peptide construct (RTL401) suitable for use in SJL/J mice that develop relapsing disease after injection of PLP-139-151 peptide in CFA. RTL401 given i.v. or s.c., but not empty RTL400 or free PLP-139-151 peptide prevented relapses and significantly reduced clinical severity of EAE induced by PLP-139-151 peptide in SJL/J or (C57BL/6ÂSJL)F1 mice, but did not inhibit EAE induced by different encephalitogenic peptides. RTL treatment of EAE caused stable or enhanced T cell proliferation and secretion of IL-10 in the periphery, but reduced secretion of inflammatory factors. In the central nervous system (CNS), there was a modest reduction of inflammatory cells, reduced expression of VLA-4, LFA-1, and inflammatory factors, but enhanced expression of Th2-related factors, IL-10, TGF-beta3, and CCR3. These results suggest that monomeric RTL therapy induces a cytokine switch that curbs the encephalitogenic potential of PLP-139-151-specific T cells without fully preventing their entry into CNS, wherein they reduce the severity of inflammation. These results strongly support the clinical application of this novel class of peptide/MHC class II constructs in patients with multiple sclerosis who have focused T cell responses to known encephalitogenic myelin peptides. Multiple sclerosis is characterized by invasion of the central nervous system (CNS) by inflammatory cells. Migration of monocytes across the bloodbrain barrier is crucial for disease onset and severity. Activated monocytederived macrophages secrete inflammatory mediators such as oxygen radicals, which contribute to axonal demyelination and damage, resulting in neurological deficits. Flavonoids are naturally occurring food compounds, with anti-inflammatory properties that scavenge oxygen radicals, implying they might suppress clinical symptoms in multiple sclerosis. We investigated this issue by treating rats sensitized for experimental allergic encephalomyelitis (EAE), with flavonoids. We demonstrate that the flavonoid luteolin is more potent than quercetin to suppress clinical symptoms in EAE in the Lewis rat and prevented leukocyte infiltration into the CNS. The therapeutic potential of luteolin was assessed in a chronic relapsing-remitting EAE model in Dark-Agouti rats. We demonstrate that luteolin substantially suppresses clinical symptoms when administered either before or after disease onset. Luteolin treatment resulted in reduced inflammation and axonal damage in the CNS by preventing monocyte migration across the brain endothelium by modulating the activity of Rho GTPases, signal transducers involved in transendothelial migration. Oral administration of luteolin also significantly reduced clinical symptoms. Our results indicate the therapeutic potential for dietary adaptations in multiple sclerosis. FTY720 is a sphingosine-1 phosphate receptor agonist under evaluation as oral monotherapy in multiple sclerosis. It acts by redistributing lymphocytes from the blood and spleen to peripheral lymph nodes (LN). We evaluated FTY720 in experimental autoimmune encephalomyelitis (EAE), focusing on total and myelin basic protein (MBP)-specific serum immunoglobulin (Ig) as an indicator for a T helper type 1 or 2 (Th1, Th2)-driven immune response. Two-week oral therapy with 0.3 mg/kg FTY720 prevented EAE onset in LEW rats. Efficacy was accompanied by N70% decrease in circulating lymphocytes, N40% reduction of splenic T cells, and downregulation of transferrin receptor on LN B cells. Peak disease in the vehicle group 2 weeks post-immunization correlated with an increasing titer of MBP-specific IgG, which was lower in FTY720-and almost undetectable in cyclosporin A (CsA)-treated rats. Both compounds suppressed symptoms during treatment, yet their Ig profiles vastly differed. Compared to vehicle, total IgG levels were 80% lower with CsA and unexpectedly almost 40% higher with FTY720 treatment. In contrast to CsA, the IgG2b (Th1)/IgG1 (Th2) ratio was markedly reduced in FTY720treated rats, primarily due to upregulation of total but not MBP-specific IgG1. Protective effects of FTY720 may not only be based on LN retention of autoreactive effector cells but also quantitative changes of polyclonal antibody responses, possibly involving Th2 deviation. Similarly, combination of atorvastatin and GA at suboptimal doses was associated with enhanced lymphocyte secretion of IL-4, IL-5, IL-10, and TGF-beta, and decreased to secretion of IL-2 and IFN-gamma. Histopathological examination showed decreased infiltration of blood leukocytes into CNS tissue in animals treated with combination therapy. Conclusion: Combination therapy with atorvastatin and GA is effective in ameliorating clinical symptoms of EAE. Our results are extremely promising and provide a rationale for testing the combination of atorvastatin and GA in MS. Once intravenously (i.v.) or intrathecally (i.t.) injected in mice affected by experimental autoimmune encephalomyelitis (EAE), syngenic adult neural precursor cells (aNPCs) promote multifocal remyelination and functional recovery by selectively home within inflamed central nervous system (CNS) areas. Here, we explored the (immune) mechanisms sustaining this phenomenon. In C57Bl/6 and SJL mice, we found that selective aNPC homing capability was mainly mediated by the membrane expression of cell adhesion molecules (CAM) and chemokine. As such, undifferentiated aNPCs constitutively express VLA-4 (in a high affinity state), CD44 and CCR2, CCR5, CXCR3 (only in C57Bl/6 mice) and CXCR4. Chemokines, such as RANTES and stromal-derived factor (SDF)-1, modulated in a dosedependent manner aNPCs chemoattraction in vitro. Intravital microscopy showed in vivo aNPCs arresting in inflamed brain venules after injection into the carotid artery. Finally, in vivo blockage of VLA-4 significantly reduced CNS homing of aNPCs. Our results indicate that aNPCs home specifically to inflamed brain areas during EAE, once i.v. and i.t. injected, owing to the constitutive expression of the same cell adhesion molecules (CAM) and chemokine receptors used by enchephalitogenic lymphocytes to enter the CNS. Despite many claims for neuroprotection in experimental allergic encephalomyelitis (EAE), the data often indicate that the majority of neuroprotective effects occur secondary to inhibition of leukocyte CNS entry. The resultant reduction in clinical disease reduces the cascades that trigger neurodegeneration. Therefore, such agents act as immunomodulators rather than direct neuroprotectants. However, the failure to inhibit progressive multiple sclerosis (MS) despite inhibition of lesion formation and relapses has highlighted the importance of neurodegeneration in MS. Experimental autoimmune encephalomyelitis (EAE) is an instructive model for the human-demyelinating disease, multiple sclerosis. Lewis (LEW) rats immunized with myelin-basic protein (MBP) suffer from EAE, characterized by a single episode of paralysis from which they recover spontaneously and become refractory to reinduction of disease. LF 15-0195, is a novel molecule that inhibits the differentiation of autoantigen-specific CD4 T cells into effector pathogenic lymphocytes and has a potent immunosuppressive effect in several pathological manifestations including EAE. In the present study, we demonstrate that LEW rats protected from EAE by LF 15-0195 treatment benefit from a long lasting tolerance as reimmunization with MBP in CFA 4 to 7 weeks after treatment withdrawal fails to induce EAE. This long lasting tolerance is transferable by lymph node cells from protected rats into syngeneic recipients and is mediated by antigen-specific regulatory CD4 T cells that prevent active EAE. These regulatory T cells are also generated in thymectomized animals and their function is not dependent on the presence of TGF beta. Together, these data demonstrate that short-term treatment with LF 15-0195, not only prevents EAE in LEW rats, but also, permits the development of an antigen-specific tolerance mediated by regulatory CD4 T cells.
Effect of NGF and anti-NGF antibodies on bstem cellsQ and inflammatory response in the brain of EAE rats V. Triaca and L. Aloe National Research Council (CNR), Rome, Italy
Using experimental allergic encephalomyelitis (EAE) rats, we have investigated effects of nerve growth factor (NGF) and anti-nerve growth factor antibodies (anti-NGF) in the brain of these rats. NGF is a neurotrophin crucial for neuronal growth, differentiation and maintenance during development and in the adulthood after CNS injury. EAE has been shown to cause a marked circulating and brain synthesis of NGF and a higher responsiveness of brain cells to NGF. Here, we show that radiolabelled NGF, injected in the ventricle of control rats, was mainly localized in SVZ cells, which co-expressed the NGF-receptor TrkA 12 h after the treatment. After 48 h, radioactivity was virtually absent in the SVZ and almost exclusively detectable in some cells of the parenchyma adjacent the SVZ. These cells were also positive for markers of mature neurons, oligodendrocytes and astrocytes. Early neuronal markers, as neurofilament and nestin, were preminent expressed by SVZ cells of EAE brain following NGF injection. We investigated the effect of NGF and anti-NGF on SVZ cells proliferation and we found that EAE per se stimulates and NGF further enhances BrdU incorporation in the SVZ, while anti-NGF reduces the number of BrdU incorporating cells, as compared to EAE rats. Our results suggest that NGF has a protective action on EAE brain, most probably acting on both damaged brain cells and immune cells. To further explore the protecting role of adult neural precursor cells (aNPCs) in mice with experimental autoimmune encephalomyelitis (EAE), we have intravenously (i.v.)-injected syngenic aNPCs into mice with a relapsing-remitting form of the disease. SJL mice were immunized subcutaneously with proteolipid protein (PLP)139-151 and i.v. injected with 1Â10 6 millions of syngenic aNPCs both at the onset of the disease and after the first relapse. aNPCs entered both the brain and the spinal cord, selectively reached inflamed CNS areas, and showed to be able to survive for up to 120 days after transplantation. Three months after immunization, mice transplanted at disease onset or after the first relapse showed a significant ( pb0.05) decrease of the disease score, the cumulative score and of the number of relapses. A significant decrease of the extent of demyelination and axonal loss was also found. Interestingly, the majority of the transplanted aNPCs maintained over time an undifferentiated phenotype within the host CNS and accumulated mainly around brain venules where gliogenic and neurogenic factors (e.g. BMP-4)-possibly maintaining aNPCs in a quiescent state-were co-localized. Our data indicate that aNPCs-once i.v. transplanted-may survive within the CNS for long periods of time and protect from relapsing-remitting EAE owing to their capability to selectively reach perivenular inflamed area where stem cell surviving factors are expressed. Background: Although neural precursor cells remyelinate focal lesions, the mechanism of their beneficial effect in clinically relevant models of multiple sclerosis (MS) is unclear. We therefore examined the clinical, neuropathological and immunological effects of neural sphere transplantation in chronic EAE. Methods: Multipotential neural spheres from newborn GFPtransgenic mice were transplanted intraventricularly to MOG35-55-induced chronic EAE in C57Bl/6 mice. Sham-and sphere-transplanted animals were scored clinically and sacrificed at days 18 and 80 post-induction for histopathological analysis. Demyelination and axonal pathology were assessed by Kluver-Barrera staining, amyloid precursor protein immunohistochemistry and Bielschowski staining. Inflammation was quantified by counting perivascular infiltratesand by image analysis of ICAM-1 and LFA-1 immunohistochemistry. Brain CD25+,CD62L+ cells were identified by immunofluorescence. Lymphocyte proliferation was examined in vitro by 3H-thymidine incorporation. Cytokine production was examined by real time PCR and ELISA. Results: EAE-attracted transplanted cell migration into white matter tracts. Neural sphere transplantation attenuated the clinical course of chronic EAE and reduced demyelination and axonal pathology. Neural sphere transplantation inhibited the inflammatory brain process by all 3 parameters and increased brain regulatory CD25+ T-cells. Transplantation decreased the inflammatory infiltrate-associated axonal damage. Addition of neural spheres to EAE-derived lymphocytes in vitro inhibited their proliferative responses to ConA and to MOG peptide and reduced MOG-induced production of interferon-gamma. Conclusions: Neural sphere transplantation attenuates clinical and pathological features of chronic EAE. The graft effect is mediated by modulation of the autoimmune-inflammatory process.
Tolerance induction by bone marrow transplantation in a multiple sclerosis model M.M. Herrmann, S. Gaertner and R. Weissert Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany
Bone marrow transplantation (BMT) is a experimental treatment approach in multiple sclerosis (MS). MS is a chronic autoimmune disease with a T cell, macrophage and autoantibody dependent pathogenesis. Myelinoligodendrocyte-glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in rats reproduces these pathological features and is therefore used as a model for MS. There is only sparse knowledge on the impact of genetic factors, timing, tissue repopulation and immuneregulatory mechanisms by BMT in MS. We used MHC (RT1 in rats) identical EAE susceptible DA (RT1av1) and resistant ACI (RT1av1) rats and performed BMT studies before disease induction, at the peak of disease and in remission of EAE in DA rats with DA or ACI bone marrow after whole body irradiation with 10 Gy. In contrast to controls, BMT resulted in strong disease protection if applied after CNS lesions and clinical symptoms had evolved for subsequent autoantigen-induced relapses and CNS lesions. The protective effect was associated with a change in the relative size of the T and B cell compartment, a shift in the T cell recognition of different MOG epitopes, and a significant reduction in MOG-specific autoantibodies. The data indicate that the protective effect of BMT in a relevant animal model of MS is mainly due to tolerogenic effects on autoantigenspecific T and B cells with subsequent reduction of autoantibody titers. Astrocytes are able to induce regulatory T cells, which can completely abolish proliferative response of T cells in vitro. The aim of our study was to test this inhibitory capacity of astrocyte-induced regulatory T cells in vivo, using EAE model in DA rats. The rat spinal cord homogenateimmunized animals received on a day 7 p.i. lymph node cells (LNC) previously incubated with astrocytes (astro-LNC group) or in medium alone (control group). The severity of clinical signs was markedly reduced in astro-LNC group as judged by mean clinical score and cumulative disease index. Alleviation of EAE symptoms correlated with the results of pathohistological analysis. Astro-LNC were also able to interfere with the transfer of EAE. When MBP-immunized rats were treated on a day 6 p.i. with astro-LNC or control LNC and 2 days later, draining LNC were collected, restimulated in vitro and injected into naRve recipients, 9 out of 10 control animals developed EAE while none of six animals that received cells from astro-LNC group showed any sign of disease. Interestingly, no protective effect was observed when the astro-LNC were administered to naive animals simultaneously with encephalitogenic cells. Our results suggest important role of astrocyte-induced regulatory T cells in the regulation of CNS inflammation. Experimental autoimmune encephalomyelitis (EAE) is an animal model for the human demyelinating disease multiple sclerosis (MS) characterized by a CD4+ T cell mediated inflammatory response to central nervous system (CNS) autoantigens. Interaction between encephalitogenic T cells and APC in the CNS is a crucial step in disease initiation. Astrocytes have been suggested to play essential roles in initiating and maintaining CNS autoimmunity. Elevated IFN-gamma is observed in MS and EAE and is deleterious in MS-patients. We addressed the effect of astrocyte unresponsiveness to IFN-gamma in EAE by studying transgenic (tg) mice expressing a dominant-negative IFN-gamma receptor under control of the GFAP-promoter (GFAP-dIFNgR mice) in comparison to age matched wild type (wt) syngenic mice. No differences in peripheral immune responses assessed by T cell proliferation and cytokine secretion were seen between tg-and wt-mice. EAE was induced by immunization with MOG35-55 peptide/CFA and animals were scored daily for clinical symptoms. Tg-mice were as susceptible to EAE as wt-controls with the same average day of disease onset (day 13). However, GFAP-dIFNgR mice exhibited significantly more severe clinical symptoms (peak disease score 4 vs. 3, cumulative score 48 vs. 33) and higher mortality (39% vs 1%) than wtmice. Histopathological evaluation of spinal cord sections revealed slight increases in extent of cellular infiltration and severity of demyelination in tg-animals compared to wt-controls. Our findings support an important role of IFN-gamma mediated astrocyte signaling in ameliorating CNS autoimmune disease. Multiple sclerosis (MS) is a demyelinating disease of the CNS, where myelin and axons are lost in multiple foci called plaques. Sporadic repair occurs giving the appearance of dshadow plaquesT or areas of thin remyelination; however, this process is often incomplete. It is well established that inhibition of alpha 4 beta 1 integrin (VLA-4) blocks immune cell influx into the CNS and provides benefit to patients and in animal model systems. We have utilized this mechanism to examine whether the presence of inflammatory cells suppresses spontaneous myelin repair in experimental autoimmune encephalomyelitis (EAE). EAE was induced in Hartley guinea pigs, and animals were chosen for treatment based on two criteria: (1) animals were in the chronic stage of disease (Nday 40 post immunization); and (2) animals had hind limb paresis (clinical score of 2). Animals received either saline or CT301 for 10, 20, 30 or 40 days, and tissues were examined by light and electron microscopy for evidence of remyelination. We found (1) 87% of plaques showed evidence of remyelination after 40 days of treatment;
(2) myelin repair occurred in half of the total lesion area;
(3) half of the animals regained motor function. There was no significant repair or gain of motor function in vehicle treated animals. Therefore, prolonged inhibition of CNS inflammation, in the absence of targeted myelin repair, facilitated mechanisms of spontaneous remyelination. We established a cytokine-delivery system within the central nervous system (CNS) using helper-dependent adenoviral vectors (HD-Ad) engineered with the IL-4 gene and the green fluorescence protein (GFP) as reporter gene. HD-Ad vectors injected into the cisterna magna (i.c.) of mice diffused in all ventricular and subarachnoid spaces infecting ependymal and leptomeningeal cells surrounding these areas. When injected in mice affected by myelin-oligodendrocyte-glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis (EAE) the day of clinical onset, IL-4-treated mice displayed a progressive clinical recovery, and 80 days after immunization had an average EAE score of 1.0 (F1.0) vs. 1.62 (F0.4) in control mice ( pb0.05) treated with an empty vector. Motorevoked potentials (MEP) were recordable in only 50% of control mice, and in all but one of the IL-4-treated mice ( pb0.05) and central conduction time was significant decreased in IL-4-treated vs. control mice ( pb0.05).
Recovery was associated to a significantly increased number of CNSinfiltrating CD4+CD69ÀCD25+ regulatory T cells. We conclude that IL-4 gene therapy using HD-Ad vectors determines the recruitment of CD4+CD25+ T cells with suppressant function able to induce clinical and functional recovery in a murine model of multiple sclerosis.
Prevention of EAE by treatment with a neurotropic alphavirus vector expressing tissue inhibitor of metalloproteinase-2 P. Furu, S. Grfnberg, J. Heikkil7 and A. Hinkkanen Åbo Akademi University, Turku, Finland
Objective: In experimental autoimmune encephalomyelitis (EAE), enhanced activity of matrix metalloproteinases (MMPs), has been implicated in disease pathogenesis. Prompted by our recent observations of increased MMP-8 and MMP-9 with simultaneous downregulation of TIMP-2 and TIMP-3 mRNA in the CNS of mice with severe EAE, we used Semliki Forest virus (SFV) to transfer and express recombinant murine TIMP-1-3 genes in the CNS of EAE mice. Results: TIMP-1, TIMP-2 and TIMP-3 expression was confirmed in cultured cells and in the CNS of infected animals. Following intraperitoneal infection with 10 6 pfu of SFV-TIMP focal TIMP protein expression was achieved throughout the brain. Although already treatment with empty vector inhibited development of EAE to some extent, the expression of TIMP-2 by the virus significantly enhanced the inhibition. TIMP-3 administered mice also had lower disease grade but the inhibition was not statistically significant. In contrast, SFV-TIMP-1 had no effect, similar to coinfection with TIMP-2 and TIMP-3. We found TIMP-2 expression also by non-infected CNS resident cells surrounding the SFV-TIMP-2 virus positive areas suggesting a bystander TIMP-2 induction.
Conclusions: These data strengthen the view that MMPs are involved in the pathogenesis of EAE and provide clear evidence that virus-mediated delivery of their protein inhibitors can be effective in preventing the clinical disease. TIMPs might be candidates for novel treatment regimens in CNS autoimmune disorders such as multiple sclerosis. Background: Immunoglobulin for intravenous administration (IVIG) possesses multiple immunomodulatory properties and represents a way of interfering with the pathological processes in multiple sclerosis (MS). In experimental autoimmune encephalomyelitis (EAE), infusions of IVIG have been shown to significantly ameliorate the disease. However, it is at present not known whether IVIG enters the CNS in sufficient amounts to influence the local immune response, or if the treatment effects are due to peripheral actions of IVIG. Objective: The purpose of the present study was to evaluate if IVIG enters the CNS by passing through the blood-brain barrier during the acute course of EAE, and to assess if IVIG accumulates at the inflammatory foci. Methods: EAE was induced in susceptible rats, and the animals were treated with IVIG infusions during the acute attack of the disease. The immunoglobulin was radiolabelled with 99mTc-pertechnetate. After in vivo administration, the 99mTc-IVIG was localised and visualised in CNS tissue sections by means of autoradiography. Studies evaluating the 99mTc-IVIG biodistribution and plasma half-life were also performed. Results: We observed a significant increased binding of IVIG to CNS tissue in rats with EAE that correlated with the degree of inflammation as assessed by histological evaluation. Furthermore, by autoradiography the radiolabelled IVIG was localised to the regions of the brain where inflammatory infiltrates and areas of demyelination were also found.
Lack of apolipoprotein E (ApoE) exacerbates experimental autoimmune encephalomyelitis M. Nakanishi a , J.-I. Satoh b , T. Aranami b , G. Hirose a and T. Yamamura b a Kanazawa-Medical University, Ishikawa, Japan; b NCNP, Tokyo, Japan
Recent studies have shown that MS patients having the 4 allele exhibit rapid progression and severe disability than those with non-4 alleles. We studied an immunoregulatory role of ApoE in the development of EAE, an immune-mediated demyelinating disease by using ApoE-knockout (KO) mice as a model system. EAE was induced in 6-to 9-week-old female ApoE-KO mice and wild type (WT) B6 mice by immunizing with myelin oligodendrocyte glycoprotein (MOG)35-55. The clinical score, antigenspecific proliferation response and cytokine production of regional lymph node (LN) cells, anti-MOG35-55 IgG antibody titers, and histopathology of the spinal cord were studied. Cannabinoids may alleviate symptoms of multiple sclerosis. Furthermore, cannabinoids might also modulate neuroinflammation. We saw that delta9tetrahidrocannabinol (D-THC) was ineffective in the passive variety of experimental autoimmune encephalomyelitis (EAE) of Lewis rats; however, R(+)-WIN 55,212-2, a synthetic cannabinoid, significantly syppressed clinical signs. As cannabinoids may exert either pro-or antiapoptotic effects, depending on cell types, we investigated possible changes in T cell apoptosis, related to the protective of this cannabinoid. By means of flow cytometry, we found that R(+)-WIN 55,212-2 induced a profound increase of apoptosis in an encephalitogenic T cell line, time and dose dependent. To ascertain type of cannabinoid receptor involved, the T cell line was cultured with CB1 and CB2 receptor antagonists (SR141716A and SR144528 respectively), and S(À)-WIN 55,212-2 (the inactive isomer). Whereas SR141716A had no effects, SR144528 and S(À)-WIN 55,212-2 prevented the induction of apoptosis in a dose-dependent way, indicating the involvement of CB2 but not CB1 receptors. However, the concentration of R(+)-WIN 55,212-2 required and the moderate effect of S(À)-WIN 55,212-2, strongly suggest that the observed effects might be in part cannabinoid receptor independent. As R(+)-WIN 55,212-2 has more affinity for CB2 receptors than D-THC, these findings may help explain the observed discrepancies in protection against EAE.
Immunoregulation of a model of multiple sclerosis using the synthetic cannabinoid R(+)WIN55,212 J.L. Croxford and T. Yamamura National Institute of Neuroscience, Tokyo, Japan
Multiple sclerosis (MS) is a human autoimmune demyelinating disease of the central nervous system, whereby the immune system attacks myelin. Symptoms include spasticity, tremor, bladder dysfunction and pain. Current therapies, beta-interferon and copaxone decrease the MRI-lesion load and relapse rates. However, disease progression is largely unaffected. Therefore, novel therapeutic agents are required for MS treatment. Anecdotal evidence and clinical surveys suggest that many MS patients derive subjective improvements in spasticity, chronic pain and spasms from smoking marijuana. Cannabinoid agonists mediate their effects through cannabinoid receptors (CB). CB1 is mainly expressed on CNS neurons and is involved in the regulation of neurotransmission. In contrast, CB2 is expressed only peripherally, and is thought to have immunomodulatory functions. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS mediated by myelin-specific CD4+ T cells. Therefore we investigated the use of a synthetic cannabinoid agonist, R(+)WIN55,212 with which to treat ongoing EAE disease. We demonstrate that treatment with R(+)WIN55,212 at the onset of clinical symptoms can significantly inhibit disease severity and progression. This was associated with the inhibition of myelin specific T cell proliferation, differentiation to Th1 phenotype and autoimmune humoral responses. Furthermore, this therapeutic effect was absent in IFN-gamma-deficient mice. In conclusion, cannabinoids are potent therapeutic agents which may be beneficial in MS as a symptomatic treatment for spasticity and as immunosuppressive agents. Rationale: Phospholipase A2 (PLA2) hydrolyzes phosphatidylcholine to lysophosphatidylcholine and arachidonic acid (AA), which can cause myelin breakdown and induce inflammation, respectively. There are two major forms of PLA2: cytosolic (cPLA2) and secreted (sPLA2). cPLA2 is thought to be the major form contributing to AA release. We therefore studied the role of cPLA2 in the pathogenesis of EAE, an animal model of multiple sclerosis. Methods: EAE was generated in C57BL/6 mice, which have a naturally occurring null mutation for a major form of sPLA2, and SJL/J mice, which contain all forms of PLA2. We blocked cPLA2 using arachidonyl trifluromethyl ketone (AACOCF3), a selective inhibitor of cPLA2. To assess the role of cPLA2 in the onset and progression of disease, we inhibited this enzyme at the time of EAE induction or after the first paralytic attack. Results: C57BL/6 mice and SJL/J mice treated with a cPLA2 inhibitor at the time of EAE induction showed a remarkable decrease in the incidence of the disease. Delaying treatments after the peak of the first paralytic episode in C57BL/6 mice also resulted in significant reduction in inflammatory burden and recovery from the relapsingremitting from of the disease. Conclusions: These findings suggest that inhibiting cPLA2 may play an important role in the onset and progression of EAE. Estrogen treatment has been shown to exert a protective effect on experimental autoimmune encephalomyelitis (EAE), and is under clinical trial for multiple sclerosis (MS). Whilst it is commonly assumed that estrogens exert their effect by modulating immune functions, we show here that 17h-estradiol (E2) treatment can inhibit mouse EAE without affecting autoantigen-specific T cell responsiveness and type-1 cytokine production. Using mutant mice in which estrogen receptor alpha (ERalpha) has been unambiguously inactivated, we found that ERalpha was responsible for the E2-mediated inhibition of EAE. We next generated irradiation bone marrow chimeras in which ERalpha expression was selectively impaired in inflammatory T lymphocytes or was limited to the radiosensitive hematopoietic compartment. Our data show that the protective effect of E2 on clinical EAE and CNS-inflammation was not dependent on ERalpha-signaling in inflammatory T cells. Likewise, EAE development was not prevented by E2-treatment in chimeric mice that selectively expressed ERalpha in the systemic immune compartment. In conclusion, our data demonstrate that the beneficial effect of E2 on this autoimmune disease does not involve ERalpha-signaling in blood-derived inflammatory cells, and indicate that ERalpha expressed in other tissues, such as CNS-resident microglia or endothelial cells, mediates this effect. N-acetylcysteine (NAC) is a well-known anti-oxidant that acts as a scavenger for reactive oxygen species and as a precursor for reduced glutathione (GSH). It has recently been reported that NAC exerts also an immunomodulatory activity, such as induction of Th2 polarization of T cell response and inhibition of B cell and dendritic cell function by downregulation of costimulatory molecules. Therefore, we tested whether NAC was capable of preventing MOG35-55-induced experimental autoimmune encephalomyelitis (EAE), an established model of multiple sclerosis. NAC was administrated orally at a daily dosage of 10 mg/ml in drinking water starting at the day of immunization and it significantly prevented EAE progression as compared to control mice. This was associated with a reduction of CNS infiltrating T cells and macrophages as well as decreased demyelination. We then tested NAC in a therapeutic protocol and disease progression was again inhibited when compared to vehicle-treated controls. We found that NAC inhibited the stimulation of encephalitogenic T cells only by MOG35-55 but not by anti-CD3 plus anti-CD28 mAbs. This was probably due to an immmunosuppressive effect of NAC at the APC level. We also show that NAC inhibits T cell migration towards CXCL10 and CCL5, chemokines involved in T cells homing to the CNS. Our data indicate that NAC can effectively interfere with the autoimmune reaction associated with EAE. Attempts to exploit the powerful immunoregulatory properties of transforming growth factor-beta (TGF-beta) for autoimmune disease therapy have been compromised by its stimulatory effects on fibrogenesis and extracellular matrix generation. TGF-beta-functions are locally regulated through activation of latent TGF-beta and subsequent intracellular signalling via the Smad proteins. Smad7 blocks TGF-beta-signalling and is upregulated by TGF-beta and proinflammatory cytokines. We hypothesized that Smad7 inhibition should enhance TGF-beta-signalling at sites of local inflammation such as the central nervous system (CNS). Administration of Smad7-antisense-oligonucleotides (Smad7-as) in vivo ameliorated clinical signs of active and adoptively transferred experimental autoimmune encephalomyelitis and diminished CNS inflammation. Smad7-as-treatment did not result in increased extracellular matrix formation. Autoreactive T cells exposed to Smad7-as during antigenic priming or secondary activation showed considerably reduced encephalitogenicity. In conclusion, Smad7 inhibition represents a novel systemic treatment strategy targeting autoimmune inflammation, augmenting TGF-beta-signalling at sites of TGFbeta-activation without TGF-beta-associated toxicity. T cell activation is a critical step in the development of an autoimmune attack against the brain. Methylation are emerging as a novel posttransductional mechanism involved in the regulation of cell signalling. We have found previously that 5'-methylthioadenosine (MTA), an inhibitor of methylation reactions, prevented macrophage activation in a murine sepsis model. Methods: Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats immunized with myelin basic protein (MBP). Animals were treated with MTA (100 microM) intraperitoneal (i.p.) daily or placebo after immunization. Clinical and histological scores were compared between groups. Results: We found that animals treated with MTA have a milder disease (mean clinical score MTA group: 0.12, placebo group: 1.09. p=0.002) and a delayed onset of the disease. In addition MTA-treated animals have less inflammatory infiltrates and demyelinating areas compared to placebo animals. Conclusion: MTA, a potent inhibitor of methylation reactions, prevents the development of a full autoimmune response in the EAE model. The therapeutic efficacy of M2000 as a novel immunosuppressive and anti-inflammatory agent in T cell-mediated autoimmune disease was tested. The influence of M2000 on myelin basic protein (MBP)-induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis was assessed. The onset and symptoms of EAE in Lewis rats could be suppressed by intraperitoneally administration of M-2000 during the induction phase of disease. Disease suppression was associated with a marked suppression of MBP-specific T cell reactivity in vitro, without any evidence for a generalized impairment of T cell activity. Collectively, our data suggest that M2000 may provide a novel therapeutic option for T cell-mediated autoimmune diseases in animal models and possibly in man. The aim of the present study was to evaluate the effect of murine interferonbeta (mIFN-beta) or dexamethasone (DEX) on myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in female C57BL/6 mice, a murine model for the human disease multiple sclerosis (MS). EAE was induced by a subcutaneous injection of MOG(35-55)-peptide in incomplete Freund's Adjuvant mixed with Mycobacterium tuberculosis, followed by intraperitoneal injections of pertussis toxin. The mice were treated with subcutaneous injections of mIFN-beta in doses of 5000 or 15,000 units/mouse or DEX in doses of 1 or 10 mg/kg body weight. The treatment started at day 7 after EAE-induction with administration once every second day until day 23, and the course of disease was followed up to day 31. The results demonstrate a significant reduction of the individual average and maximum clinical score in mice treated with 1 or 10 mg/kg of DEX. Treatment with DEX also effectively reduced the inflammation and demyelination in mice with EAE. However, the only effect of mIFN-beta was a significant reduction of the individual maximum clinical score in mice that received 5000 units of mIFN-beta.
Effects of EGIS-8332, an AMPA antagonist, in transient focal cerebral ischemia and multiple sclerosis in rats K. Mó ricz, G. Gigler, M. Albert, É . Matucz, M. Á goston, A. Simó, A.
Benedek, J. Barkó czy, L.G. Hársing, G. Lévay and G. Szénási EGIS Pharmaceuticals Ltd., Budapest, Hungary
A role for AMPA receptors in the pathogenesis of neurodegenerative disorders is well established. EGIS-8332 is a negative allosteric modulator of AMPA receptors with good anticonvulsant and neuroprotective effects in animal models of global and permanent focal cerebral ischemia. In the present study we compared the neuroprotective effects of EGIS-8332 and GYKI 53405, a structurally analogue compound, in transient focal cerebral ischemia and experimental autoimmune encephalomyelitis (EAE) tests. The middle cerebral artery was occluded intraluminally for 60 min using a monofilament, and the effects of the two compounds administered 60 min after occlusion at 20 mg/kg i.p. were evaluated after a 24-h reperfusion. EGIS-8332 and GYKI 53405 reduced both the core (56%, 50%, respectively) and total brain infarctions (33%, 28%, respectively). Male Lewis rats were treated with guinea pig myelin basic protein for the induction of EAE, and both compounds were administered at 3 mg/kg i.p. twice daily for 7 days starting on day 10 after immunization. EGIS-8332 reduced cumulative neurological score and partially reversed histopathological damage while the effect of GYKI 53405 was not significant. The favourable neuroprotective effect of EGIS-8332 indicates good perspectives for further development for the treatment of stroke and multiple sclerosis. Object of study: The study was undertaken to enlighten influence of major effectors of stress response-endocrine catecholamines (CA) and neuronally released noradrenaline on development of EAE. Method: Adult DA rats were treated with either saline or propranolol chloride (0.4 mg/100 g/ day, PT) s.c. and on day 4 after the beginning of treatment were immunized by guinea pig spinal cord homogenate in complete Freund's adjuvant with B. pertussis vaccine. When the clinical sings of disease were at maximum the expression of CD4/CD8/TCRalpha/beta on thymocytes, thymocyte proliferative and apoptotic indexes and expression of CD4/25 and CD8/25 on peripheral blood lymphocytes (PBL) were analyzed by FCA. Results: PT decreased morbidity and mortality rate of disease. In propranolol-treated rats the maximal sign of disease, as well as its duration, were reduced. PT did not affect the thymocyte yield, but altered thymocyte phenotypic profile. The percentages of CD4-8-TCRalpha/betaÀ cells, that of CD4+8+TCRalpha/betaÀ cells, and the relative proportion of all subsets of thymocytes expressing TCRalpha/beta+ (except CD4-8À) were decreased, while the percentage of CD4+8+TCRalpha/betaF cells was increased. The relative number of CD8+ PBL was decreased; while that of CD4+ cells remained unaffected (although the percentage of CD4+25+ PBL was reduced) in propranolol-treated rats. Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system in which it is widely accepted that autoimmunity plays a significant role. The animal model equivalent of MS, experimental autoimmune encephalomyelitis (EAE), implicates an autoimmune T cell response as the initiating factor. There is now substantial evidence that other components of the immune system are recruited in MS and in EAE to cause myelin damage and neuronal/axonal injury. The complement (C) system has been implicated as a myelinolytic agent in both man and experimental animals. The fluid-phase fragment C5a is chemotactic for phagocytic cells and activating for many other cell types. We here describe a study of the effects of C5aR blockade using AcF- [OPdChaWR] in the well-documented Lewis rat models of EAE and an antibody-mediated demyelinating form of EAE (ADEAE). Disease was monitored clinically and by using multiple pathological parameters. AcF-[OPdChaWR] treatment had no effect on the clinical course or pathological outcome in either the primarily inflammatory EAE model or the profoundly demyelinating ADEAE model. IL-12 is a heterodimeric cytokine which plays an important role in driving the differentiation of T helper type 1 (Th1) cells. Such cells have been considered a key factor in the initiation of autoimmune inflammatory demyelination in experimental autoimmune encephalomyelitis (EAE). However, recent evidence showed that IL-12 is dispensable in the pathogenesis of, and that severe EAE can develop in the absence of IL-12 or IL-12 responsiveness. These data raised the possibility of an immunomodulatory action of IL-12 in this disease model. The object of this study was to test the ability of exogenous IL-12 to modulate chronic EAE induced by MOG35-55 in the C57BL/6 mouse. We used T-cell proliferation assays, ELISA, flow cytometry, real-time PCR, and histopathology to characterize the autoimmune response in IL-12-treated and control mice. We found that IL-12 suppressed EAE clinically and pathologically when administered during the early induction phase of the disease. The suppression of EAE was accompanied by induction of IFNgamma and TNF-alpha and inhibition of MOG-induced T cell proliferation in IL-12 treated mice. IFN-gamma-deficient mice were resistant to disease suppression. IL-12 also inhibited mRNA expression of the proinflammatory cytokine IL-17. We conclude that exogenous IL-12 inhibits EAE by a premature induction of autoreactive effector Th1 cells and that increased IFN-gamma has an inhibitory effect on such cells. In addition, anti-inflammatory effects may be mediated by inhibition of IL-17.
Disruption of the TGF-beta-signaling pathway in the macrophage compartment and its impact on immune-mediated diseases U. Malipiero a , F. Ackermann a , S. Karlsson b , K. Buerki a , U. Koedel c and A. Fontana a a University Hospital Zurich, Zurich, Switzerland; b Lund University, Lund, Sweden; c Ludwig-Maximillians-University, Munich, Germany
The TGF-beta family of proteins are secreted molecules with potent immunoregulatory properties and they control many activities of macrophages such as expression of MHCII, chemotaxis, production of cytokines and their receptors, production of reactive oxygen species and nitric oxide. Activation and infiltration of macrophages is seen in various disease models including experimental allergic encephalomyelitis (EAE) and bacterial meningitis. The latter are blocked by TGF-beta. The current study investigates the role of TGF-beta on the macrophage compartment in inflammatory and autoimmune disease models. In order to interrupt the TGF-beta-signaling pathway in macrophages, we delete the TRII by breeding floxed TRII mice with mice expressing Cre recombinase under the macrophage-specific mouse lysozymeM promoter. Cultured bone marrow cells from mice devoid of TRII in macrophage/monocyte lineage cells show a deletion of exon 4 in the TRII gene. The LPS-induced production of TNFalpha is not inhibited by TGF-beta in Cre-expressing macrophages. Macrophages of wildtype animals show a 50-80% reduction of TNF-alpha secretion in the presence of TGF-beta. The results show disruption of the TGF-beta-signaling pathway in macrophages. TGF-beta macrophage receptor knockout mice are currently tested in EAE and bacterial meningitis to analyze disease progression with disturbed TGF-beta-signaling in the macrophage compartment. 2002; Bechtold et al, Ann Neurol, 2004, in press ) have led us to question whether such agents might affect T cell activation. In vitro, rat lymph node cells stimulated with phytohemagglutinin in the presence of flecainide, lamotrigine and phenytoin (5 Ag/ml) exhibited a 20-30% reduction in the proportion of ICAM+ CD4+ and CD8+ T cells. In the same culture conditions lamotrigine also caused a 35% decrease in cell proliferation: this decrease was not observed with flecainide or phenytoin. The effects of flecainide on T cell activation were also examined following its subcutaneous injection in animals with EAE induced by immunisation with spinal cord homogenate. Administration of flecainide (30 mg/kg/day) between 7 and 12 days post-immunisation did not alter the percentage of T cells positive for CD44 or CD62L, but it did result in a 20% reduction in the proportion of ICAM+ CD4+ and CD8+ T cells. In summary, the results show that sodium channel blocking agents can affect T cell function in vitro and in vivo.
Myelin-specific T cell receptor transgenic T cells Vaccination with DNA encoding myelin oligodendrocyte glycoprotein (MOG)-peptide 91-108, pMOG91-108, suppresses experimental autoimmune encephalomyelitis (EAE) induced with the same peptide. EAE is an animal model for multiple sclerosis (MS) and can be actively induced by immunization with myelin proteins or peptides. The CpG DNA content of the plasmid DNA backbone is necessary for efficient DNA vaccination to EAE. Recently, we demonstrated that the suppressive mechanism involves induction of interferon-beta. These data are of great interest, since interferon-beta is used as a MS-therapy in humans. Here, we study the transcription of over 6000 genes using cDNA microarrays in spleens from DNA vaccinated LEW.1AV1 rats relative to control rats during peak of disease. Plasmacytoid dendritic cell (pDC)-associated genes, as defined in mice and human pDCs, were differentially expressed in DNA-vaccinated rats compared to controls. In contrast to human systemic lupus erythematosus (SLE)-where pDCs have been suggested to be involved in the pathogenesis-our data may indicate that pDCs can mediate protection from EAE, possibly via endogenous production of interferon-beta. Th1-cell activation is crucial in EAE and MS development. TIRC7 is a membrane molecule expressed early in lymphocyte activation and anti-TIRC7 therapy inhibits Th1-cell antigen-responsiveness. Here, we studied the role of TIRC7 and therapeutic potential of TIRC7 mAb in EAE and MS. We used mAb to characterize cell-type specific TIRC7-expression in MS lesions. mAb effects on EAE in mice immunized with peptide (PLP139-151) were studied by comparing clinical signs, brain immunohistochemistry, lymphocyte proliferation, and cytokine levels in mAb-treated and untreated animals. TIRC7-positive T-cells were the dominant cell-type in both acute and chronic MS lesions. At 28 days, 5 mAb-pretreated EAE mice had a lower mean clinical score of 0.6F0.6 (S.E.M.) compared with 12 EAE controls (1.75F0.58; p=0.19) suggesting mAb inhibition of EAE development. In addition, mAb halted the clinical progression of established EAE: at 14 days, mAb-treated mice scores were 0.5F0.5 (n=4) compared with 1.7F0.73 ( p=0.08) for controls (n=5). Clinical improvements were typically accompanied by reduced mononuclear cell infiltration in EAE lesions in mAb-treated compared with untreated mice. mAb effects were immunologically-selective. Treated mice had lower splenocyte IFN-gamma and IL-6 expression and reduced T-cell proliferation compared with controls. These pilot data suggest that TIRC7 is involved in the pathogenesis of EAE, and possibly of MS, and that TIRC7 mAb is a potential MS therapy with selective immunomodulatory effects. Phenotype and kinetics of CSF B cells in inflammatory CNS diseases S. Cepok, F. Vogel, V. Grummel, S. Hofmann and B. Hemmer Clinical Neuroimmunology Group, Department of Neurology, Philipps University, Marburg, Germany B cells are frequently found in the cerebrospinal fluid (CSF) of patients with neuroinflammatory diseases like multiple sclerosis (MS) and various infectious CNS diseases. The objective of the present study was to characterize the phenotype and kinetics of B cell subpopulations in the CSF and blood of patients with MS and infectious diseases in order to dissect differences between chronic and acute inflammatory CNS diseases. For this purpose, we performed serial analyses of CSF and blood cells by flow-cytometry in patients with MS and acute infectious diseases such as lyme meningoradiculitis or viral meningitis. Plasma cells and B cells were detected in most patients with MS or acute infectious diseases in the CNS. In contrast to peripheral blood, most of the B cells in the CSF showed a memory phenotype with expression of CD27. CSF B cells also downregulated several B cell marker, like CD21 and CD22. Plasma cells upregulated LFA-1 compared to peripheral plasmacells. Interestingly, as determined by serial analysis a differential kinetic pattern in MS patients was observed compared to acute infectious diseases. In MS patients, more than 30% of CD19+ cells were plasma cells, which remained stable over years accompanied by a stable IgG synthesis. In contrast, in infectious diseases the plasmacells dropped immediately within weeks after the pathogen was removed. The data demonstrate that in MS a permanent and stable activation of the humoral immune system occurs. Multiple sclerosis (MS) is characterized by increased intrathecal synthesis of immunoglobulins of unknown specificity and by the presence of B-cell clonal expansions in the central nervous system (CNS). Because ectopic lymphoid tissue developing at sites of chronic inflammation is thought to sustain local immune processes, we have investigated whether abnormal B-cell follicles could be identified in the CNS of MS patients. Sections from post-mortem MS brains and spinal cords were examined by immunohistochemistry for the presence of CD20+ B cells, CD138+ plasma cells, CD3+ T cells and CD21+, CD35+ follicular dendritic cells, and for expression of the lymphoid chemokines CXCL13 and CCL21. Lymphoid follicle-like structures containing B cells, T cells, plasma cells and follicular dendritic cells producing CXCL13 were observed in the cerebral meninges of two out of three patients with secondary progressive MS, but not in relapsing-remitting and primary progressive MS. Proliferating B cells were detected in intrameningeal follicles, a finding which is suggestive of germinal center formation. Despite accumulation of numerous B cells and plasma cells around blood vessels, no folliclelike structures were identified in white matter lesions. The formation of ectopic lymphoid follicles in the meninges of patients with MS could play a role in maintaining humoral (auto)immunity and in disease progression.
A full maturation process of B cells into the CSF suggests the existence of an intrathecal lymphoid-like compartment in subjects with MS and other inflammatory disorders of the CNS A. Uccelli a , S. Casazza a , E. Ferretti b , D. Giunti a , E. Zappia a , A. Pistorio b , C. Gambini c , G.L. Mancardi a and V. Pistoia c a Neuroimmunology Unit, Department of Neurosciences, Ophthalmology and Genetics, Genoa, Italy; b Laboratory of Oncology, G. Gaslini Institute, Genoa, Italy; c Laboratory of Pathology, G. Gaslini Institute, Genoa, Italy
Clonally expanded populations of hypermutated B cells have been detected in the CNS of MS subjects suggesting that a process of B cell affinity maturation may occur inside the CNS. We have characterized by flow cytometry the B cell subsets identified within the CSF of MS patients and of individuals with other inflammatory neurological disorders (OIND). CD19+CD38+CD77+, Ki67+, Bcl-2À centroblasts, a subset exclusively represented within lymphoid organs, were detected in the CSF, but not in peripheral blood (PB), from both patient groups. CD27+IgDÀ memory B cells, which represent hypermutated B cells, were significantly increased in the CSF vs PB from both groups. Memory B cells expressed high level of CD80 and CD86 costimulatory molecules and up-regulated CCR1, CCR2 and CCR4 suggesting their activated state. Lymphotoxin-alpha, CXCL12 and CXCL13, key players in lymphoid neogenesis, were present in the CSF from MS and OIND patients and expressed in MS brain tissue providing a favourable microenvironment for B cell maturation. This study suggests that a compartimentalized B cell response occurs within the CNS during an ongoing inflammatory reaction, evolving through all stages of B cell differentiation observed in secondary lymphoid organs.
BAFF production in human brain by astrocytes: the The brain in multiple sclerosis (MS) provides a long-term survival niche for B cells and plasma cells, which give rise to persisting oligoclonal Ig. B cell activating factor of the TNF family (BAFF) plays a crucial role in B cell survival. We report that BAFF is expressed in the normal human brain and is produced by astrocytes in vitro. BAFF transcripts found in gray and white matter amounted to about 9% of that in lymphatic tissue (tonsils and adenoids). The splice variant delta-BAFF was expressed at a lower level than the full-length variant in both brain and lymphatic tissue. BAFF was a regular component of the normal spinal fluid, and was demonstrated in brain astrocytes by double-fluorescence microscopy. Cultured human astrocytes secreted high amounts of BAFF after stimulation with IFNgamma and TNF-alpha, which was mediated by a furin-like protease. The quantity of BAFF secreted per astrocyte was manifold higher than that of monocytes or macrophages. In MS lesions, the expression of BAFF was elevated as seen by quantitative PCR; BAFF was localized on astrocytes, and the BAFF receptor BAFF-R was found on infiltrating immune cells. This study establishes, we believe for the first time that a non-immune cell type can produce substantial amounts of BAFF. BAFF production in the CNS might contribute to the long term survival of B cells in MS brains.
A panel of novel candidate antigens in multiple sclerosis revealed by phage cDNA display V. Somers, C. Govarts, J. Raus and P. Stinissen Biomedisch Onderzoeksinstituut (BIOMED), Limburgs Universitair Centrum, University Limburg, Diepenbeek, Belgium
Cerebrospinal fluid (CSF) of patients with clinically definite multiple sclerosis (MS) is characterized by the presence of oligoclonal antibodies, which is used as a diagnostic test. However, despite intensive research, the antibody specificities remain unknown. To identify immunogenic targets recognized by the humoral immune response in MS, we focus on a novel method called bserological antigen selection (SAS)Q, which is based on phage display. First, we constructed a cDNA display library by cloning a normalized cDNA library prepared from active chronic MS plaques, with varying degrees of demyelination and inflammatory activity (Soares et al, 1994) for expression as a fusion protein with a filamentous phage minor coat protein, pVI. This cDNA display library was then enriched on pooled CSF (n=10) from relapsing-remitting MS patients. We identified a panel of nine different clones reactive with the CSF pool. A detailed serological analysis of the nine different antigens on a large panel of individual patient and control CSF showed exclusive reactivity to MS patient CSF for eight different antigens. Sequence analysis revealed that these clones have never been associated with MS. In conclusion, our findings demonstrate that this novel molecular approach is useful to identify novel candidate antigens in MS that can be used as disease markers, and can be used to study the humoral immune response in MS.
An approach to the identification of targets of autoantibodies in multiple sclerosis E.T. Littleton, J. Palace and A. Vincent Oxford University, Oxford, UK
The finding in multiple sclerosis (MS) patients of oligoclonal IgG bands upon isoelectric focusing of cerebrospinal fluid and the partial clinical response to immunosuppressive drugs support the theory of a role for antibodies in the disease. However, previously employed candidate antigen-based approaches, usually involving ELISA assays, have failed to identify pathologically relevant antibodies and antigens. We hypothesise that the antigenic target(s) for a relevant antibody would be located on the cell membrane of neuronal or oligodendroglial cells. We have developed an approach that can be used to identify target antigens, first applying it to demonstrate that muscle-specific kinase (MuSK) is a muscle cell target in myasthenia gravis, and then applying it in the context of MS. Using immunocytochemistry with flow cytometry we showed that myasthenia gravis patients' sera, containing antibodies against MuSK, bind MuSK-expressing TE 671 rhabdomyosarcoma cells, and that MuSK can be immunoprecipitated from TE 671 cell extracts by these sera and identified on Western blots. Applying the same approach, we found that about 10% of relapsing-remitting and secondary progressive MS patients' sera contained IgG antibodies, which bound SK-N-SH neuroblastoma cells. We then used these neuronal antibody-containing MS sera to immunoprecipitate antigens from biotin-labelled SK-N-SH cell extracts, isolating the antigens through Western blotting of the immunoprecipitates. Immunoprecipitated antigens are being identified by protein sequencing. Guillain-Barré syndrome (GBS) is a post infectious polyradiculoneuropathy characterized by demyelination and axonal degeneration of peripheral nerves. Phagocytes and glycolipid (ganglioside)-specific antibodies, are thought involved in the pathogenesis. We recently documented the inflammatory capacity of GBS-associated anti-GM1 IgG, as measured by leukocyte degranulation and phagocytosis. Additional experiments showed that sera containing GM1-IgG (n=41), GQ1b-IgG (n=32), GD1a-IgG (n=5) and GM2-IgG (n=2) induced leukocyte activation as measured by degranulation in 59%, 84%, 80%, and 100%, respectively, of the patient sera investigated. Leukocyte activation was IgG-receptor (FcgammaR) dependent, as shown by blocking studies using FcgammaR-specific antibodies. The addition of pooled gammaglobulin (IVIg), used as treatment for GBS, reduced ganglioside-specific IgG-induced leukocyte activation in a dose-dependent way. These results indicate that gangliosidespecific IgG contains pro-inflammatory activity, and that IVIg may reduce its functionality.
New genes-new targets for therapy of myasthenia gravis M. Souroujon a , T. Feferman b , A. Revital b , P. Maiti b , S. Berrih-Aknin c and S. Fuchs b a The Open University of Israel, Tel Aviv, Israel; b Weizmann Institute of Science, Rehovot, Israel; c Centre National de la Recherche Scientifique, UMR-8078, IPSC, Hopital Marie Lannelongue, Le Plessis Robinson, France New genes involved in the pathogenesis of experimental autoimmune myasthenia gravis (EAMG) were identified by DNA microarray technology. Out of the differentially expressed genes associated with myasthenia we focused on genes coding for phosphodiesterases (PDE) and chemokines-which are, respectively, involved in lymphocyte activation, differentiation and migration to the target tissue. Microarray analysis supported by real time RT-PCR revealed a significant increase in mRNA levels of specific PDE subtypes in lymph node cells and muscles of myasthenic rats. Pentoxifylline (PTX), a general PDE inhibitor suppressed ongoing EAMG and led to downregulation of PDE subtypes 1, 4 and 7, TNF-alpha, IL-12 and IL-10 with no effect on IL-4, IFN-gamma and TGF-beta. These results suggest that PTX may in the future serve as a potential drug for immunotherapy of myasthenia gravis in humans. The mRNA levels of the chemokines IP-10 and Mig and of their receptor CXCR3 were upregulated in myasthenic rats and reduced after EAMG suppression. A significant increase in IP-10 and CXCR3 mRNA levels was also observed in thymuses and muscles of myasthenic patients. These results suggest that IP-10/CXCR3 signaling is a potential target for immunotherapy of myasthenia gravis.
Deletion of Fc-receptor-gamma abrogates encephalitogenicity of effector T cells in EAE E. Urich and B. Becher Department of Neurology, Neuroimmunology Unit, University of Zurich, Zurich, Switzerland
The role of B-lymphocytes and auto-antibodies in experimental autoimmune encephalomyelitis (EAE) remains heavily debated. While some have reported that B cells are irrelevant for the development of EAE using B cell-deficient mice, others have shown that antibodies are a central requirement. Furthermore, mice deficient in Fc-receptors (FcR) have been shown to be resistant to develop EAE. FcRs recognize immunoglobulins and thus the requirement of FcRs to develop EAE is in apparent conflict with the findings obtained using B cell-deficient mice. To determine the underpinnings of this paradox, we used B cell-and FcR-gamma-deficient mice to establish the functional role of these molecules in the induction of EAE. We confirmed that B cell deficient mice are fully susceptible to the development of MOG-induced EAE regardless of whether peptides or recombinant MOG was used to immunize. In contrast to these findings, mice deficient in FcR-gamma were shown to be EAE-resistant. Preliminary data indicate that by combining both mutations, the FcR-gamma phenotype is dominant and that B-cell/FcR-gamma deficient mice are EAE-resistant. Our data further show that the FcR-gamma chain is recruited into the TcR complex of effector T cells and that the ablation of FcR-gamma lesions the T cell compartment rather than Fc-receptor bearing cells. Taken together, our data dismiss any involvement of humoral immunity in MOG-induced EAE and demonstrate that FcR-gamma-deletion affects T lymphocytes rather than FcR-bearing accessory cells.
Induction and effector phase of experimental allergic encephalomyelitis are independent of the Fc gamma chain E. Breij a , D. Heijnen a , R. Vloet a , J. Van Immunoglobulin (IgG) receptors (FcgammaR) have been implicated in the induction of experimental allergic encephalomyelitis (EAE). We addressed the role of FcgammaR in the effector phase of EAE. Using the MOG35-55 induction protocol, we were able to reproducibly induce chronic EAE in C57BL/6 FcR gamma-chainÀ/À mice, lacking both stimulatory Fcgam-maRs FcgammaRI and FcgammaRIII. Disease incidence and severity were similar to EAE in wild type C57BL/6 (wt) mice. However, disease onset was significantly delayed, suggesting a role for FcR gamma-chain signalling in the early immunological events resulting in EAE. The model we used is B-cell independent, therefore interactions between myelinspecific antibodies and FcgammaR are unlikely to contribute to disease induction. The FcR gamma-chain is involved in a number of antibodyindependent signaling pathways. We propose that delayed onset of EAE in FcR gamma-chainÀ/À mice results from disturbance of one of these pathways, rather than from deficient antibody-FcgammaR interactions. Subsequently, we investigated the role of FcgammRs in antibody-enhanced EAE. The monoclonal anti-MOG antibody Z12 (mAb Z12) was previously shown to enhance clinical EAE. EAE was induced in wt and FcR gamma-chainÀ/À mice, and both groups were injected with 1 mg mAb Z12 at disease onset. In both wt and FcRg amma-chainÀ/À mice, mAb severely enhanced EAE, as revealed by clinical scores and immunocytochemical analysis of the CNS. Our results demonstrate that anti-myelin antibodies can enhance clinical EAE in absence of stimulatory FcgammaR. Discriminant patterns of IgG self reactivity against brain antigens have been described in multiple sclerosis (MS) patients, highlighting the possibility of B cell involvement. To define the sequence of humoral events involved, self-reactive IgG repertoires were sequentially compared in experimental autoimmune encephalomyelitis (EAE) mice and in control mice. EAE was induced by immunizing SJL/J mice with proteolipid protein (PLP139-151), (n=37). Control groups comprised either mice receiving complete Freund adjuvant (CFA) and Pertussis toxin (n=18) or healthy mice (n=19). Sera were collected weekly and tested by immunoblotting assays using healthy mice brain homogenates. With statistical procedures a linear discriminant analysis indicated that a selfreactive IgG repertoire was significantly different in each group of mice at day 14 (k=0.942), and more so at day 28 (k=0.974). Characterization of the discriminant antigens involved might provide new tools for understanding the mechanisms underlying the initiation and progression of EAE and MS.
Intracerebral expression of CXCL13 and BAFF and formation of Bfollicle-like structures in the meninges of mice with experimental autoimmune encephalomyelitis S. Columba-Cabezas, R. Magliozzi, B. Serafini and F. Aloisi Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy
Given the abnormalities in B-cell activity occurring in the central nervous system (CNS) of multiple sclerosis patients, we investigated whether autoimmunity triggers CNS expression of molecules regulating the development and functional organization of lymphoid follicles, the sites where B-cell responses are initiated. We found that gene expression of CXCL13, a chemokine facilitating B-cell recruitment into follicles, and BAFF, a regulator of B-cell survival, is markedly upregulated in the CNS of SJL mice with PLP 139-151-induced, relapsing-remitting experimental autoimmune encephalomyelitis (EAE). Using immunohistochemistry, follicle-like structures containing B cells and a reticulum of CD35+, FDC-M1+ and CXCL13+ follicular dendritic cells were identified in the brain stem meninges of a proportion of relapsing mice. Ki67+ proliferating B cells were also detected in the inflamed meninges. Intracisternal injection of the lymphotoxin-beta receptor-Ig fusion protein which blocks the action of lymphotoxin alpha/beta, a key cytokine in lymphoid organ development, either delayed or completely suppressed EAE relapses, and reduced B-cell proliferation in the CNS. In line with our recent observations in MS, these findings indicate that the less immune privileged meningeal compartment might promote autoreactive B-cell responses. They also suggest that local targeting of molecules involved in B-cell immunity or lymphoid tissue development should be considered as a possible therapeutic strategy to control CNS autoimmune disease. Background: Mast cells and other elements of allergic immune responses can participate in the regulation of the immune response associated with pathogenesis of EAE. We have shown in a previous study that patterns of self-IgG reactivity could model pathological processes underlying the various forms of multiple sclerosis and the sequence of events involved in EAE. In this work, we evaluate the incidence of anti-allergic drugs on IgG reactivity against brain homogenates. Method: EAE was induced by immunization of SJL/J mice with PLP139-151. One group of mice (n=8) were treated daily after immunization with pyrilamine, an histamine receptor 1 (HR1) antagonist and an other group of mice (n=8) with CV6209, a platelet activating factor receptor (PAFR) antagonist. Control EAE group (n=6) received PBS. Sera were collected weekly and sequential IgG immunoreactivity profiles were performed by western blots on brain homogenate and analyzed with statistical procedures. Results: As expected, clinical signs of EAE were delayed and reduced in mice treated with pyrilamine and CV6209. Analysis of antibody patterns shows that they evolved distinctly between the three groups of mice. Moreover, discriminant analysis indicates that a self-reactive antibody repertoire differed significantly from each group of mice. Conclusion: Pyrilamine and CV6209 reduced significantly clinical signs of EAE. Our data show analysis of the immune profiles could be an indicative marker for evaluation of disease progression or disease resolution in autoimmune disorders as EAE. To study the nature of oligoclonal IgG, single-cell RT-PCR was used to characterize Ig repertoires of both CD19+ B-lymphocytes and CD138+ plasma cells from MS CSF. Sequence analysis of amplified heavy-and light-chain variable (V)-regions identified rearranged germline segments, extent of somatic mutation, and clonal relationships within and between both cell populations. Expanded B-lymphocyte and plasma cell clones were detected in each CSF, including CSF examined during a clinically isolated syndrome. Comparison of CD138+ and CD19+ repertoires in each CSF showed that CD138+ repertoires were more restricted and contained larger numbers of clones that shared little sequence overlap with corresponding CD19+ repertoires. To identify putative antigenic targets of MS oligoclonal IgG, we developed a transient mammalian expression system to produce recombinant antibodies that utilize rearranged V-region sequences of plasma cell clones. Recombinant IgG will be used in various immunoassays, including screening of cDNA expression libraries and random peptide libraries to identify potential targets of MS oligoclonal IgG.
Antibodies to native myelin-oligodendrocyte-glycoprotein in multiple sclerosis patients S. Gaertner, K.L. de Graaf and R. Weissert Hertie Institute for Clinical Brain Research, Tübingen, Germany
The best characterized autoantigen for autoantibody mediated myelin destruction in multiple sclerosis (MS) is myelin-oligodendrocyte-glycoprotein (MOG). MOG is a glycoprotein (218 aa) of the immunoglobulin superfamily expressed on the surface of the myelin sheath. Native MOG was purified from MOG-transfected cells by affinity purification. IgM and IgG MOG-specific antibodies from sera of MS patients and controls were determined by ELISA. IgM antibodies to native MOG were elevated in patients with a first demyelinating event compared to controls ( Pb0.001). We detected high anti-MOG IgG responses in patients with acute disease exacerbations ( Pb0.001) and secondary chronic progressive disease compared to controls ( P=0.001). In contrast, patients in remission did not have elevated IgG antibody titers. Thess data underscore the relevance of conformation and glycosylation of MOG for recognition of the full repertoire of MOG-specific Ig in MS patients. We propose that antibodies against native MOG may have a potential as a diagnostic test for MS and as a possible surrogate marker for therapy. We have determined the three-dimensional structure of the extracellular domain of MOG (MOGex) and of MOGex in complex with the antigen binding fragment (Fab) of 8-18C5 using X-ray crystallography. 8-18C5 is a demyelinating monoclonal mouse antibody that recognises a purely conformationdependent epitope as the vast majority of other demyelinating MOGspecific antibodies do. The complex structure, the first structure of a classical hidden autoantigen complexed with an antibody, reveals the highly discontinuous epitope at the membrane-distal side of MOG. Homologues of MOG are expressed outside the central nervous system, able to induce B-cell tolerance. Interestingly, those residues bound by 8-18C5 are least conserved between MOG and its relatives. Despite of the epitope's discontinuity one loop of MOG dominates the interaction. The conformation of the amino acids that form this loop, however, is very strained and maintained by surrounding loops of the structure providing a simple explanation for the failure to detect this region by peptide mapping with linear peptides. It is widely accepted that an autoimmune response to myelin components plays an important role in the pathogenesis of multiple sclerosis (MS). The aim of the current project is to find new myelin antigens, which are immunogenic in MS. We purified human myelin glycoproteins by sucrose gradient centrifugation and subsequent lentil-lectin affinity chromatography. Sera from MS patients and controls were screened in ELISA and 1D western blots (WB). In addition, immunoadsorption eluates from 5 MS and 2 control patients were used for screening of 2D western blots. In the ELISA experiments, we found an increased reactivity against human myelin glycoproteins in MS patients versus control patients. In 1D WB, a series of proteins with different molecular weights was recognized by MS patients and controls. In the 2D WB, 2-3 protein spots were recognized by the immunoadsorption eluates from MS patients. The identity of these protein spots could be identified by mass spectrometry. Thus, by using a proteomic approach, we could identify some novel candidate autoantigens of the humoral immune response in MS. Ongoing studies with recombinant proteins will clarify if these proteins are predominant targets of the immune response in MS. Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous (CNS) system. Increasing evidence, both from studies in experimental autoimmune encephalomyelitis (EAE) and MS suggests an involvement of autoreactive antibodies in the immunopathogenesis of CNS demyelination. To further our understanding of the involvement of CNSreactive autoantibodies, we are studying immunoglobulin gene rearrangements and the respective antigen specificities of individual B-and plasma cells in MS lesions. Immunoglobulin heavy (H) and light (L) chain genes of individual microdissected cells will be amplified by RT-PCR and rearranged H and L chain genes cloned into vector systems suitable for the expression of recombinant antibodies, which then will be screened for antigen specificity. In order to identify conformation-dependent epitopes, we are currently establishing eukaryotic expression libraries of human brain tissue. Using highly purified mRNA of different CNS regions we have generated cDNA libraries containing between 1.9 and 3.9Â10 7 independent clones with a high proportion of full-length cDNA inserts ranging from 0.4 to 5.6 kb. These cDNA-libraries will be cloned into a viral vector system for the surface expression of CNS antigens on mammalian cells. Providing the correct folding and glycosylation, these eukaryotic expression libraries should provide an essential tool to screen for the identification of novel target autoantigens in multiple sclerosis and further our understanding of the role of autoantibodies in the pathogenesis of MS. The structure-based designed glycopeptide CSF114(Glc) is able to recognize specific auto-Abs in sera of an important subgroup (37%) of MS patients (250), but not in other autoimmune conditions (90). Auto-Abs can be detected only using glycosylated antigens (Ags) recognizing conformational epitopes. Glycopeptide libraries (based on amino-acid and structural diversity) screened by competitive ELISA, in MS patients' sera and controls, demonstrated that the relevant epitope should contain the Asn(Glc) moiety exposed on the tip of a beta-hairpin structure. Moreover, the amino-acid sequence is important for a correct epitope accessibility. Isolated anti-CSF114(Glc) Abs recognized myelin and oligodendrocyte Ags, and are instrumental for isolation of the corresponding native Ag. A proteomic approach based on high-resolution mass spectrometry will lead to its characterisation at molecular level. The value of CSF114(Glc) appeared in a longitudinal study of untreated MS patients (40) We report preliminary data from an ongoing trial of rituximab in relapsing multiple sclerosis (MS) patients with suboptimal response to standard therapy. Rituximab is a chimeric monoclonal antibody that depletes circulating CD20-positive B cells. Objectives: Assess safety and effects on B cells and antibodies in MS patients. Number of enhancing lesions on MRI (primary endpoint) is still blinded and will not be presented. Results: Of 11 patients treated, two discontinued drug: one due to fever and one for shortness of breath. Nine patients were treated with four doses of 375 mg/m 2 without serious side effects. At 20 weeks post-treatment, no changes in cerebrospinal fluid (CSF) oligoclonal band number, IgG concentration, or serum antibodies to myelin oligodendrocyte glycoprotein and myelin basic protein were seen. The proportion of B cells in CSF was reduced in six of eight patients and CSF IgG synthetic rate decreased in five of eight. Two patients had detectable neutralizing antibodies to beta interferons; the titer decreased in one (from 1:72 at baseline to b1:20) and became positive in one (from b1:20 to 1:64) after treatment. In eight patients reaching week 24, 25-ft timed walk was improved in four, stable in three, and worse in one. Conclusion: Rituximab appears safe in MS patients, and depleted circulating B cells with less robust effects on CSF B cells and IgG synthesis. [supported by NMSS USA, Genentech].
Turner syndrome associated with Guillain-Barré syndrome and a complex autoimmune serological profile. A case report S. Losi a , S. Matà b , S. Lori a , R. Salti a and S. Stagi a a Ospedale Pediatrico A. Meyer, Florence, Italy; b Azienda Ospedaliera di Careggi, Florence, Italy
Turner syndrome (TS) is associated with autoimmune disorders, such as thyroiditis, celiac disease, inflammatory bowel disease, juvenile rheumatoid arthritis, and type I diabetes mellitus. Celiac disease, in turn, is associated with several autoimmune disorders, and with neurological diseases, in particular peripheral neuropathy and cerebellar ataxia. We report a young female patient diagnosed in 1998 with a TS (karyotype 45, X0). Subsequent laboratory examinations revealed the presence of serum antibodies against gliadin, endomisium and tissue transglutaminase (2001), nucleus and smooth muscle (2002) and microsomal and thyroglobulin (2003) . HLA analysis revealed the presence of HLA-DQ8. In February 2002, a duodenal biopsy showed increased intraepithelial lymphocytes, and a potential celiac disease was diagnosed. She had a normal thyroid function. In August 2002, she was admitted to the hospital for a 2-week history of increasing weakness of four limbs. On the basis of laboratory and electrophysiological examination, she was diagnosed with an acute motor axonal neuropathy (AMAN), a pure motor axonal variant of the Guillain-Barré syndrome, and treated with intravenous immunoglobulin. She had high titers of anti GD1a, sulfatide and phosphatidylinositol antibodies, and serological evidence of recent coxsackie B virus infection. This is the first description of an association between TS and AMAN in a patient with a complex serological profile of autoimmunity.
316 Acetylcholinesterase used to screen drug treating patients should be from human brain It has been proved that the anionic subsite of acetylcholinesterase (AchE) from Torpedo constructs a part of conformational epitope directed by McAb 3F3 against this enzyme. In this paper, the cross immunoactivity between McAb 3F3 and AchEs (bovine erythrocyte AchE, human erythrocyte AchE and human brain AchE) was detected by enzyme-linked immunosorbent assay and dot-blot immunoassay. The results showed that each AchE reacted well with antibody against itself but McAb 3F3 did not react with any AchEs except Torpedo AchE. It suggests that the anionic subsite of AchE from bovine erythrocyte, human brain and erythrocyte are different from that of Torpedo AchE although all their esteric subsites contain the same active serine. Thus, it is recommended that the AchE from human brain should be used as mark enzyme when it is applied to select a drug to treat patient with Alzheimer's disease or with organophosphate poisoning, in order to get a more effective drug in man.
Natural autoantibodies against neurofilament protein alpha-internexin in human sera K. Teesalu a , T. Rajasalu a , P.A. Janmey b and R. Uibo a a University of Tartu, Tartu, Estonia; b University of Pennsylvania, Philadelphia, USA Serum autoantibodies (AAbs) to pituitary antigens have been associated with autoimmune hypophysitis, idiopathic hypopituitarism, and other autoimmune endocrinopathies. The aim of the present study was to characterize the main AAb reactivities against pituitary antigens in patients with idiopathic hypopituitarism and type 1 diabetes using immunoblot method. Sera from six patients with idiopathic hypopituitarism, 60 patients with type 1 diabetes, and 74 healthy controls were studied. Frequent patient serum IgG reactivity was observed against a 60-kDa human pituitary antigen, and the cross-reactive 62 kDa protein from rat brain was identified as alpha-internexin (alpha-INX) by proteomic methods. By using alpha-INX containing cytoskeletal extracts for AAb screening, IgG and IgM AAbs to this neuron-specific type IV intermediate filament (IF) protein were found in most patient sera as well as in healthy subjects with no significant differences in frequencies between the groups, but the levels of IgM alpha-INX AAbs were higher in patients with hypopituitarism as compared to healthy controls ( p=0.032, Mann-Whitney U-test). In addition to the first demonstration of common occurrence of alpha-INX AAbs in human sera, AAbs were also frequently found against neurofilament heavy subunit (NF-H), a known target of natural AAbs, and relatively rarely against other neuronal IF proteins. We conclude that alpha-INX AAbs belong to the natural AAb repertoire and their physiologic function needs further elucidation.
Mechanism of immune responses in myasthenic thymus I. Kamo a , H. Tomoyasu b , T. Yamamoto c , M. Kusakabe d and A. Kikuchi e a School of Medicine Toho Univ. Sagamihara, Japan; b Ohmori Red Cross Hospital, Ohmori, Japan; c Institute of Medical Science, Univ. of Tokyo, Tokyo, Japan; d ANB Tsukuba Institute, Tsukuba, Japan; e National Institute of Neuroscience, Tokyo, Japan MG is an autoimmune disease induced by autoantibodies against self-AChR. Thymectomy results in clinical effectiveness, suggesting that this thymus plays a central role in antibody production. However, the immunopathological mechanism of effectiveness has not been elucidated. We aimed to understand this mechanism. MG thymi were used for immunohisthochemical staining against B-cell marker, IL-2, IL-4 and IFNgamma, an 80-and 100-kDa haemopoietic factor and nude mouse splenocytes for in vitro immune response against SRBC. Hyperplastic MG thymi commonly contain many IL-2 producing cells, B-cells, but essentially lack IL-4 and INF-gamma producing cells. MG thymi also contain many myoid lineage cells that produce the 80-and 100-kDa haemopoietic factor. Thus, in MG thymi, a new cytokine network is developed. We designed in vitro immune response system consisting of nude mouse splenocytes and soluble factors to mimic the microenvironment of hyperplastic MG thymus. IL-2 alone induced very small numbers of anti-SRBC-antibody producing cells. However, when myoid cell 80-and 100-kDa haemopoietic factor, were added together, many anti-SRBC producing B-cells were detected. IgG subclasses were also induced in the secondary responses. These results suggest that hyperplastic MG thymus generates a new B-cell stimulatory mechanism not found in the periphery.
Anti-MOG autoantibodies in sera of patients with myasthenia gravis and thymoma C. Cappelletti, P. Cristaldini, P. Bernasconi, F. Andreetta, O. Simoncini, F. Baggi, F. Cornelio and R. Mantegazza National Neurological Institute bCarlo BestaQ, Milan, Italy
Thymomas are neoplasms of thymic epithelial origin, commonly associated with myasthenia gravis (MG). All MG patients with thymoma have antibodies against postsynaptic nicotinic acetylcholine receptors; anti-titin and anti-ryanodine receptor antibodies have an 84% and 74% positive predictive value of thymoma detection, respectively. It seems that autoantibodies are not produced within thymoma; nevertheless, antibodies to different antigens are associated with thymoma. We tested by anti-human recombinant MOG antibody-specific ELISA, 49 MG sera: 39 positive for anti-AChR antibody (10 with thymic hyperplasia and 10 with thymoma) and 10 seronegative. Ten out of 49 (20.4%) patients were positive for anti-MOG antibody; when the positive ones were stratified according to thymus histology, six patients (60%) were positive for thymoma. We then analyzed MOG-specific transcript expression in MG thymus specimens. A positive signal was found in the different pathological thymic alterations (hyperplasia, involuted and thymoma) with variable intensity, differently from data previously reported (Eur. J. Immunol. 2002; 32:2737) ; no MOG expression was observed in non-pathological young thymuses. These results further suggest that a thymoma-associated immune dysregulation underlies the generation of antibodies towards different self antigens, whose pathogenetic role remains to be defined. Measurement of autoantibodies to a muscle specific tyrosine kinase receptor (MuSK) may be useful in the assessment and management of patients with acetylcholine receptor antibody (AChRAb) negative myasthenia gravis (MG), and we have developed a new and convenient assay for MuSKAb measurement based on 125I-labelled purified recombinant MuSK. In addition, the use of rat or human recombinant MuSK in this assay was assessed. In the assay serum samples (5 Al) were incubated with 50 Al of 125I-MuSK (rat or human) and any immune complexes formed precipitated with anti-human IgG (100 microlitres). MuSKAb were detected in 8/33 (24%; range 1.1-1.4 nmol/l MuSK bound) sera from AChRAbnegative MG patients studied. In contrast, no MuSKAb were detected in 53 AChRAb-positive MG patient sera and furthermore, 0/18 Lambert-Eaton myasthenic syndrome sera, 0/5 non-MG neuromuscular disease sera, 0/95 control autoimmune disease sera and 0/50 healthy blood donor sera contained detectable MuSKAb. Inter-assay coefficients of variation (CV, n=5) were 6.8%, 5.9% and 4.1% for samples with MuSKAbs at 0.73, 0.32 and 0.11 nmol/l, respectively; and intra-assay CV (n=12) were 2.1%, 4.3% and 7.3% for samples with MuSKAbs at 0.77, 0.32 and 0.11 nmol/l, respectively. Similar results were obtained with 125I-labelled rat and human MuSK. Overall, the 125I-MuSK immunoprecipitation assay we have developed provides a simple, specific and precise procedure for detecting MuSKAb. In continuation of our attempts for antigen-specific suppression of the immune system (Urbatsch, I.L., Sterz, R.K.M., Peper, K., and Trommer, W.E. (1993) Eur. J. Immunol. 23, 774-779) a fusion protein composed of amino acids 4-181 of the extracellular domain of the human acetylcholine receptor from human muscle and the plant toxin gelonin was expressed in E. coli. Gelonin is a potent single chain type I ribosome-inactivating protein. Due to lack of the B chain of type II toxins it is not active against intact cells. The recombinant gelonin was not glycosylated and was about twice as active in an in vitro translation assay as the native toxin. The fusion protein formed inclusion bodies but could be solubilized in the presence of guanidinium chloride. It exhibited a native structure as shown by antibodies recognizing a conformational epitope. Half maximal inhibition of translation was achieved at 46 ng/ml as compared to 4.6 ng/ml for native gelonin and 2.4 for the recombinant product. Its use as therapeutic agent for the treatment of myasthenia gravis is currently investigated in myasthenic rats.
Autoimmune agrypnia is due to autoantibody against GABAB receptor G. Frisullo, G. Della Marca, M. Mirabella, M. Caggiula, G. Mennuni, P. Tonali and A.P. Batocchi Institute of Neurology, Catholic University, Rome, Italy
We have described the clinical feature of a woman with several relapsingremitting episodes of agrypnia and continuous 9-11 Hz activity pattern in EEG recording, dysautonomic symptoms and nocturnal respiratory failure. Plasma exchange and high-doses of corticosteroid treatment induced a remission of clinical symptoms and recovery of REM and NREM sleep. Immunohistochemistry showed, after incubation with patient serum and CSF, an intense immunoreactivity over brainstem, hippocampus and cerebellum granular layer. Using Western blotting, patient IgG recognized a 108 KDa protein on mouse cerebellum, cortex and brainstem. Immunohistochemical analysis of patient purified IgG showed a binding on GABAergic synapse-rich neuronal cells. Preincubation of mouse cerebellum sections with patient IgG completely abolished the staining obtained with the antiGABABR1 antibody but not using antiGAD antibody. To evaluate the pathogenicity of patient antibodies, her purified IgG, at different concentration, was injected into the cisterna magna of C57BL/6 mice, pre-implanted with two EEG electrodes. We observed, 12-20 h after injection, a severe respiratory failure followed by death in the mice injected with high dose, a severe ataxia followed by breathing depression in mice injected with middle dose, and a progressive truncal ataxia in mice injected with low dose. These symptoms disappeared completely after 12 h. EEG showed a strong reduction of delta EEG activity that started after 1 h of IgG injection and persisted along 14 h. The mice injected with IgG from healthy subject did not show any neurological symptom. Our study demonstrated that anti-GABABR1 antibody causes autoimmune agrypnia. Antibodies against glutamic acid decarboxylase (GAD) have been described in a number of autoimmune and some neurological diseases (e.g., stiffperson syndrome, ataxia). The observation that sera from 4/5 patients with stiff-person syndrome, in our study, had circulating anti-gliadin antibodies led us to investigate the relationship between anti-GAD antibodies and gluten sensitivity. Using ELISA, we investigated the prevalence and levels of anti-GAD antibodies in patients with gluten ataxia (the most common neurological manifestation of gluten sensitivity) and patients with coeliac disease only in comparison to controls. The effect of gluten-free diet was also investigated. The prevalence of anti-GAD antibodies in patients with gluten ataxia and patients with coeliac disease only, not on gluten-free diet, was 56% and 59%, respectively, which was higher than healthy controls (10%). Antibody levels of both groups were significantly reduced after the introduction of gluten-free diet. Confirmation of ELISA results was achieved using Western blotting. GAD is an important antigen in gluten sensitivity-related diseases and may provide the link between gluten sensitivity and neurological dysfunction. Elimination of anti-GAD antibodies by gluten-free diet may have important therapeutic implications in other anti-GAD-associated autoimmune diseases. Gluten ataxia is the most common neurological manifestation of gluten sensitivity. In common with other forms of gluten sensitivity, gluten ataxia is believed to have an immune-mediated pathogenesis. All patients possess circulating anti-gliadin antibodies (IgG and/or IgA) and a significant proportion also possess antibodies directed against tissue transglutaminase and/or glutamic acid decarboxylase (GAD). In addition, we have previously reported the presence of circulating anti-Purkinje cell antibodies in these patients. Identification of the proteins targeted by these antibodies may facilitate further understanding of the pathogenesis of this condition. The aim of the current study was to investigate the avidity and cerebellar target(s) of these antibodies using sodium thiocyanate elution assays, 2D gel electrophoresis, Western blotting and mass spectrometry in comparison to patients with coeliac disease only and appropriate control groups. Preliminary results have suggested no apparent difference in the avidity of either anti-gliadin or anti-GAD antibodies between groups. Sera from patients with gluten ataxia appeared to react more frequently with proteins within certain molecular weight ranges (24-28 kDa and 56-59 kDa) than sera from other groups. Analysis of these proteins by MALDI-TOF will allow their identification. The use of GlycoChip(R) microarray technology for identification of anti Glc(alpha 1,4)Glc(alpha)-serum antibodies as a specific biomarker for multiple sclerosis M. Schwarz a , L. Spector a , M. Gortler a , L. Glass-Marmor b , A. Karni c , N. Dotan a and A. Miller b a Glycominds Ltd., Lod, Israel; b Carmel Medical Center, Haifa, Israel; c Sorarsky Medical Center, Tel Aviv, Israel
There is an unmeet need to develop specific biomarkers for multiple sclerosis (MS) to improve the management of patients and the monitoring of the effectiveness of treatment. Our objective was to identify anti glycans antibodies that can serve as biomarkers. We have screened serum from 107 patients with relapsing-remitting MS (RRMS) against a library of glycans on a glycan chip, and have found significantly higher levels of IgM anti-Glc(alpha 1,4)Glc(alpha) antibodies (anti-Ga4Ga antibodies) than in 77 control patients suffering from other neurological diseases ( pb0.001) and other autoimmune diseases ( p=0.03), and higher levels than in 20 patients with primary progressive MS (PPMS, p=0.06). We suggest that the level of anti-Ga4Ga antibodies is a specific biomarker for RRMS, with potential clinical utility for MS prognosis and disease management. Background: Several recent reports identified high titres of anti-glutamic acid decarboxylase autoantibodies (GAD-Ab) in association with cerebellar ataxia. The aetiological link remains unclear, as well as the frequency and level of GAD-Abs in an unselected ataxic population. We have explored their significance in a population-based, idiopathic LOCA (ILOCA) patients in South Wales and provide phenotypes. Methods and materials: Sera of 84 patients (mean disease duration: 7.8 years; range: 1 to 35) were analysed for anti-GAD Ab using a commercial GAD-65Ab radioimmunoassay. Results were interpolated in the standard curve constructed using dilutions of standard controls and healthy sera. Results: Three patients (3.6%) showed highly elevated Ab titre with background history of type I diabetes mellitus (DM) and type II DM, n=1 and n=2, respectively. Three other ILOCA patients with co-morbid DM (both types I and II) showed a normal Ab titre. Clinically, all patients showed relatively pure cerebellar syndrome with few extra-cerebellar neurological manifestations. There were no significant phenotypic differences observed with other ILOCA patients. Discussion: Up to 4% amongst our population-based ILOCA patients were positive for GAD-Ab. Our data emphasise the need for a routine screening of GAD-Ab in patients with ILOCA as it improves the diagnostic accuracy for ILOCA cases as well creating opportunity for therapeutic interventions. Circulating specific CD8+ T cell responses are unknown in autoimmune diseases with central nervous system (CNS) involvement. Twenty-five HLA-A2 patients (13 with relapsing-remitting MS, 4 with a clinically isolated syndrome (CIS) at high risk for MS, 2 neurolupus and 3 SLE without CNS involvement, 3 healthy volunteers) were labelled on blood with PE-HLA-A*0201 tetramers loaded with myelin specific peptides according to previously published CD8+ T-cell responses to human myelin protein-derived peptides (iTAg, Beckman Coulter, Europe): MBP110-118: SLSRFSWGA; PLP80-88: FLYGALLLA; MAG556-564: VLFSSDFRI; along with CD3-FITC and CD8-PerCP antibodies, and analysed on a flow cytometer. CD3+CD8+ T-cells specific for myelin epitopes were expanded in 6 (35%) of HLA-A2 patients with MS or CIS and in both patients with clinically active neurolupus: two CIS and one MS patient specific for MBP only (respectively, 0.13%, 0.30%, 0.58% of CD3+CD8+ T cells), one (MS) for MBP (0.38%) and PLP (0.35%), one (CIS) for MBP (0.76%) and MAG (0.36%), one MS and both neurolupus for MBP (respectively 0.53%, 6.97%, 2.01%), PLP (0.40%, 2.39%, 0.20%) and MAG (1.15%, 3.81%, 0.41%) and none of the healthy volunteers or SLE patients devoid of neurolupus (b0.15%). MBP110-118 seems the main peptide targeted by the class I-restricted cellular response against myelin specific determinants. Early events in the development of MS lesions are the formation of cellular infiltrates, consisting of monocyte-derived macrophages and T lymphocytes. Sequencing of cDNA from MS brain lesions and control brains has revealed that osteopontin (OPN) is the most abundant cytokine-encoding transcript unique to MS plaques. OPN induces chemotaxis of immune cells and promotes the induction of Th1 cytokines and inhibits the release of Th2 cytokines. Previous studies demonstrated that an imbalance between Th1 and Th2 type cytokine mRNA level in PBMC of RR MS patients preceded the occurrence of increased disease activity. To determine whether OPN may contribute to this phenomenon, the relation between OPN serum levels and disease activity was investigated in a longitudinal study of sixteen RR MS patients undergoing monthly clinical and MRI examinations. A trend was observed for OPN serum levels in relation to clinical exacerbations with elevated levels 2 months prior to the occurrence of a relapse. Moreover, OPN protein levels were significantly elevated 1 month prior to increase of lesion number with a median increase of 30% compared to baseline levels, whereas no relation was observed between OPN levels and increase in gadolinium (Gd)-enhancing lesion volume. These data indicate that OPN plays a role in increased disease activity in RR MS patients.
Systemic vs. central nervous system cellular immunity with relevance to relapsing-remitting multiple sclerosis M. Matsui, S. Araya, H.-Y. Wang, K. Ozawa and T. Saida Utano National Hospital, Kyoto, Japan
To clarify which disturbances in cell-mediated immunity in blood and the central nervous system (CNS) are associated with the active phase of multiple sclerosis (MS), paired peripheral blood and cerebrospinal fluid (CSF) samples were obtained from 36 patients with relapsing-remitting MS (RRMS), and examined for the percentages of lymphocyte subsets and levels of soluble immune mediators. Active RRMS patients (n=27) were characterized by an increase in CD4 + CXCR3 + Th1 cells in blood, which was inversely correlated with plasma levels of IL-10 and IL-12p70. Further, an increase in CD4 + CD25 + cells and a decrease in CD8 + CD11ahigh cells were found in the CSF of those patients, while CSF CD4 + CD25 + cells showed a positive correlation with leukocyte counts as well as to albumin and CXCL10 levels. In addition, CSF CD8 + CD11a high cells showed a negative correlation with CSF cell count and a positive correlation with CSF IL-4 levels. We also studied the clinical relevance of these cellular immune parameters by determining their percentages in another group of 31 non-treated inactive RRMS patients, and found that an increase in CSF CD4 + CD25 + cells predicted an exacerbation of MS within 3 months. These findings suggest that flow cytometric analysis of CSF CD4 + CD25 + cells provides a good measure of ongoing inflammation in the CNS as well as indication of aberrant immunity leading to MS relapse in the near future.
Probing the specificity of recombinant antibodies generated from multiple sclerosis cerebrospinal fluid with a random peptide library X. Yu, G. Owens, M. Burgoon, A. Ritchie, K. Keays and D. Gilden University of Colorado Health Science Center, Denver, USA Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). The most common laboratory abnormality associated with MS is the intrathecal synthesis of oligoclonal immunoglobulin (Ig) G in brain and cerebrospinal fluid (CSF). The specificity of oligoclonal IgG in MS is unknown. To determine its specificity, single cell RT-PCR was used for the amplification of IgG heavy-and light-chain V-regions (VH and VL) from plasma cells of MS CSF. The paired VH and VL regions from over-represented IgG sequences were selected to generate recombinant antibodies (rABs). The specificity of the rABs was probed by panning a phage-displayed random peptide library with 12-mer peptides. After three rounds of panning, affinity-selected peptides were identified, and ELISA confirmed the binding specificity to MS rABs. Sequence alignments to the Swiss-Prot database revealed a plethora of putative targets, including some peptides that are highly homologous to proteins of pathogens. To refine the many potential antigens under study, alanine scanning is being used to identify the amino acids in each peptide that are important for antibody binding. Phage-displayed random peptide libraries have the potential to identify continuous epitopes or mimotopes of MS antigens. Reliable, and easy to measure, immunological markers able to denote disease characteristics in multiple sclerosis (MS) patients are still lacking. We applied a multivariate statistical analysis on results obtained by measuring-by real-time RT-PCR-mRNA levels of 25 immunological relevant molecules (e.g., cytokines, chemokines and chemokine receptors) in peripheral blood mononuclear cells (PBMCs) from 198 MS patients. The combined measurement of mRNA levels of IL-1beta, TNF-alpha, TGFbeta, CCL20 and CCR3 was able to distinguish MS patients from healthy individuals (MS pattern). CXCR5, CCL5, and CCR3 combined mRNA levels identify primary progressive MS patients (PP-MS pattern) while TNF-alpha, IL-10, CXCL10 and CCR3 differentiate relapsing MS patients (RR-MS pattern). Finally, a significant correlation was found between mRNA levels of CCR3, CCR4, CCR7, and TNF-alpha measured in PBMCs and in mononuclear cells obtained from the cerebrospinal fluid. On the other hand, we did not find any correlation between immunological markers and gadolinium-enhancing magnetic resonance imaging (MRI)detectable lesions or T1 and T2 MRI lesion load. Our results indicate that multi-parametric analysis of mRNA levels of immunological relevant molecules in PBMCs may represent a successful strategy for the identification of putative peripheral markers of disease state and disease activity in MS patients. The frequency of anti-GM1 ganglioside antibodies in neuropathies and other neurological and inflammatory diseases varies considerably and largely depends on the technique used. In our study, we extended the validation of the standardized ELISA developed by the European Inflammatory Neuropathy Cause and Treatment (INCAT) group. Anti-GM1 IgG and IgM antibodies were determined by the INCAT-ELISA in large groups of patients and controls. Correlation of variance (CV%) was calculated by determining standard deviation divided by the mean of optical densities in 48 wells of one serum sample. Using a positive control sample the CV% was 4% for anti-GM1 IgG and 7% for anti-GM1 IgM. The variation could be attributed to variation between wells, plates, days and technicians. Anti-GM1 antibodies were found in 97 (21%) of 462 patients with Guillain-Barré syndrome (GBS), 2 (5%) of 41 chronic demyelinating inflammatory polyneuropathy, 5 (50%) of 10 multifocal motor neuropathy, 9 (6%) of 143 inflammatory polyneuropathy (I-PNP), 2 (3%) of 72 noninflammatory polyneuropathy (NI-PNP), 7 (9%) of 78 motor neuron disease, none of 20 multiple sclerosis, 3 (2%) of 142 other forms of autoimmune diseases and 3 (2%) of 124 healthy controls. IgG antibodies were only found in patients with GBS, I-PNP and NI-PNP. In conclusion, the INCAT-ELISA has a good reproducibility and the presence of IgG anti-GM1 antibodies is almost specific for inflammatory neuropathies. Object: To correlate the lymphocyte network in the peripheral blood, with neurological status (EDSS) and MRI activity in newly diagnosed untreated MS patients. Methods: Twenty patients presenting with a first CNS inflammatory event suggestive of MS were enrolled in the study. Every 45 days, for 1 year, they underwent clinical and MRI evaluation, and flow cytometry analysis of a broad-spectrum of lymphocyte subsets. Results: Twelve patients showed MRI activity throughout the follow-up. Most of the patients had a significant reduction of blood CD8+ alpha-beta T cells compared to reference subjects. Concomitant with MRI activity, we observed a decreased frequency of the following lymphocyte subsets: CD4+CD25high and CD8+CD28À alpha-beta T cells; CD4+CCR5+ CXCR3+ T cells; NKT cells, NK-like T cells and CD8dim NK cells. In a subgroup of patients, we observed increased CD8dim gamma-delta T cells and CD8+CD45RA+CCR7À alpha-beta T cells at the beginning of MRI activity. Conclusions: Newly diagnosed MS patients display, concomitantly with MRI activity, increased terminally differentiated CD8+ alpha-beta T cells in presence of a reduction of defined regulatory T cells subsets. These preliminary data indicate that the assessment of peripheral immunophenotype traits, consisting of the above pattern of lymphocyte subsets, may be useful in the clinical management of MS patients. Understanding the mechanisms that sustain the effects of disease modifying drugs in MS may help refine current therapies and our knowledge of disease pathogenesis. We investigated gene expression, by microarrays, in peripheral blood mononuclear cells (PBMC) of 7 MS patients at baseline, and after 1 and 3 months of interferon-beta (Rebif 44 mcg). Blood sampling and gadolinium-enhanced MRI were obtained 36 h after the last injection. The influence of IFN-beta on gene expression was more pronounced after one than after 3 months of therapy, involving genes of immunological significance and others related to energy metabolism, cell cycle and cytoskeleton dynamics. Genes validated by RT-PCR included IL-10, FILAMIN-B (increased) and IL-16, RAB7 (decreased). RAB7 represents a novel finding and was further investigated given its role as modulator of class-II antigen presentation. In accord with previous reports on a reduced expression of HLA-DR on PBMC in MS and in other autoimmune diseases, RT-PCR showed that RAB7 was decreased in untreated MS patients compared to healthy controls (including three disease discordant monozygotic twin sets). The global impact of IFN-beta treatment on gene expression tends to decrease after the first months of therapy. The decreased expression of RAB7 may contribute to the downregulation of MHC class II, one of the known immunomodulatory actions of IFN-beta. In vivo cell imaging by magnetic resonance (MR) may be a powerful tool to reveal the pathogenic events of cell migratory processes in multiple sclerosis. For this purpose, cells have to be isolated, labeled ex vivo with MR contrast agents and reintroduced into the body. In this study, we aimed to develop an efficient method for labeling primary monocytes. As suitable contrast agents we used two differently sized ultra small particles of iron oxide (USPIO's), Sinerem and Endorem. Beneficial effects of preincubation with transfection agents (TA's; Poly-L, Fugene and Superfect) were evaluated. Labeling efficiency was determined by T2-relaxation time MRI and Perls' stain for iron. Cell viability and function were constantly monitored. Incubation of monocytes with Endorem resulted in a labeling efficiency higher than 80%. T2-relaxation time decreased up to 40%. Superfect was the only TA that further increased the uptake of endorem. In conclusion, endorem preincubated with Superfect is most efficient in labeling primary monocytes. Currently, we are planning experiments to track primary monocytes in an animal model of multiple sclerosis. Multiple sclerosis (MS) is a demyelinating disease characterized by inflammation in brain and spinal cord. There is a wide variability in symptoms, disease course, progression rate, and areas of CNS involved. Based on the autoimmune nature of MS and migration of immune cells to and from the brain, we reasoned that an imprint of the disease would be present in peripheral blood (PB) cells. In order to identify molecular differences between relapsing-remitting (RR) MS patients and healthy controls (HC) and RRMS patients in different phases of disease, we investigated gene-expression profiles in whole blood from HC and RRMS patients, using cDNA micro arrays with a complexity of 43,000 cDNAs. A total of 1201 genes differed significantly in expression between RRMS patients and HC. The differentially expressed genes represent several distinct functional categories. Most interestingly, a large number of genes reflect differences in the activity of G protein signaling between MS patients and HC. These preliminary data revealed indications for important molecular and biological variation between RRMS patients and HC, and RRMS patients in different phases of the disease that provide a lead to relevant biological pathways and could ultimately lead to identification of novel criteria for (sub) classification. Immune-mediated mechanisms play a crucial role in the patogenesis of multiple sclerosis (MS). Though neurodegeneration with axonal loss is an early event in MS, the therapeutic approach remains restricted to antiinflammatory drugs. Responsivness of the patients to all available drugs is limited. To detect if this responsiveness is generated by the shift in cytokine production, we studied a group of MS patients with monthly pulse methylprednisolone (MP) and cyclophosphamide (CPA) therapy. Patients and methods: 40 progressive MS patients (27 females, 13 males, age 46.9F9.2 yrs, duration of MS 14F6.9 yrs) were divided into two groups according to clinical response (7 good responders, 19 stable patients versus 14 non-responders). Peripheral blood was taken 14 days after 1 g MP and 1 g CPA administration. Peripheral blood from 10 ageand sex-matched healthy controls was used to compare cytokine production. Results and discussion: Cytokine production did not differ significantly in treatment responders versus non-responders. Surprisingly, there was significantly higher expression of CCR5+CXCR3 on CD4 lymphocytes of responders which may be explained by the theory that immunosuppression is effective only where inflammation is still active. Strikingly significant difference (pN0.000001) was detected in production of IFN gamma, TNF alpha, IL2, IL10 and IL12 between MS patients and healthy controls suggesting that the immune hyperactivation is present in the disease even in the second decade of its duration and despite aggressive treatment. Aim: To investigate interactions between immunological changes and vertebral syndrome in multiple sclerosis (MS). Patients and methods: 30 patients with definite MS were examined, mean age (Fsigma) was 31.3F10.5 years; Kurtzke Expanded Disability Status Scale (EDSS) score was 4.4F1.7. Results: Chronic back and neck pain was present in 80% of patients. The mean age of onset of vertebral pain was 25.8F9.4 years, mean duration of pain 6.8F4.8 years. The impairment of vertical spinal balance (scoliosis, kyphosis) was diagnosed in 70% of patients. There were correlations (r=0.5-0.7; pb0,05) between the following vertebral signs and immunological indices: (1) high tonus of paravertebral muscles and high level of circulating immune complexes (CIC), CD8+, CD25+ T-lymphocytes; (2) presense of muscle trigger points in thoracic girdle and high levels of CIC, anti-nDNA antibodies, CD4+, CD HLADR+; (3) pronounced spondyloarthrosis and level of CIC. These relationships may be explained by immune aggression against muscle and cartilaginous cells. Correlation with the level of anti-nDNA antibodies confirms the fact of polyclonal activation. Plaques in cervical part of spinal cord were visualised by magnetic resonance imaging in 56.7% of patients. Vertebral signs (neck pain, scoliosis, high tonus of paravertebral muscles, trigger muscle points) and the level of CD8+ were more pronounced in this group versus patients without cervical lesions ( pb0.01). Conclusions: Immunological changes and cervical plaques have an influence on the developing of vertebral syndrome in multiple sclerosis.
CSF change in IFN beta-treated MS patients K. Yokoyama Juntendo University, Tokyo, Japan Object: Interferon (IFN) beta is pleiotrophic molecules with complex immunoregulatory activities. It is commonly used world wide. However, a valid definition of therapeutic response is needed for individual treatment decision. We have collected the CSF samples from MS and other neurological diseases as control. We assessed the clinical response and the mechanism of IFN beta in multiple sclerosis (MS) patients by CSF analysis. Methods: The CSF from 14 relapsing-remitting MS patients and 30 other neurological diseases (OND) were collected with informed consent. Nitric oxide (NO) metabolites and 41 amino acids were measured by HPLC. IgG index was also calculated in each patient. The change of those measures in relation to clinical disease severity (expanded disability status score; EDSS) were evaluated. We could measure and compare CSF change before and after IFN beta treatment in two patients. Summary: In relapsing MS patient with severe EDSS worsening showed increased some amino acids level. NO metabolites do not change drastically in acute phase of the disease but IFN beta treatment decreased the concentration of NO as well as IgG index. Conclusions: The IFN beta treatment might influence the NO and IgG synthesis in the CSF of MS patients. Serial measurement of IgG index might be useful as a surrogate marker for the effectiveness of IFN beta treatment. During neuroinflammatory diseases of the CNS, immune cells infiltrate both CNS parenchyma and the cerebrospinal fluid. Although these two populations may be distinct regarding their phenotype and functions, it is currently assumed that CSF cells resemble more closely parenchymainfiltrating cells than their blood counterpart. Thus, as far as CNS biopsies remains an invasive procedure, analyzing CSF immune cells represent a unique tool for understanding the pathophysiology of neuroimmune diseases. The development of techniques allowing gene array analysis from a small amount of RNA prompted us to evaluate the feasibility of analyzing gene profile of CSF cells. Our results indicate that 1 to 3 ng of mRNA can be extracted from 1 ml of CSF obtained from MS patients. This theoretically allows performing up to 40 RT-PCR from one CSF sample. However, the RNA quality as assessed by analyzing the ratio of ribosomal bands depends primarily on the time elapsed from CSF sampling to CSF processing. Indeed, only CSF cells that are immediately treated for RNA extraction display unaltered RNA profiles. Moreover, once good quality samples are selected, an amplification step is required in order to perform assay analysis. We studied by ELISA cerebrospinal fluid (CSF) and serum levels of classical soluble HLA-I (sHLA-I) and non-classical soluble HLA-G (sHLA-G) molecules in 69 relapsing-remitting (RR), 21 secondary progressive (SP) and 13 primary progressive (PP) multiple sclerosis (MS) patients, grouped according to clinical and Magnetic Resonance Imaging (MRI) evidence of disease activity, and 183 patients with other inflammatory neurological disorders (OIND) and non-inflammatory neurological disorders (NIND). An intrathecal production of sHLA-I and sHLA-G was more frequent in MS than in OIND and NIND ( pb0.001), with more consistent values in clinically and MRI active than in clinically and MRI inactive MS for sHLA-I ( pb0.02 and pb0.001, respectively) and in clinically and MRI stable than in clinically and MRI active MS for sHLA-G ( pb0.01 and pb0.001, respectively). A decrease in sHLA-I and an increase in sHLA-G levels were detected in serum of clinically active MS, while MRI active MS showed high and low CSF levels of sHLA-I and sHLA-G, respectively. Our results seems to indicate that CSF and serum sHLA-I/ sHLA-G balance could modulate MS activity in opposite directions. T cell immunoglobulin-and mucin-domain-containing molecules (TIMs) comprise a recently described family of molecules expressed on T cells. TIM-3 has been shown to be expressed on murine T-helper (Th) 1 cell clones and has been implicated in the pathogenesis of Th1-driven experimental autoimmune encephalomyelitis. In contrast, association of TIM-1 polymorphisms to Th2-related airway hyperreactivity has been suggested in mice. The TIM molecules have not been investigated in human Th1-or Th2-mediated diseases. Using real-time (TaqMan) RT-PCR, we show that human Th1 lines expressed higher TIM-3 mRNA levels, while Th2 lines demonstrated a higher expression of TIM-1. Analysis of cerebrospinal fluid mononuclear cells (CSF-MC) obtained from patients with multiple sclerosis (MS) revealed significantly higher mRNA expression of TIM-1 compared to controls. Moreover, higher TIM-1 expression was associated with clinical remissions and low expression of IFN-gamma mRNA in CSF-MC. In contrast, expression of TIM-3 correlated well with high expression of IFN-gamma and TNF-alpha. These data imply the differential expression of human TIM molecules by Th1 and Th2 cells and may suggest their differential involvement in different phases of a human autoimmune disease.
Cerebrospinal fluid tau protein in multiple sclerosis E. Pal
Multiple sclerosis is an immune-mediated demyelinating disease of the central nervous system. However, not only myelin, but axonal-neuronal damage also develops in the advanced stage, but in the early stage as well. It may be finally responsible for irreversible disability. Soluble markers, such as microtubule-associated protein, tau are suitable for detection of axonal damage and evaluation of neurodegeneration in vivo. CSF tau level was determined by enzyme-linked immunosorbent assay (ELISA) in 20 MS, 6 viral encephalitis and 7 control patients. Tau level was elevated both in MS and encephalitis cases comparing to controls. In MS group tau level correlated with disease duration and severity. Therefore, we concluded that CSF tau would be a probable marker for detection of axonal loss. The effective use of neuroimmunological tests for diagnostic and research purposes requires standardization of methods and external quality control schemes. These two requirements guarantee accuracy and reliability in diagnostic routine, and data reproducibility in research settings. AINI promoted an unprecedented consensus-based program for procedure and method standardization in neuroimmunology. Twenty-six Italian centres joined the program. Four areas were identified (approximate total number of exams/year in brackets): cerebrospinal fluid examination (7000); diagnostics for paraneoplastic neurological syndromes (1700); diagnostics for peripheral disimmune neuropathies (2500), diagnostics for myasthenia gravis (4000). A questionnaire-based survey, which was prepared by expertise-based selected centres, revealed interlaboratory variability in procedures and assay methodologies. Attempts to uniform procedures and methods were made by internet contacts and in two AINI workshops during [2002] [2003] . Contemporarily, quality assurance schemes was, and will be in the future, promoted. The results of this program were summarized in four internet-available documents (http://www.aini.it). The documents, which will be yearly updated, report tests' indications and limits, agreed common protocols, instructions for result interpretation, and essential references. They should improve diagnostic performances, and represent a premise for clinical guidelines in neuroimmunology. However, there is lack of data concerning OCB during disease-modifying drugs (DMD) therapy and changes of their pattern were not reported in the literature. The aim was to assess OCB in CSF of patients with relapsingremitting multiple sclerosis (RRMS) treated with DMD. Patients and Methods: Set of 22 RRMS patients consisted of 17 females (aged 26-51 years, average age 37.8) and 5 males (aged 19-44 years, average age 29.8). CSF samples were taken 0-42 (average 13.5) months before and 1-16 (average 6.2) months after initiation of DMD (interferon beta 1b, 1a or glatiramer acetate) therapy. The number of alkaline OCB in CSF was assessed by the method of isoelectric focusing. Paired samples test was applied when assessing statistical significance. Results: In 18 (81.8%) patients reduction of the number of OCB by 1-23 (average 7.8) was observed, in 2 (9.1%) patients the number of OCB increased by 1 and 3 (average 2) and no changes of the OCB number were present also in 2 (9.1%) patients. Results differed statistically significantly ( p=0.001). Conclusions: Our results document the immunomodulation effect of DMD in the decreased intrathecal synthesis of IgG.
Characterization of immunorelevant human brain antigens of multiple sclerosis patients by two-dimensional electrophoresis Z. Adwan Swaida Neuroimmunology Institution, Swaida, Syria Objective: To identify and characterize new immunorelevant antigens associated with multiple sclerosis. Background: Currently, none of the myelin-associated antigen targets conclusively discriminates between the immune response observed in multiple sclerosis (MS) patients and healthy subjects. A recent study in our laboratory found that the analysis of global IgG immune profiles to whole brain self-antigens discriminated MS subjects from healthy subjects, and could also differentiate between the three clinical forms of the disease. Indeed, respectively, 17 and 29 brain antigens, defined according to their molecular weight, were described to support a discriminant immune response. Design/Methods: The protein identification of these discriminant bands was performed by one-dimensional and two-dimensional immunoblotting using MS patient sera, followed by mass spectrometry analysis and a database search. Results: By this approach, a total of 29 different immunorelevant antigenic bands were detected. These bands were broken up in more than 40 spots, further selected for mass spectrometric analysis: 21 of them were annotated. Conclusions: Serological proteome analysis (SERPA) may constitute a new tool for the identification of new MS-associated antigens.
Leptin levels in subgroups of patients with multiple sclerosis A. Kurne, M. Arsava, B.I. Ç ikrikci and R. Karabudak Hacettepe University Medical Faculthy, Ankara, Turkey Introduction: Leptin is primarily synthesized in the adipose tissue and is responsible for the control of appetite and energy metabolism. Apart from the effects on metabolism, leptin has been shown to play an important role in inflammatory conditions. Objective: To measure serum leptin levels among different subgroups of patients with multiple sclerosis, a predominantly Th1 mediated inflammatory condition. Methods: Fourteen recently diagnosed relapsing-remitting multiple sclerosis (RRMS) patients, ten (10) RRMS patients on immunomodulatory drug therapy and thirteen primary progressive multiple sclerosis patients and age-matched nine control subjects were included in the study. Body mass index (BMI) was calculated for each patient. Serum leptin levels were measured with ELISA. Results: The mean leptin level were 2.9, 3.0, 3.1 and 3.9 ng/ml in controls, newly diagnosed RRMS, RRMS on immunomodulatory treatment and primary progressive MS patients respectively. There was no statistically significant difference among the study groups, after adjustment for height, weight and BMI values. Conclusion: Leptin is a peptide influencing immune system modulation and has a crucial role in triggering the acute inflammation. There is no sufficient data on its role in central nervous system inflammation and demyelination. In our study, no difference was detected in serum leptin levels neither between the control and MS patients, nor among the MS subgroups. The disruptiom of the blood-brain barrier, which is composed of endothelial cells, accompanied by infiltration of macrophages and T lymphocytes is implicated in the pathogenesis of multiple sclerosis (MS). Vascular-endothelial cadherin (VE-cadherin), a major protein component of interendothelial adherens junctions, has an important barrier function regulating leukocyte transendothelial migration. We determined serum levels of VE-cadherin to investigate the significance of VE-cadherin in the formation of MS lesions. The subjects were 38 patients with relapsingremitting MS and 32 healthy controls. Serum concentrations of VEcadherin were measured by ELISA method. We measured plasma activities of von Willebrand factor (vWF) and serum levels of soluble thrombomodulin (sTM) as markers for endothelial injury and plasma levels of hepatocyte growth factor (HGF) as a marker for endothelial repair in patients with MS. Serum VE-cadherin levels tended to be higher in MS patients during exacerbation than in the age-matched controls or MS patients during remission, but were not significantly different among the groups. Serum VE-cadherin levels had a tendency to be associated with plasma HGF levels in MS patients, while serum VE-cadherin levels were not significantly associated with plasma vWF activities and serum sTM levels in MS patient. The findings suggest that serum concentration of VEcadherin may increase as a reflection of the endothelial repair and adherens junction formation in the blood-brain barrier in patients with MS. Object: To evaluate the gene expression profiles of PBMC in newly diagnosed untreated MS patients compared to healthy subjects. Methods: Twenty patients with a first event of MS underwent clinical and MRI evaluation, and PBMC collection every 45 days, for 1 year. RNA was extracted from PBMC of nine MS patients with no MRI activity and ten sex and age-matched healthy subjects. Fluorescent cDNA probes were prepared by reverse transcription and hybridised to cDNA arrays of 6000 human genes prepared using a Lucidea Microarray Spotter (Amersham-Pharmacia Biotech). Array included genes involved in apoptosis, cell proliferation and inflammation. cDNA reference consisted of a mRNA pool from 10 human cell lines. After normalisation, sample-to-reference log ratios were calculated. Only spot signals above the background in at least 90% of the samples in each group were considered for statistical analysis. Results: Following RNA integrity test, probe preparation and chip hybridisation, high-quality fluorescent spots were obtained. Ten percent of evaluated genes showed differential expression upon comparison between MS and controls. Conclusions: Although statistical analysis is still in progress, our preliminary observations support the use of cDNA microarray technology for detection of altered gene expression profiles in the periphery of MS patients during active disease. Multiple sclerosis is widely believed to be an autoimmune disease driven by a relative dominance of Th1 cells over Th2 and regulatory T cells. We sought to identify lineage and activation markers of human Th subtypes that might serve as biomarkers of disease activity, diagnostic aids or guides to therapeutic efficacy. We tested murine T cell subset markers, derived from SAGE libraries, in human systems. Polarised Th1, Th2 and Tr1 PBMC cultures were produced in vitro by defined cytokine cocktails and validated by ELISA and real time PCR. The putative marker genes from SAGE included GM2 ganglioside activator protein, and integrin(E)beta(7) (CD103) for regulatory T cells (Tr1). Differentially expressed genes for Th1 were two proteins with four transmembrane domains related to the CD20 family. GATA 3 was used as a marker for Th2 cells. We found that GM2 was expressed by Tr1 cells and GATA3 was induced by PMA stimulation of Th2 and Tr1 subsets. Most marked was the specific expression on activated Th1 cells of a four transmembrane protein previously identified as Chandra. We are currently testing expression of these molecules as markers of disease activity in multiple sclerosis. Objective: To correlate MxA and IL-10 production with the response to treatment, dose of IFN-beta, and NAb occurrence. Methods: After 6 months of IFN-beta-1b 250 mcg treatment, patients with persisting disease activity were randomized to either continue on 250 mcg dose or receive an increased dose (375 mcg). MxA was determined by immunochemiluminescence in whole blood samples; IL-10 by ELISA in PBMC supernatant; NAb by the MxA-induction assay. Results: We present the preliminary data concerning 34 patients. MxA production peaked at month 3 of treatment ( pb0.001), with a greater increase in patients who responded well ( p=0.01). Also, IL-10 peaked at month 3, remaining higher than baseline only in responders ( p=0.02). The increase of the IFN-beta dose was associated with a renewed MxA and IL-10 increase ( p=0.03). NAb occurred in four patients. NAb positivity was associated with a decrease in MxA but no changes in IL-10 production. Conclusions: MxA and IL-10 levels correlated well with treatment response and IFNbeta dose.
Laser microdissection and single-cell RT-PCR analysis of IgG repertoire in SSPE brain M. Burgoon, K. Keays, G.P. Owens, A. Ritchie, X.-L. Yu and D.H. Gilden University of Colorado Health Sciences Center, Denver, CO, USA A prominent immunologic feature of subacute sclerosing panencephalitis (SSPE), a chronic fatal measles virus encephalitis, is the presence of increased IgG and oligoclonal bands (OGBs) in brain and CSF. The oligoclonal IgG is antibody directed against measles virus. To determine the fine specificity of individual immune cells in SSPE brain, and to develop techniques to analyze IgG repertoires in other chronic inflammatory CNS diseases, we microdissected individual plasma cells from SSPE brain and identified the specific IgG sequences expressed by each cell. Plasma cells were identified in frozen 7-micron sections of postmortem SSPE brain by immunostaining for CD138. Individual cells were microdissected with a nondestructive infrared laser. cDNA was synthesized and the sequences of the specific IgG heavy/light chain pairs expressed by each plasma cell were determined by nested RT-PCR amplification. A repertoire of 50-70 plasma cells from a single SSPE brain revealed features of antigen-driven selection and affinity maturation, including clonal expansion and somatic hypermutation. The variable region sequences can be inserted into expression vectors to produce large quantities of functional recombinant IgG (rIgG). Studies of specific rIgG reactivities have the potential to identify the antigens in other chronic inflammatory CNS diseases of unknown cause, such as multiple sclerosis and Behcet's disease. Neuro-Behcet's disease (n-BD) is a chronic inflammatory disease of the central nervous system (CNS). Although the exact etiology of n-BD is unknown, it is generally accepted that autoimmunity is involved and that the auto antigen(s) probably reside in CNS, the target of immune response. Current studies argue for some protein, especially Heat shock protein (HSP)-60, as an initiating or enhancing factor for Behcet's disease. In this regard, levels of HSP-60 and TNF-alpha, which is one of the important cytokine in neuroimmune disorders, in cerebrospinal fluids (CSF) with n-BD patients was examined. The CSFs were collected from n-BD patients, acute and chronic states at St. Marianna university Hospital. HSP-60 and TNF-alpha were analyzed at the message and protein levels by RT-PCR and ELISA. There was a relationship between HSP-60 and TNF-alpha in acute and chronic states CSFs obtained from n-BD patients. Thus, these results indicated that HSP-60 may be one of the initiating or enhancing factor for n-BD.
Unexplained cases of CNS disorders with evidence of Chlamydia pneumoniae DNA C. Contini a , S. Seraceni a , R. Cultrera a , M. Castellazzi a , E. Fainardi b , D. Marchetti c and E. Granieri a a University of Ferrara, Ferrara, Italy; b S. Anna Hospital, Ferrara, Italy; c Bellaria Hospital, Bologna, Italy Chlamydia (C.) pneumoniae has been linked to a number of CNS chronic diseases including MS, and to few individually described cases of neurologic disorders such as encephalitis, meningoencephalitis, Guillain-Barré syndrome and lumbosacral meningoradiculitis. Methods: Cerebral spinal fluid (CSF) and serum specimens, collected from 48 patients with undefined meningitis, or meningoencephalitis without respiratory symptoms and neurological and magnetic resonance imaging abnormalities, were retrospectively analysed by a n-PCR btouchdownQ with specific primers for C. pneumoniae 16sRNA, MOMP and HsP 70 genes. The specific IgG/IgA antibody response was evaluated by ELISA. Results: 6/48 (12.5%) patients were positive by C. pneumoniae PCR in either CSF or serum for MOMP gene (8, 17%), 16S RNA (11, 23%) and HSP-70 (5, 10%). An anti-Chlamydial IgG antibody response was found in two patients. Serological tests for bacteria and virus were negative. C. pneumoniae infection should be included in the differential diagnosis of meningoencephalitis, even if there are not associated respiratory symptoms. Of interest, the high quote of PCR positivity for Hsp-70 gene (a primary immunogen which elicits the inflammatory response during chlamydial disease) in these patients and previously demonstrated in MS disease (Contini et al., Multiple Sclerosis, in press) may be interpreted as a part of a stress response which can trigger an autoimmune reactivity. Studies to quantitate relative transcript levels of Endogenous vasoactive neuropeptides act as hormones, neurotransmitters, and immune modulators and they have neurotrophic actions. They are readily catalysed to small peptide fragments. They and their binding sites are immunogenic and are known to be associated with a range of autoimmune conditions. This theoretical paper describes a biologically plausible mechanism for chronic fatigue syndrome and sudden infant death syndrome. Most utilised anti-gliadin antibodies (Ab) using such methods and patients were considered to have gluten sensitivity (gluten ataxia) leading to dietary treatment regime. We have explored its significance amongst ILOCA patients in our populationbased study using both ELISA and an alternative immunoassay. Methods/ Materials: Sera of 84 patients (mean disease duration: 7.8 years; range: 1 to 35) were analysed using Western blotting (WB) for anti-gliadin IgA/IgG Ab, followed by quantitation using densitometer. Results of assay were compared with a group of sex-and age-matched elderly in-patients as a non-neurological control group as well as positive controls. Results: In the initial WB step, 23/84 (27%) ILOCA, 6/34 (18%) elderly and 9/12 (75%) CD sera showed probable immunoreactivity to gliadin. Further validation of these findings on quantitative densitometer revealed 8/9 CD, 2/23 ILOCA and none amongst elderly control with significant immunoreactivity. Discussion: Our data suggests that anti-gliadin Ab detection may be dependent on the assay employed. In our WB/densitometer approach, 8% ILOCA patients showed significant levels of anti-gliadin IgA/IgG Ab and may have gluten ataxia, contrary to previous reports suggesting levels up to 40% on ELISA.
A study on serum autoantibodies in post-infectious acute cerebellar ataxia A. Uchibori a , M. Sakuta a , S. Kusunoki b and A. Chiba a a Kyorin University, Tokyo, Japan; b Kinki University, Osaka, Japan Acute cerebellar ataxia (ACA) is characterised by an acute onset of selflimiting ataxia following a systemic viral infection. An autoimmune process has been suggested and some anti-neuronal antibodies were reported in ACA, but their antigen molecules have not been identified. We searched for serum antibodies in patients with ACA by Western blot (W/B) using a total protein fraction extracted from the cerebellar tissue with 2% SDS as the antigen. We found an IgM antibody that strongly reacted with a protein of approximately 28 kDa. The cerebellar cortex showed the strongest antigenicity among various nervous and non-nervous tissues based on the same amount of protein, and the strongest reaction was detected in the cytoplasmic fraction. The antigen protein was separated by 2D-electrophoresis and subjected to N-terminal amino acid sequencing. The sequence of 11 amino acid residues was shown to be identical with the N-terminal sequence of triose phosphate isomerase (TPI). Further, in 8 of 23 serum samples from patients with ACA, bands of the same mobility were detected by W/B. Anti-TPI IgM antibody titers were measured quantitatively using ELISA and the same 8 patients showed a significantly high antibody titer above mean +3SD of healthy controls (n=45), which decreased over time in patients checked in a time course evaluation. These findings suggest that the anti-TPI IgM antibody may be related to ACA. Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS) refers to a proposed subgroup of childhood onset Obsessive Compulsive Disorder (OCD) and/or Tourette Syndrome, in which onset or exacerbations of illness are triggered by Group A Streptococcal pharyngitis. These children present with sudden and dramatic symptoms of OCD, have an episodic or sawtooth course of illness, and often show subtle neurological signs such as tics, choreiform movements, sudden clumsiness and worsening of handwriting. This presentation will update current research comparing general immune function, cellular markers, and autoantibodies in children presenting with OCD/tic exacerbation to those children that are healthy. A correlation of immune findings in the PANDAS subtype will be made to those children with OCD/tics without PANDAS subtype. Increased concentrations of several inflammatory cytokines have been observed in brain tissue and cerebrospinal fluid (CSF) of several neurodegenerative diseases. They include Alzheimer disease, Down syndrome, epilepsy, Parkinson's disease, and multiple sclerosis. Whether these inflammatory molecules are causal in the neurodegenerative process or secondary to it and which is their role in neuronal survival and damage is still not known. One distinctive feature of these immune molecules is that they act in cascade stimulating and inhibiting one another production and action. A crucial question concerns the initial stimuli triggering the production of different cytokines that are simultaneously present in neurodegenerative conditions. IL-6 was measured by highly sensitive sandwich enzyme immunoassays. IL-6 was detectable in serum from control and PD patients. The concentration of IL-6 in serum from patients with PD was higher than those from control patients. These results agree with previous reports, in which the cytokine levels were elevated in the striatal dopaminergic region of the brain from patients with PD. Because cytokines play an important role as mitogens and neurotrophic factors in the brain, the increases in cytokines as a compensatory response may occur in the brain of patients PD during the progress of neurodegeneration. Increase in cytokines may contribute not only as a compensatory response but as a primary initiating trigger for the neurodegeneration.
Acute brain ischemia and inflammation. An outcome study S. Sotgiu a , B. Marchetti b , M.L. Fois a , G. Arru a , B. Zanda a , A. Pirisi a , A. Sanna a and G. Rosati a a Istituto di Clinica Neurologica, University of Sassari, Sassari, Italy; b Dipartimento di Neurofarmacologia, OASI (IRCCS), Troina, Italy
Although numerous examples of failed surrogate markers are provided in literature, metalloproteinases, adhesion molecules and IL-6 serum levels seem to predict neurological deterioration in patients after ischemic stroke. Accumulating evidences suggest however that inflammation includes both detrimental and protective components thus contributing to both neurotoxicity and neuroprotection in the central nervous system. With the aim of investigating this issue we evaluated a series of 49 consecutive patients with acute ischemic stroke, and, within 24 h from stroke onset, the serum concentrations of a panel of different biomarkers previously reported to be associated with stroke: TNF-alpha, IL-1beta, IL-6, IL-8, MCP-1, sVCAM-1, sICAM-1, MMP-1, MMP-2/9 and BDNF. Neurological impairment was scored by using GCS, GOS and NIH scales, both at stroke onset and after a three month follow-up. TNFalpha, BDNF and VCAM-1 and particularly ICAM-1 ( pb0.0001) and MMP-2/9 ( pb0.001) were associated with the clinical severity of the ischemic stroke, showing a linear correlation with the initial and final NIH scale. On the contrary, and in contrast to previous reports, IL-6 showed a significant inverse correlation ( pb0.001) being up-regulated in patients who developed a milder severity score after three months. We suggest that IL-6 may have neuroprotective actions in patients with ischemic brain injury in the context of the complex cascade of a proinflammatory response. It has been implied that inappropriate haemostasis contributes to ischemic damage in a number of issues. It is not known if the risk factors for developing vascular dementia differ from those found in stroke. Because vascular dementia (VaD) is a preventable type of dementia, the determination of risk factors for VaD is extremely important. To evaluate the role of humoral parameters in the genesis of VaD in elderly patients, we investigated a group of 30 patients with VaD and compared the results with those of 30 cerebrovascular non-demented patients (VaND) and 30 healthy controls subjects without cerebral ischemic episodes. A significant increase of Hcy, Fg, CRP, slCAM-I, SvCAM-I, IL-Ib, IL-6 and IL-10 was observed in VaD patients compared to that found in cerebrovascular ischemic patients and controls, respectively (56.5F22.4 mmol/l, 415F68.5 mg/dl, 6.7F2.55 mg/l, 315F75 mg/l, 975F125 mg/l, 2.05F1.15 pg/ml, 1.75F1.25 pg/ml and 1.45F1.05 pg/ml vs 26.5F12.5 mmol/l, 385F70.5 mg/dl, 3.7F2.35 mg/l, 275F75 mg/l, 635F105 mg/l, 1.50F1.35 pg/ml, 1.25F1.05 pg/ml and 1.15F1.11 pg/ml vs 7.5F3.5 mmol/l, 224F64.5 mg/ dl, 0.85F0.60 mg/l, 157F39 mg/l, 637F99 mg/l, 1.04F0.65 pg/ml, 0.75F0.55 pg/ml and 0.45F0.60 pg/ml respectively; pb0.001 Mann Whitney U-test). Our findings demonstrate that Hcy and humoral inflammatory factors are very elevated in individuals suffering from VaD after ischemic stroke. Our study confirm that an increased systemic levels of proinflammatory and immunoregulating cytokines may be found in patients with VaD.
Prognostic significance of antibodies to acetylcholine receptor (AchR) in myasthenia gravis (MG) G. Galassi, P. Faglioni and A. Ariatti
Objective: to evaluate significance of antibodies to AchR in MG. Methods: A retrospective study was conducted on 67 MG patients (mean age 63.9 years): 30 males, mean age 59.1, 37 females, mean age 59.6. Mean duration of illness was 66.9 months. At time of diagnosis,19 patients were AchR antibody negative whereas 48 patients had detectable antibodies to AchR. Disease severity (Osserman's classification) was graded I in 11 AchR antibody positive and in 13 seronegative, IIa in 21 seropositive and in 6 seronegative, IIb in 16 seropositive patients. No subjects were classified as III and IV. With regard to actual severity, among 48 patients AchR antibody positive 7 were asymptomatic; 11 were graded I, 23 IIa and 7 II b. Among 19 seronegative subjects, 14 were classified I and 5 IIa. Degrees of improvement was computed in AchR antibody positive (N=48) and in seronegative (N=19). Results:1). At time of diagnosis, percentages of AchR antibody positive patients were significantly higher among subjects IIa (78%) and IIb (100%) as compared to those graded I (46%), Chi square(2)=14.70; exact P=0.0004. 2). Percentages of AchR antibody positive patients was significantly higher among patients who showed clinical improvement (94%) as compared to those either unchanged or worse, Chi square(1)=4.279; exact pb0.03. 3). Degrees of improvement between baseline and actual severity did not differ significantly between AchR antibody positive and seronegative subjects; Chi square (2)b1.
Immunosuppressive treatment modifies both the number and phenotype of circulating but not of intrathymic CD4+CD25+ T regulatory cells in myasthenia gravis A. Fattorossi, A. Battaglia, F. Ciaraffa, C. Di Schino, G. Scambia and A. Evoli Catholic University, Rome, Italy
We analyzed immunosuppressive CD4+CD25+ T (Treg) cells in 42 MG patients. The number of Treg cells in the peripheral blood of untreated patients was significantly lower than in healthy subjects (HS) and increased after immunosuppressive (IS) therapy. IS therapy-induced Treg cells expressed less HLA-DR which is indicative of an expansion of newly generated Treg cells. The increase in Treg cell number following IS therapy was lowered but not suppressed by thymectomy, suggesting that the thymus contributes to the peripheral pool of these cells. The number of circulating Treg cells did not differ in patients with thymomatous and nonthymomatous thymuses, but the enhancing effect of IS therapy was considerably more prominent in patients with a non-neoplastic thymus. This suggests that normal thymic epithelium can be relevant in maintaining peripheral Treg cell homeostasis. Thymus function, however, appeared dispensable in governing the peripheral pool of Treg cells, as thymectomized patients had a number of circulating Treg cells within the normal range. Intrathymic Treg cell frequency was not related to thymus histology and was not influenced by IS therapy. Moreover, there was no correlation between intrathymic and circulating Treg cell frequencies.
Collectively, these findings are consistent with the possibility that Treg cell deficiency has a role in MG development and that up-modulation of these cells contributes to the therapeutic effect of IS treatment. (veab) , and 2 main clinical presentations: ocular (oMG) and generalised (gMG). Objectives. To compare age/sex distributions of all MG variants by onset of symptoms rather than ab positivity, and determine the age specific incidence of AChR ab+ve MG. Methods. We studied 141 patients with clinically validated gMG or oMG. All were tested for AChR and, if negative, MuSK abs. Results. 58% had AChR abs (gMG [75%], 24m 36f; oMG [25%], 12m 8f), 42% were ab-ve (gMG [36%], 1m 20f; oMG [64%] 15m 24f), and 1 gMG man was MuSK ab+ve. Here, age/sex distribution and specific incidence for all AChR ab+ve MG increased with age with two main peaks at the fourth and ninth decades caused respectively by younger gMG women and older oMG men. Ab-ve gMG started in women at all ages. Ab-ve oMG increased steadily to peak in the eighth decade and affected both sexes. Conclusions. Ab -ve MG is probably a pathogenetically-heterogeneous disorder and our data suggests it is relatively common. It presents as generalized weakness in women of all ages and ocular weakness in older women and men. Our AChRab+ve MG data is similar and representative to those reported for AChR ab positivity in the UK. To clarify the distinct immune balance in the cerebrospinal fluid (CSF) compartment, we investigated intra-and extracellular levels of various cytokines and chemokines in CSF in chronic inflammatory demyelinating polyneuropathy (CIDP) and vasculitic neuropathy (VN). Sixteen cytokines, IL-1h, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17, IFN-gamma, TNF-alpha, G-CSF, MCP-1 and MIP-1beta, in CSF supernatant were measured by a multiplexed fluorescent-bead based immunoassay; and intracellular cytokine production of IFN-gamma and IL-4 in CSF CD4+ T cells by flowcytometry, in 14 patients with CIDP, 8 patients with VN and 24 other non-inflammatory neurologic diseases (OND). In the CSF supernatant, significant increases of IL-8 and IL-17 and significant decreases of IL-4, IL-5, IL-6 and IL-7 were detected in pretreated CIDP as compared with OND; whereas significant increases of IL-6, IL-8 and IL-10 were found in pretreated VN. In CSF CD4+ T cells, IFN-gamma+ IL-4cell percentages were markedly elevated in CIDP compared with OND, but not in VN, resulting in a significant increase of intracellular IFN-gamma/IL-4 ratio in CIDP even in the absence of CSF pleocytosis. Non-responders to intravenous immunoglobulins showed significantly lower IFN-gamma-IL-4+ CD4+ T cell percentages and tended to have higher intracellular IFNgamma/IL-4 ratios in CSF than responders. Upregulation of Th1 and downregulation of Th2 cytokines are pathognomonic for CIDP, while some Th2 cytokines are upregulated in CSF in VN.
Allergic bronchial asthma induced in NGF-antibody producing rats: evidence for a non pro-inflammatory role of NGF B. Stampachiacchiere, A. Micera, S. Bonini and L. Aloe National Research Council (CNR), Rome, Italy Both in animal models and humans allergic bronchial asthma (ABA) can increase Nerve growth factor (NGF) levels in the blood serum and bronchial alveolar lavage (BALF). The NGF inflammatory role in ABA is not clear and matter of discussion. In the present study, we used an animal model of ABA with and without circulating levels of NGF antibodies (Ab-NGF) to investigate the role of NGF in neuroinflammatory disorders. ABA was induced in adult female rats by ovaalbumina (OVA) immunization as previously described. Rats with circulating Ab-NGF were obtained by injecting purified NGF in Freund's adjuvant. NGF, TNF-alpha and IgE were measured in the serum, BALF and lung tissues, using ELISA kits. OVA treated rats revealed significant increase in IgE, TNF-alpha levels, mast cells in the lung and eosinophils in both BALF and lung tissues. These animals showed also an increase in circulating NGF in the serum and BALF. NGF administration in normal adult rats did not lead to an increase in eosinophils and IgE in the BALF and lung of allergic rats. This findings suggest that NGF does not stimulate ABA or inflammatory response and that the increase in NGF during allergic responses may be associated with regulation of neuroallergic and/or neuroimmuno protective action. The study's aim was to evaluate whether serum levels of the matrix metalloproteinase MMP-9 and its inhibitor TIMP-1 correlate with symptomatic versus asymptomatic carotid stenosis, and restenosis following endarterectomy. Serum from patients (n=48; 26 symptomatic) with severe carotid stenosis, confirmed by duplex, was collected pre endarterectomy, and a year post-surgery. CRP, MMP-9, and TIMP-1 levels were measured. Pre endarterectomy levels of CRP, MMP-9, and TIMP-1 were similar in symptomatic and a-symptomatic patients. CRP levels did not change following surgery. Overall, MMP 9 levels decreased (-26.5%) a year post surgery, and TIMP 1 levels increased (+9.8%). When patients were stratified according to presence or absence of concomitant ischemic heart disease (IHD) a significant decrease in MMP 9 levels was observed (33.6%) only in the IHD-free group. In asymptomatic patients, a trend for a decline in MMP-9 levels (-50%) was observed, and in parallel, a significant increase in TIMP-1 (+14%). Similar changes were observed in symptomatic patients, but failing to reach statistical significance. In eleven patients that developed restenosis, MMP-9 was significantly decreased (-50%), while in the no-restenosis group a significant increase in TIMP-1 levels was observed (+9.9%). The results of our study indicate that a year post-carotid endarterectomy, the MMP 9/TIMP 1 ratio is reduced. Further studies are required to support the use of these enzymes as bio-markers of atherosclerotic activity and impending stroke. Vesicle shedding mediates IL1-beta release from ATP stimulated microglia cells F. Bianco a , E. Pravettoni a , U. Schenk a , M. Matteoli b and C. Verderio a a CNR-Institute of Neuroscience, Milan, Italy; b University of Milan, Milan, Italy ATP has been indicated as a primary factor in microglial response to brain injury and inflammation. By means of different P2 receptors, ATP is known to induce chemotaxis and stimulate the release of several cytokines from these cells. The activation of P2 receptors in microglia can be triggered either by ATP deriving from dying cells, at sites of brain injury (Ferrari et al. 1997a) , or by ATP released from astrocytes to mediate cell-to-cell signaling, in the absence of cell damage (Verderio and Matteoli, 2001; Shipke et al., 2002) . By the use of a biochemical approach integrated with videomicroscopy experiments, we investigated the functional consequence triggered in microglia by ATP released from mechanically stimulated astrocytes, in mixed glial co-cultures. Astrocyte-derived ATP induced in nearby microglia the formation and the shedding of membrane vesicles. Vesicle formation was inhibited by the ATP-degrading enzyme apyrase or by P2X7 receptor antagonists. Shed vesicle isolation, followed by IL-1beta evaluation by a specific ELISA assay revealed the presence of the cytokine inside the vesicular organelles and its subsequent efflux into the extracellular medium. IL-1beta efflux from shed vesicles was enhanced by ATP stimulation and inhibited by pre-treatment with the P2X7 antagonist oATP, thus indicating a crucial involvement of the pore-forming P2X7 receptor in the release of the cytokine. Toll-like receptors (TLRs) have evolved as conserved pattern-recognition receptors that are central to innate immunity. Signaling through TLRs may also regulate endogenous responses to injury. In the central nervous system (CNS), microglia respond rapidly to axonal injury. Transection of axons in the entorhinal cortex causes anterograde axonal degeneration and induces microglial reactivity in the hippocampus, accompanied by local cytokine and chemokine production and later macrophage and T cell infiltration. TLR2 and the LPS-receptor CD14 were induced in the lesionreactive hippocampus within 3 h of injury, coincident with glial cytokine and chemokine responses, but before detectable leukocyte infiltration. TLR1 and TLR4 were not significantly induced until 24 h. Induction of MyD88 and IkBa transcripts suggested signal transduction via TLRs in reactive glial cells. Macrophage and T cell infiltration were reduced in MyD88-deficient mice. T cell infiltration was delayed following axonal lesions in TLR2-deficient mice. The microglial expansion that occurs 5 days post-lesion was reduced in TLR2-deficient mice, although macrophage influx was unaffected. No inhibition of leukocyte infiltration or microglial expansion was observed following axonal lesions in TLR4deficient mice. Our findings suggest that induction of and signaling through TLRs is an early innate response to CNS axonal injury that selectively regulates later cellular responses implicated in regeneration and repair. In contrast to other tissues, the central nervous system (CNS) is essentially devoid of MHC expression and shielded from antibodies by the blood-brain barrier. Therefore, a rapid local innate immune response by resident brain cells is required to effectively fight infectious agents. The recognition of microbial infections is mediated by invariant receptors called Toll-like receptors (TLR). Upon ligand binding, immune cells mount a direct response to the pathogen and additionally activate the adaptive immune system. This study analyzed the expression of the ten TLRs by quantitative PCR in human astrocytes. Only TLR3, the receptor responsible for the recognition of viral double-stranded RNA, had a constitutive expression. Its levels could be enhanced by IFN-gamma, IL-1beta and IFN-beta. The other TLRs were barely detectable and inducible. The TLR3 ligand poly (I: C) triggered production of the cytokines IL-6 and TNF-alpha, and of the chemokines CCL5/RANTES, CXCL10/IP-10, CCL20/MIP-3alpha and CCL2/MCP1. The TLR adapter molecules MyD88 (full length and short isoform), TIRAP/Mal, and TICAM-1/TRIF were expressed by astrocytes. In summary, human astrocytes may sense viral infections and respond with a proinflammatory activation program that will be amplified by the recruitment of immune cells into the tissue. Hydroxychloroquine (HQ), an anti-malarial agent, is used as an immunomodulator in lupus and rheumatoid arthritis. It interferes with antigen processing and inhibits cytokine production by peripheral blood mononuclear cells. We studied the effect of HQ on cultured microglia, the activation of which is thought to be crucial to the pathogenesis of multiple sclerosis (MS), and on the course of mice afflicted with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Human fetal and adult microglia were pretreated with HQ followed by lipopolysaccharide (LPS), a monocytoid activator. The overnight culture medium was then collected and subjected to cytokine measurement using ELISA. HQ (3-15 microM) significantly decreased TNF-alpha and IL-10 levels and was superior to minocycline, a known microglia modulator, in inhibiting TNF-alpha production; a pretreatment period was more effective than without. Using U937 cells, the effect of HQ on transcripts encoding matrix-metalloproteinases (MMPs) was evaluated. HQ reduced the mRNA for MMP-1, 9 and 12, which are incriminated in the pathogenesis of MS and EAE. Finally, we tested HQ in monophasic EAE and found that 100 mg/kg given as a pretreatment completely prevented the development of disease; this was correlated with lack of signs of microglia activation in treated animals. We conclude that by interfering with microglia activity and MMPs, HQ deserves to be tested further as a disease modifying therapy in EAE and MS.
Dept. of Pharmacology, University of Washington, Seattle, USA Cells produce endocannabinoids that activate cannabinoid receptors, the molecular target for marijuana's bioactive ingredient 9-tetrahydrocannabinol. The molecular mechanism underlying the production of the most abundant endocannabinoid, 2-arachidonoylglycerol (2-AG), is unclear. Here we show that ATP increases 2-AG production by mouse microglial cells in culture, a response mediated by phosphatidylinositol-specific phospholipase C (PI-PLC) and diacylglycerol (DG) lipase. However, efficacious activation of metabotropic P2Y purinergic receptors coupled to PI-PLC does not increase 2-AG production. This suggests that ionotropic, and not metabotropic, purinergic receptors control 2-AG production at an unexpected enzymatic step of its metabolic pathway. We show that activation of P2X7 ionotropic receptors, which are highly permeable to calcium, is necessary and sufficient to increase 2-AG production in microglial cells. We also show that the sustained rise in intracellular calcium induced by activation of P2X7 receptors directly increases DG-lipase activity while inhibiting the activity of monoacylglycerol (MG) lipase, the enzyme that degrades 2-AG. This inverse sensitivity of DG-lipase and MG-lipase to calcium constitutes an original and efficient modality for sustained accumulation of 2-AG. Finally, overnight activation of microglial cell with interferon-gamma disrupts the ATP-induced 2-AG production. Because prolonged increases in 2-AG amounts in brain parenchyma are thought to orchestrate neuroinflammation, the enzymatic steps involved in 2-AG synthesis and degradation by microglial cells constitute appealing targets for therapy aimed at controlling exacerbated neuroinflammation. Nicotinic acetylcholine receptors are a family of ligand-gated pentameric ion channels whose main function is to transmit signals for the neurotransmitter acetylcholine in the peripheral and central nervous system. Recently, the nicotinic acetylcholine receptor alpha7 subunit (alpha7) was found in peripheral macrophages. Its interaction with specific agonists strongly reduced the release of the cytokines IL-1beta and TNF-alpha. Epidemiological studies showed that nicotine may be protective in some neurodegenerative pathologies, such as Alzheimer and Parkinson diseases, in which chronic inflammation, sustained by microglial cells, plays a crucial role. In the present study, we investigated the expression of alpha7 and its functional role in purified rat microglial cultures. RT-PCR analysis revealed the presence of alpha7 mRNA in microglial cells. Strong binding on microglial surface of FITC-alpha-bungarotoxin, which specifically binds to alpha 7 subunits, confirmed the presence of the receptor. Nicotine slightly reduced the release of nitric oxide and IL-10 by LPS activated microglia, whereas COX-2 mRNA and synthesis of PGE2, a lipid mediator with potential anti-inflammatory effects, were enhanced. Our study is in line with the hypothesis of an anti-inflammatory effect of nicotine and suggests that the development of molecules able to stimulate the alpha7 receptor represent potential therapeutic tools for the treatment of several inflammatory neuropathologies. Supported by ISS, grant n. C3A4.
Adaptive immunity instructs microglia to protect against aggregated Bamyloid and glutamate toxicity O. Butovsky, E.A. Talpalar, K. Ben-Yaakov and M. Schwartz Weizmann Institute of Science, Revohot, Israel bProtective autoimmunityQ refers to the well-controlled T-cell-mediated anti-self response that helps the body resist neurodegenerative disorders. We show that this response, through the cytokines interferon (IFN)-gamma (Th1) and interleukin (IL)-4 (Th2), renders microglia protective. Relative to microglial activation by IFN-gamma, IL-4-elicited activation was delayed and was effective over a wider concentration range. Microglia responded to disease-associated self-compounds (aggregated beta-amyloid, glutamate) as aggressively as if they were bacteria (e.g., bacterial cell wall-derived lipopolysaccharide). Their cytotoxicity down-regulated MHC-II expression through the MHC class-II transactivator and the invariant chain. Partial protection was achieved by down-regulation of TNF-alpha and upregulation of insulin-like growth factor (IGF-I) induced by IL-4. These findings suggest that whether the local immune response in the damaged CNS will be beneficial or harmful depends on how the microglia interpret the threat, and that a well-regulated T-cell-mediated response serves as a self-contained mechanism of repair. Microglia are the resident tissue macrophage of the central nervous system (CNS). Their activation is an early and common feature of nearly all CNS neuropathologies. We and others have demonstrated that microglia are phenotypically and functionally distinct from other tissue and CNS-infiltrating macrophage populations. Here, we sought to define microglial phenotypes on a molecular level. Using an open profiling method (TOGA) to screen gene expression, we have detected over 19,000 novel and known molecules expressed in microglia (adult and neonatal), several populations of macrophages and dendritic cells. From these studies, we have identified patterns of gene expression that 1) distinguish microglia from other myeloid populations, 2) reveal microglia are highly heterogeneous in the healthy CNS, and 3) indicate microglial responses to pathologic stimuli are heterogeneous even within a single pathology. We are currently examining to what extent these multiple microglial phenotypes are a consequence of different lineages versus different environmental cues.
Oligodendrocyte recruitment in multiple sclerosis (MS): a role for CXC chemokines K.M. Omari a , G. John b and C.S. Raine a a Albert Einstein College of Medicine, NY, USA; b Mount Sinai School of Medicine, NY, USA Subsequent to demyelination in MS, oligodendrocytes initially retain the capacity to remyelinate but this diminishes as lesions age. To further elucidate factors affecting oligodendrocyte recruitment, we investigated expression of CXC chemokines and their receptors in MS lesions. Chemokines were detected by immunohistochemistry and western blotting with antibodies against CXCL1, CXCL8 and CXCL10, and their receptors, CXCR1, CXCR2 and CXCR3. CXCL1, CXCL8 and CXCL10 were found to be strongly induced on hypertrophic astrocytes at the edge of active MS lesions, weakly expressed in silent lesions, and absent in controls. Interestingly, specific expression of the corresponding receptors was found on oligodendrocytes in both MS and non-MS tissue. Human astrocytes and oligodendrocytes were also studied in vitro. Chemokines were measured by sandwich ELISAs in astrocyte supernatants after stimulation, RNA by northern blots and quantitative-PCR, and chemokine receptors by immunofluorescence. At protein and RNA levels, IL-1beta strongly induced CXCL1 and CXCL8 by astrocytes, while IL-1beta and IFN-gamma induced CXCL10. Astrocytes in culture media alone produced limited amounts of CXC chemokines. Moreover, immature (A2B5+) and more mature (CNPase+) oligodendrocytes displayed surface expression of CXCR1, CXCR2 and CXCR3. Based on the concurrent expression of these chemokines on hypertrophic astrocytes, and their receptors on oligodendrocytes at lesion margins, we propose that CXC chemokines play an important role in recruitment of oligodendrocytes to lesions in MS.
ATP regulates oligodendrocyte progenitor migration and proliferation via P2Y receptor activation C. Agresti a , M.E. Meomartini b , S. Amadio b , E. Ambrosini a , B. Serafini a , C. Volonté b , S. Visentin a and F. Aloisi a a Istituto Superiore di Sanità , Rome, Italy; b CNR-Fondazione Santa Lucia, Rome, Italy ATP released in high amounts during inflammation may influence cell function as well as damage and repair processes via the activation of ionotropic (P2X) and metabotropic (P2Y) receptors. To better understand the consequences of purinergic receptor stimulation in oligodendrocytes, we have investigated the expression of P2X and P2Y receptor subtypes and their functional activity in oligodendrocyte progenitors (OPs) purified from rat brain cultures. Using western blot analysis, we demonstrate that OPs express several P2X and P2Y receptor subtypes. Analysis of Ca2+ transients induced by P2 agonists and by the specific antagonists oxATP and MRS2179 revealed that Ca2+ signalling in OPs is mediated mainly by P2X7 and P2Y1 receptor activation. As a functional correlate of these findings, we show that ATP, ADP and the more selective P2Y1 receptor agonist ADPbetaS, but not UTP, induce OP migration. Moreover, ATP and ADP, but not UTP, are able to inhibit OP proliferation induced by plateletderived growth factor and to promote oligodendrocyte differentiation. The effects of ATP and ADP on OP migration and proliferation are prevented by the P2Y1 antagonist MRS2179. By confocal laser scanning microscopy, we also show that P2Y1 receptors are expressed in NG2+ OPs in the developing rat brain. Our results suggest that ATP, by regulating OP mobilization and differentiation, could affect remyelination in inflammatory demyelinating diseases of the central nervous system like multiple sclerosis.
There is interest in Schwann cells (SC) as a possible source of myelinating cells for transplantation into the CNS of patients with multiple sclerosis (MS) and spinal cord injury. Migration of SC into the CNS is limited, but even when injected directly CNS glia may interfere with SC survival and maturation. To investigate the effects of CNS glia on SC, we cultured neonatal rat SC and exposed them to serum free supernatants (Sup) obtained from rat mixed glial cultures. Sup from 1 and 3 day glial cultures induced proliferation of SC assayed at 5 days in a concentration dependent manner. Sup did not induce SC differentiation as measured by induction of surface expression of galactolipids (GalL) as assessed by indirect immunofluorescence (IF) with a monoclonal antibody to GalL. Sup did not inhibit cyclic AMP (cAMP)-induction of SC differentiation. Sup had no apparent effect on SC viability at 48 h. We previously demonstrated that incubation of SC with transforming growth factor-beta (TGF-beta) + tumor necrosis factor-alpha (TNF-alpha) induces SC death via apoptosis. Sup inhibited TGF-beta+TNF-alpha -induced SC death. Our data suggest that soluble products of CNS glia have the potential to alter SC functions and may effect the outcome of SC transplantation or migration into the CNS.
The chemokines SDF-1 alpha and IL-8 stimulate oligodendrocyte precursor proliferation and enhance myelin formation in vitro L. Kadi a , R. Selvaraju a , P. De Lys a , A. Proudfoot a , T. Wells b , Y. Chvatchko a and U. Boschert a a Serono Pharmaceutical Research Institute, Geneva, Switzerland; b Serono International SA, Geneva, Switzerland
Chemokines are a group of cytokines involved in immune system signaling, which have recently been postulated to regulate central nervous system (CNS) development and function. In particular, chemokines may play a role in regulating oligodendrocytes, the cells myelinating axons within the CNS. Here we have chosen to study the role of several chemokines in mediating myelination, the process that leads to axonal ensheathment. We have used a mouse oligodendrocyte precursor-like cell line, Oli-neu, to assess the activities of chemokines on oligodendrocyte development. The recombinant human chemokines GRO-alpha, IL-8, and SDF-1alpha dose-dependently increased proliferation of mouse Oli-neu cells, while MCP-1 did not. Oli-neu cells express CXCR4, the receptor for SDF-1alpha, and SDF-1alpha-mediated induction of proliferation could be inhibited by addition of an anti-CXCR4 blocking antibody. The activities of GRO-alpha, IL-8, and SDF-1alpha were also studied using primary mixed cortical cultures from embryonic mouse brain, an in vitro model of myelination. Treatment with these chemokines stimulated myelin basic protein (MBP) synthesis in a dose-dependent manner, and also enhanced myelin segment formation, visualized via anti-MBP immunocytochemistry. In keeping with the results obtained using Oli-neu cells, these chemokines were shown to stimulate proliferation of various cell types in mixed cortical cultures. These cultures were shown to contain a sub-population of cells expressing both the CXCR4 receptor and markers of oligodendrocyte precursor cells (OPCs) such as platelet-derived growth factor receptor alpha (PDGFaR).
Several clinical and experimental lines of evidence indicate that glycolipids (GL) may serve as autoantigens in demyelinating autoimmune disease of the peripheral nervous system. In multiple sclerosis (MS), both humoral and cellular immune mechanisms directed to GL have been suggested to be play a pathogenetic role; however, the cellular distribution of GL on glial cells in situ has not been clearly assessed, thus hampering pathogenetic correlations with autoimmune responses. The aim of this study was to assess the pattern of expression in situ of GL on glial cells in adult normal central nervous system (CNS) and in chronic MS lesions, focusing on mature oligodendrocytes and their precursors (OP). We performed double staining with specific glial cell markers and mAbs for GM1, GM2, GD2, GD3, GD1a, GD1b, A2B5 and GT1b on frozen sections. In normal conditions, most GL were preferentially expressed on astrocytes, with the exception of GD2 being present on oligodendrocytes and GD1a on OP. In MS lesions, most gangliosides were diffusely down-regulated on astrocytes and upregulated on NG2-positive OP. In particular, at the edge of MS lesions, OP showed high levels of expression with mAb A2B5 and for GD1a and GT1b, while GD2 and GD3 were present on oligodendrocytes and myelin in the surrounding white matter. These results may provide interesting insights on the pathogenic role of GL as autoantigens in MS and in CNS autoimmune disease. Sonic Hedgehog (SHH) and downstream molecules Olig1 and Olig2 may be dysregulated in MS, a disease in which selective depletion of oligodendroglia occurs. In this study, MS lesions at different stages and CNS tissue from normal and disease controls were evaluated for the expression of SHH, its receptor Patched (PTC), Olig1, and Olig2 by immunocytochemistry and Western blot. Oligodendrocytes at the edge of chronic active MS lesions were intensely immunoreactive for SHH and PTC. Westerns confirmed increased SHH and PTC levels in expanding, and decreased levels in silent MS. In contrast, Olig1 stained oligodendrocytes in normal-appearing white matter (NAWM), and cells with both astrocytic and oligodendroglial features at the active lesion edge. Westerns showed no difference among the groups. Olig2 selectively stained oligodendrocytes in all tissues examined. In active MS, a gradient in immunoreactivity was seen, with oligodendrocytes in NAWM staining most intensely, and those near the lesion edge, less intensely. Interestingly, Westerns revealed markedly increased Olig2 in chronic active MS. Upregulation of SHH and Olig2 in oligodendrocytes around chronic active lesions suggests these genes to be active during ongoing demyelination. Downregulation of SHH and Olig2 may explain progression from chronic active to chronic silent lesions. (Supported by NS 07098 and NMSS CA 1022-A-1).
Adult human oligodendrocytes proliferate in long-term culture A. Boullerne a , D. Frim b and B. Arnason c a Dept. Neurology, University of Chicago, Chicago, USA; b Section of Neurosurgery and the Brain Research Institute, University of Chicago, USA; c Dept. Neurology and the Brain Research Institute, University of Chicago, Chicago, USA Human oligodendrocytes (OLG) were isolated from resection specimens of epilepsy patients and maintained in primary culture to assess their proliferative capacities in various culture conditions. Immunostaining of cell preparations showed that all surviving cells were of OLG lineage. At the time of isolation, staining for the OLG progenitor marker NG2 showed between 5% and 20% progenitor cells. Co-labeling with the microglia marker CD11b was negative confirming that the NG2 labeled cells were OLG. At several time points over two months in culture, continuous evidence of cell division by bromodeoxyuridine incorporation along with an increased cell density was documented by confocal imaging. Proliferative capacity of OLG from adult brains was found to be dependent upon a low insulin concentration (15 Ag/ml) inhibitory at 50 Ag/ml insulin despite the presence of growth factors. The maximum proliferation rate of OLG progenitors was 9%. OLG maintained on laminin, an extracellular matrix protein of axons in the central nervous system, retained their proliferative capacity. These findings are relevant for the development of therapeutic strategies to promote remyelination in human central nervous system degenerative diseases.
Lymphocytes are present within ethidium-bromide (EB)-demyelinating lesions in the Central Nervous System (CNS) and the possibility of their participation in possible immune-mediated responses to the detached myelin sheaths can not be ruled out. This study aimed to investigate the consequences of lymphocytic interference with the immunosuppressive agents cyclophosphamide (Cy) and cyclosporine (CsA) in CNS repair after local EB injection. Adult Wistar rats received 10 microlitres of 0.1% EB solution into the cisterna pontis. Some were treated intraperitoneally with Cy (50 and 30 mg/kg, twice a week, group I) or Csa (10 mg/kg/day, group II) during the experimental period; others were not immunosuppressed (group III). Three animals from each group were perfused with 4% glutaraldehyde at 7,11,15,21 and 31 days following EB injection. Brainstem slices were collected and processed for transmission electron microscopy studies. Rats from group I showed greater amounts of myelin-derived membranes than non-immunosuppressed rats, suggesting a delay in the macrophage activity of removing myelin debris. Rare lymphocytes were found. Oligodendrocyte remyelinating activity also showed a delayed pattern, with clear predominance of naked axons. Although neovascularization was decreased, astrocytic reaction did not appear to be affected. In rats from group II, the most important finding was the presence of a higher density of oligodendrocytes near remyelinated axons at the periphery. Lymphocytes were still present. Results from group II suggest that IL-2 suppression by CsA had a proliferative effect on oligodendrocyte progenitors. Tumour necrosis factor (TNF) is a major immunomodulatory proinflammatory cytokine that's been identified as having a pathogenic role in a number of inflammatory diseases within the central nervous system including multiple sclerosis and the animal model experimental autoimmune encephalomyelitis (EAE). TNF is synthesised as a membrane bound protein that is cleaved into a soluble form. The proteinase responsible for this cleavage has been identified as TNFa-converting enzyme (TACE or ADAM-17). We've shown ADAM-17 to be expressed by both the endothelial and astrocyte cells in white matter of normal human control brain. Here we report the distribution and level of ADAM-17 expression within spinal cord white matter of naRve Lewis rats as well as in rats with EAE assessed by immunocytochemistry, western blotting and realtime RT-PCR (qRT-PCR). ADAM-17 is associated with the blood vessel endothelium in the naRve and pre-disease spinal cords, whereas there is an abundance of astrocytic and inflammatory cells expressing ADAM-17 at peak-disease and in recovery. This apparent up-regulation of ADAM-17 protein is coupled with a four-fold decrease in mRNA levels of its natural inhibitor TIMP3 (tissue inhibitor of metalloproteinase 3) as determined by qRT-PCR.
The Coxsackie Adenovirus receptor expressed by ependymal cells is a key component of the brain stem cell niche M. Hauwel, C. Canova and P. Gasque
In the adult vertebrate brain, the subventricular zone (SVZ) is one of the rare neurogenesis areas. Progenitors from the SVZ actively proliferate to generate neuronal precursors that migrate to the olfactory bulb and to sites of injury. The ependymal cells, which cover the inside of the ventricles, are in direct contact with neural stem cells (NSCs). Interestingly they express specifically the Coxsackie-Adenovirus Receptor (CAR), a 46 kDa transmembrane protein member of the immunoglobulin super family whose function remains unknown. CAR is abundantly expressed in the brain during embryogenesis but is restricted to ependymal cells in the adult brain. We found that CAR immunoreactivity was significantly modified following a broad range of brain infarcts. Mass spectrometry analysis and co-immunoprecipitation of CAR from new born SVZ cell lysate revealed several associated proteins which may be involved in stem cell differentiation. Furthermore, the proliferation of NSCs in vitro was modified by the presence of CAR in the substrate. In conclusion, CAR is expressed by ependymal cells in the SVZ as an element of regulation of the NSCs activation. Ependymal cells create a micro-environment in the SVZ maintaining the pluripotentiality of NSCs and controlling their proliferation. Objective: To investigate the mechanisms of human NSC (hNSCs) migration to sites of CNS injury. Methods: Migration studies were performed in Boyden chambers. For in vivo studies, hNSCs were labeled with fluorescent cell tracker and injected in the ventricle of stroke animals that were euthanized 2-10 weeks later. 3D Confocal reconstructions were used to correlate the migration of hNSCs to foci of pathology with expression of SDF-1. In vitro, we measured the directional development of chain migration from neurospheres in the presence of ischemic and normal rat brain organotypical explant and with anti-CXCR4 antibody. Results: hNSCs proliferate and migrate incrementally to in vitro addition of hSDF-1alpha ( pb0.001) and express cxcr4 mRNA and protein. Exposure of hNSCs to hSDF-1alpha triggers phosphorylation of p38MAPK, p90RSK, ERK, IkappaB and Paxilin. In vivo, hNSCs implanted in periventricular areas migrated towards the stroke regions, where astrocytes and endothelium upregulate SDF-1alpha (r=+0.6079, pb0.0001). In vitro, neurospheres elaborate chains of nestin+cells with no apparent directionality. When co-cultured with ischemic brain explants, 80% of the neurospheres elaborated, robust migratory chains of nestin+NSCs directed towards and into the explant compared with only 12% of the neurospheres co-cultured with non-ischemic explant and this directionality was inhibited by blockade of CXCR4. Conclusions: Our data implicate SDF-1/CXCR4 as a chemoattractant pathway for mammalian neural stem cells migration and provide new insights into the signals that activate neural stem cells molecular programs. Bone marrow transfer experiments have shown that microglia is constantly renewed by precursor cells belonging to the hematopoietic system. However, the exact identity of such precursors remains enigmatic. We developed a culture system allowing the in vitro generation of microgliallike cells from bone marrow-derived myeloid precursors expanded in the presence of M-CSF. We found that a subset of CD34+/CD11b+/MHC class II-cells give rise to microglial-like cells in the presence of glial cell conditioned media. The differentiated cells present with phenotypical and morphological features of microglia as assessed by flow cytometry and scanning electron microscopy. Interestingly, we observed that microglia derived from mixed glial cell cultures or from bone marrow myeloid precursors express CD34 (5% to 10% of cells) and the neural stem cell marker nestin (20% to 30% of cells). Using a model of intracerebral viral infection with the canine distemper virus (CDV), we found that bone marrow derived microglial precursors are expanded in the bone marrow of infected versus control animals. Moreover when these microglial precursors were labelled with CFSE and injected i.v. to CDV infected mice, they could be detected as isolated cells in brain parenchyma.
TREM-2 in the central nervous system C. Buonsanti, M. Mariani, F. Benigni, P. Di Lucia, S. Smiroldo, L. Adorini and P. Panina-Bordignon Bioxell SpA, Milan, Italy TREM-2 is a cell surface molecule of the immunoglobulin superfamily that is expressed on monocyte-derived dendritic cells (DC) in association with DAP12. TREM-2 cross-linking promotes DC migration and activation in vitro, suggesting that it may have a role in antigen presentation and T cell stimulation in vivo. A genetic defect of human TREM-2 results in a rare genetic syndrome, the Nasu-Hakola disease, characterized by pre-senile dementia and bone cysts. Furthermore, DAP12-deficient mice show decreased susceptibility to EAE induction and exhibit functional defects of oligodendrocytes and osteoclasts. These observations have suggested that the array of myeloid cells regulated by TREM-2 may extend beyond DC, including microglial cells and osteoclats. Here, we show that the TREM-2/DAP12 complex is selectively expressed on human and mouse microglia. The expression of TREM-2 is down-regulated in brain upon intra-cerebral injection of LPS in mice. Conversely, TREM-2 transcripts and protein are up-regulated in the spinal cord of mice with EAE induced by immunization with MOG peptide. Work is in progress to evaluate the effect of TREM-2 blockade during EAE and LPS-induced encephalitis. Staining with a soluble form of TREM-2 has revealed that both primary astrocytes and astrocytoma cell lines, either of mouse or human origin, express detectable levels of TREM-2 ligand. Current data suggest that TREM-2 might modulate differentiation and activation of microglial cells, possibly by modulating responsiveness to certain cytokines or reinforcing signalling pathways triggered by cytokine receptors, integrins or chemokine receptors.
Evidence for distinct expression of pro-and anti-inflammatory cytokines within subsets of activated brain macrophages C.-F. Calvo, E. Amigou and J. Glowinski INSERM U114, College de France, Paris, France The brain macrophage (BM) cell population already considered heterogeneous in terms of phenotype expression pattern also exert, upon activation, dual functions resulting in either beneficial or detrimental effects on neuron survival. Our interest was to further characterize BM functional heterogeneity, by trying to show that production of pro-and anti-inflammatory cytokines would segregate in distinct BM cell subsets. BM isolated from newborn (P1) mouse brain cell cultures were examined under bacterial lipopolysaccharide stimulation by intracellular immunofluorescence staining with specific antibodies to TNFalpha and IL10, as candidates of the proand anti-inflammatory families of cytokines, respectively. By confocal microscopy, we showed that only 8% of the total cell population produced both cytokines, whereas 35% and 8% produced exclusively TNFalpha or IL10, respectively. Using specific MAP kinase inhibitors, we showed by immunofluorescence and ELISA that these cell populations could be regulated independently. Indeed, inhibition of the p38 MAP kinase by SB203580 inhibited both TNFalpha and IL10 production, while blockade of the p42/44 MAPK pathway by U0126 abolished the TNFalpha production but was without effect on that of IL10. Altogether, these results show that BM response to a given stimulus represents the sum of independent cell subset responses. We therefore postulate that the neurotrophic or neurotoxic function of BM is supported by the inflammatory nature of cytokines secreted by distinct subpopulations. Microglial cells are bone marrow-derived antigen presenting cells involved in CNS immune surveillance. Microglial function in the brain microenvironment comprises a wide range of activities from phagocytosis of apoptotic/necrotic cells to antigen processing and presentation to patrolling T cells. Secreted cytokines can tune the functional capability of microglial cells by shaping them in fully competent antigen presenting cells through a bclassical activationQ or inducing a phenotype mostly associated with antiinflammatory process, healing and induction of tolerance which is often called balternative activationQ. GM-CSF is a growth factor present only during inflammatory conditions in the adult brain which is also known to regulate the antigen presenting cell function of microglia. In the current study, the regulation by GM-CSF of several proteins associated with either classical or alternative microglial activation was investigated. Our results show that in murine microglia, GM-CSF induces not only primarily markers of classical activation but also some molecules involved with alternative activation, underlining its unique involvement in both pathways of APC activation.
Influence of PPAR-gamma agonists on prion-protein induced microglia activation S. Wagner, R. Mfssner, S. Walter and K. Fassbender University of Göttingen, Göttingen, Germany A pathophysiological hallmark of prion disease is chronic microglial activation, which is considered to contribute to progressive neuronal injury by release of neurotoxic products. In Alzheimer's disease, also characterized by a microglial-induced proinflammatory response, the use of agonists to peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to reduce amyloid-beta stimulated cytokine expression by microglia. We therefore analyzed the effect of drugs of the thiazolidindione class like rosiglitazone and troglitazone on the prionprotein-induced microglial activation. Murine microglia or peritoneal macrophages were stimulated with fibrillar Prion protein (PrP)106-126. The fibrillarity of the peptide was controlled by electron microscopy. Control experiments consisted of stimulation with non-fibrillar PrP106-126 and PrP scrambled. Stimulation of the cells with fibrillar PrP106-126 resulted in strong induction of expression of the pro-inflammatory cytokines interleukin 6 (IL-6) and tumor-necrose-factor alpha (TNFalpha) as measured by ELISA, whereas non-fibrillar and scrambled peptide had no effect on microglial cells. Using PPAR-gamma agonists like Troglitazone, we were able to reduce up to 60% the PrP-induced expression of IL-6 and TNF-alpha by macrophages/microglia in a dosedependent manner. These results indicate the use of PPAR-gamma agonists as a potential new therapeutic approach in prion-protein induced neuroinflammation.
Early cytoskeletal rearrangements occurring in activated microglial cells C. Gallotti, L. Grandbarbe, E. Morga and P. Heuschling
After stimulation with pro-inflammatory signals, microglia, the brain endogenous myeloid cell population, rapidly change their cell morphology in order to adopt the shape of phagocytic cells. Using a cell line as well as primary rodent microgliocytes, we have studied the early modifications occurring in vitro at the level of the actin cytoskeleton, on actin-binding proteins, as well as on focal adhesion spots. Morphometric analysis documents the rapid change in cell morphology over the first 6 h after activation with LPS. Actin-fiber bundles as well as the actin-associated proteins l-plastin and alpha-actinin have been studied under fluorescence microscopy. A link between cell morphology and the intracellular distribution of these proteins is observed. The focal adhesion spotassociated proteins integrin beta1 and alpha6 as well as vinculin have been studied. The disposition of adhesion spots changes after activation. lplastin, its phosphorylated form, and alpha-actinin are seen in close association with the focal adhesion spots. Immuno-blot-analysis as well as RT-PCR has been used to study the kinetics of expression of these proteins. The synthesis of l-plastin and especially its phosphorylation are tightly regulated during the first 30 min after activation, indicating that l-plastin and its post-translational changes are key elements during the morphological changes occurring after microglial activation.
Ischemia/reperfusion injury is associated with inflammatory responses that may play both deleterious and benefic role in neuronal damage. FK506 (Tacrolimus) is an immunosuppressant that blocks an activation of immune system cells and exerts neuroprotective and neurotrophic action in traumatic brain injury, sciatic nerve injury, focal and global ischemia. We found that neuroprotectant FK506 administered at 1 mg/kg, 60 min after MCAo (induced in male Wistar rats using the suture model) produced a significant reduction of IL-1beta, IL-6, and TNFalpha mRNA levels observed 12 h after reperfusion. It was associated with a significant down-regulation of IL-1beta protein expression in microglia in the injured side. We studied the influence of FK506 on microglia morphology and cytokine expression in LPS-activated microglial cells. LPS-induced changes in microglia morphology were completely inhibited by 20 AM FK506. Cultured microglia constitutively express cytokines; a basal level of cytokine mRNAs was observed in untreated cultures. However, stimulation with LPS induced an expression of studied cytokines at 6 h and the most evident changes were observed in the levels of TNFalpha and IL-6 mRNAs. Addition of 10 or 20 AM FK506 blocked the LPS-induced increases in cytokine mRNA levels. Our findings suggest that microglial cells are targets for FK506 and modulation of inflammation may be a mechanism of FK506-mediated neuroprotection in ischemia.
In most neurodegenerative disorders, including multiple sclerosis, Parkinson's disease, and Alzheimer's disease, a massive neuronal cell death occurs as a consequence of an uncontrolled inflammatory response, where activated microglia and its cytotoxic agents play a crucial pathologic role. Activated microglia secrete inflammatory mediators such as cytokines and chemokines, which contribute to the pathophysiological changes associated with several neuroimmunologic disorders. Vasoactive intestinal peptide (VIP) is neuropeptide that function as potent anti-inflammatory factor in the periphery. We investigated the effects of VIP on chemokine production by activated microglia. VIP inhibited the expression of the microglia-derived CXC chemokines MIP-2 and KC, and of the CC chemokines MCP-1, and RANTES. The inhibition of chemokine gene expression correlates with an inhibitory effect of VIP on NFkB binding. The VIP inhibition of both chemokine production and of NFkB binding is mediated through the specific receptor VPAC1 and involves a cAMP-dependent intracellular pathway. Of biological significance is the fact that the inhibition of chemokine production by VIP leads to a significant reduction in the chemotactic activity generated by activated microglia for peripheral leukocytes, i.e., neutrophils, macrophages, and lymphocytes. VIP has a clear neuroprotective effect on inflammatory conditions by inhibiting the production of microglia-derived proinflammatory factors (tumor necrosis factor alpha, interleukin-1beta, nitric oxide). Therefore, VIP represents an important factor in the control of inflammation in the central nervous system and emerges as a valuable neuroprotective agent for the treatment of pathologic conditions of the central nervous system where inflammationinduced neurodegeneration occurs. Microglial cells are the intrinsic immunocompetent cells of the central nervous system. Following brain injury, these cells are rapidly activated and transform from a resting into an activated state. It is widely believed that substances released from damaged cells within the brain trigger this process and consequently lead to the long-term changes of microglial gene expression and reorganization of the cell phenotype. Here, we have investigated how immunoglobulins activate microglial cells. We found that immunoglobulins trigger dose-dependent microglial proliferation as well as nitric oxide release. Furthermore, immunoglobulins led to cytokine release (TNF-alpha, IL-6) as well as changes in surface antigen expression. Immunoglobulins are found in high concentrations in serum and could enter the CNS during periods of blood-brain barrier breakdown.
Microglia are innately refractory to nitric oxide-inducing stimuli, but acquire responsiveness following differentiation by colony-stimulating factors C.A. Brannan and M.R. Roberts Department of Microbiology and Medicine, University of Virginia, Charlottesville, VA, USA Microglia are the immunoregulatory cells of the central nervous system (CNS), and share many characteristics with resident macrophages in extracerebral tissues. Macrophages secrete nitric oxide (NO) following NF-kappaB/IFN-dependent transcriptional activation of the NO synthase gene (NOS2) by stimuli elicited during a T cell response or by microbial products. NO plays a key role in regulating both adaptive and innate immune responses, including inhibition of T cell proliferation and elimination of intracellular pathogens. Regulation of NO production by microglia is poorly understood, however. We find that microglia from healthy adult mice produce negligible amounts of NO compared to resident macrophages during restimulation of peptide-specific CD8 T cells, and therefore cannot block T cell proliferation. The impaired NO response extends to all other NOS2-inducing stimuli, including cytokines, CD40 ligation, and lipopolysaccharide, and operates at the level of NOS2 RNA. Microglia do produce proinflammatory cytokines in response to these stimuli however, and therefore possess a relatively selective block in NOS2 expression. Furthermore, microglia acquire profound responsiveness to NO-inducers following differentiation by GM-CSF or M-CSF. These factors are constitutively required for differentiation of macrophage populations in vivo, and increase in the CNS during inflammation and disease. We therefore propose that microglia in the healthy adult brain exist in an bNO-incompetentQ state that may be reversed in the setting of CNS disease, thus facilitating temporal modulation of CNS immune responses. There is increasing evidence that inflammatory activation of microglia has pathogenic influence on Alzheimer's disease (AD). According to in vitro studies, microglia activated by amyloid-beta (Abeta) peptides have been reported to damage or kill neurons by the release of neurotoxic molecules such as tumor necrosis factor-alpha (TNF-alpha), nitric oxide or reactive oxygen species. Whereas it has been reported that phagocytes which engulf apoptotic cells actively suppress the inflammatory response by releasing anti-inflammatory mediators such as prostaglandin E2 (PGE2). Thus, it is tempting to speculate that activated microglia may change from proinflammatory to anti-inflammatory after phagocytosis of apoptotic cells. In this study, we have attempted to elucidate effects of PS-liposomes, mimicking apoptotic cells, on the inflammatory response of Abeta-activated microglia. Pretreatment of PS-liposomes significantly inhibited the release of both TNF-alpha and NO from microglial cells activated by Abeta combined with interferon-gamma (IFN-gamma). PS-liposomes also reduced the expression of mRNA encoding TNF-alpha in Abeta/IFNgamma-activated microglia. Furthermore, pretreatment of PS liposomes substantially decreased the generation of superoxide radicals associated with microglia activated by lipopolysaccharide (LPS) combined with phorbol 12-myristate 13-acetate (PMA) in electron spin resonance (ESR) spectra. These results strongly suggest that PS-liposomes can be applied to the treatment of AD. The stress hormones, glucocorticoids (GCs), and the key reproductive hormone, estrogen (E2), play a central role in neuroendocrine-immune crosstalk and have a major impact in astroglial cell function. In the context of innate inflammatory mechanisms, a dysfunction of the astroglial cell compartment is believed to contribute to the selective degeneration of DA neurons in the substantia nigra pars compacta (SN) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's like syndrome. Here, we show a chief role of hormonal programming of glial response to inflammation and oxidative stress in MPTP-induced loss of DA neuron functionality. Studies carried out in transgenic (Tg) mice constitutively expressing a GR antisense RNA from early embryonic life establish a link between GRs and iNOS-derived NO and demonstrate that lack of GR regulation of NO exacerbates experimentally induced parkinsonism. Studies carried out in female mice deprived of endogenous ovarian hormones show exacerbation of MPTPinduced loss of DA neuron function preceded by a strong astroglial activation and iNOS expression, resulting in massive death of astrocytes and neurons, whereas E2 replacement therapy sharply inhibits iNOS expression, counteracts astrocyte apoptosis and restores neurochemical parameters of DA neuron functionality, pointing to endogenous steroid hormones as prime modulators of iNOS inducibility and crucial checkpoints in MPTP-induced neuroinflammation. The Notch pathway is a fundamental cell interaction mechanism that influences cell fate decision by interactions between cellular neighbours. In recent years, it has been shown that the Notch signaling plays a crucial role in the differentiation process of monocytes, macrophages and dendritic cells. In the normal brain tissue, microglial cells are found as quiescent cells. Upon appropriate stimulation though, they continue the previously halted differentiation process to become immunocompetent phagocytic cells. In the present study, we examine the involvement of Notch pathway in the microglial activation. Modulation of Notch signaling by a soluble form of ligand (Jagged1) indicates that Notch signaling is functional in a murine microglial cell line as well as in primary microglia. Our data show that Notch activation modulates the expression of proinflammatory cytokines such as TNF-alpha. Moreover, in an attempt to modulate these neurotoxic properties of activated microglial cells, we have undertaken the characterization of pharmacological properties of hybrid compounds combining an alkanol structure and an antioxidant moiety. This compound modulates, via the Notch pathway, the pro-inflammatory activation of microglial cells. Together, these results suggest that Notch signaling, has important immunomodulatory functions in the brain.
Regulation of astrocytic cell response by P2Y2 nucleotide receptors N. Chorna a , G.A. Weisman b and A.F. Gonzalez a a University of Puerto Rico, San Juan, Puerto Rico, USA; b University of Missouri-Columbia, Missouri, USA Immuno-regulatory properties of astrocytes and glial cells and their role in neurogenesis in response to tissue damage remain unknown. It is recognized that activation of G-protein coupled P2Y2 nucleotide receptors by ATP or UTP upon injury or inflammation stimulate intense signaling between astrocytes, microglia, oligodendrocytes, and neurons (Neary et al., 1996 (Neary et al., , 1999 Burnstock and Williams, 2000; Chorna et al., 2003) . We found that activation of P2Y2 receptors in 1321N1 astrocytoma cells transfected with human P2Y2 receptor, coupled to the p38 and ERK1/2dependent phosphorylation of CREB kinase triggered CRE-dependent upregulation of cell survival genes bcl-2 and bcl-xL. Additionally, using cDNA microarray analysis and RT-PCR, P2Y2 receptors were found to up-regulate the expression of genes for neurotrophins, neuropeptides and growth factors including nerve growth factor-2; neurotrophins 3,4,5 and glia-derived neurite-promoting factor. Moreover, conditioned media from UTP-treated 1321N1 cells expressing P2Y2 receptors stimulated the outgrowth of neurites in PC-12 cells. Nucleotides mediated release of neurotrophins and activation of CRE-dependent cell survival response may be assumed as a solid rationale for the possible use of P2Y2 receptors in the development of new therapeutic strategies in immune and neurological disorders.
The calcium channel TRPC4 co-localizes with ZO-1 in human fetal astrocytes X. Song, Y. Zhao and C.F. Brosnan Albert Einstein College of Medicine, New York, USA Members of the mammalian transient receptor potential (TRP) family form cation-permeable channels at the plasma membrane implicated in capacitative calcium influx following activation by either second-messengermediated pathways and/or by store depletion. We now show by RT-PCR, Western blotting and immunohistochemistry that resting astrocytes express TRPC4, particularly at sites of cell-cell contact. By confocal imaging and immunoelectron microscopy, we detected co-localization of TRPC4 with the junctional protein ZO-1 and demonstrated association by co-immunoprecipitation. It has been proposed that the targeting of TRPC4 to the cell membrane is dependent upon the interaction of the C-terminal TRL motif with PDZ domains. Using transfection of astrocytes with myc-tagged TRPC4 or TRL-motif truncated TRPC4 (deltaTRL), we found that deltaTRL localized to a juxtanuclear compartment, whereas the wild-type channel showed prominent cell surface distribution. Deletion of the TRL motif also reduced plasma membrane expression as assessed by cell surface biotinylation experiments. Using GST fusion proteins, we found that TRPC4 interacted with the PDZ1 domain of ZO-1 and that this interaction was lost in constructs missing the TRL motif. Thus, our data demonstrate that the PDZ-interacting domain of TRPC4 controls its localization and surface expression. Since ZO-1 also interacts with the gap junction protein 43, these data implicate TRPC4 as part of the signaling complex that forms between astrocytes and which is lost in astrocytes activated with the cytokine IL-1.
Characterisation of the Jagged-Notch-Hes pathway on activated astrocytes E. Morga, L. Grandbarbe, K. Hemmer and P. Heuschling Université du Luxembourg, Luxembourg, Luxembourg
It has been shown that the Jagged-Notch-Hes pathways are implicated in many aspects of the CNS development and functions, including differentiation and myelination. During development Jagged1 is down-regulated, oligodendrocyte precursors mature and myelination begins. Human oligodendrocyte maturation is blocked in the presence of cells transfected with Jagged1. In inflamed MS tissue, Jagged1 is expressed by reactive astrocytes whereas astrocytes in myelinated areas do not show Jagged1 immunoreactivity. In an attempt to modulate the expression of Jagged1 on astrocytes, we have undertaken the characterization of different elements of the Jagged-Notch-Hes pathway. In this part of our work, we studied the expression of these different elements after activation of the astrocytes. To induce the activation of the astrocytes, we used LPS or a simultaneous incubation of IFN-gamma and TNF-alpha. LPS induces an up-regulation of ligands like Jagged1 and DeltaII but down-regulates the expression of Notch1, interestingly LPS also down-regulates Hes1. In the presence of IFN-gamma and TNF-alpha astrocytes inhibit their expression of Notch1 and Notch2. The simultaneous incubation with these two cytokines does not induce a clear up-regulation of the ligands. IFN-gamma+TNF-alpha also inhibits the expression of Hes1. Taken together, these preliminary results could have interesting issues on the understanding of this pathway on astrocytes. In fact, a better understanding of the expression of Notchligands can be a helpful tool for demyelinating diseases.
Dexamethasone increases astrocyte immunoreactivity to GFAP following ethidium bromide injection in the brainstem of Wistar rats M.A. Lallo and E. Bondan Universidade Bandeirante de Sao Paulo, Sao Paulo, Brasil Ethidium bromide (EB) is a gliotoxic agent that causes focal astrocytic and oligodendroglial disappearance. As immunosuppressive and anti-inflammatory drugs are known to modify glial expression, this study was designed to investigate astrocyte immunoreactivity to Glial Fibrillary Acidic Protein (GFAP) after EB injection in animals submitted to dexamethasone (DX) treatment. Adult Wistar rats were injected into cisterna pontis with 0.1% EB and received DX (3 mg/kg/day, intraperitonial route-group 1) or not (group 2). Some were injected with 0.9% saline solution and were also treated with DX (group 3) or not (group 4). Brainstem samples were collected from 24 h to 31 days post-injection for GFAP immunohistochemical staining using avidin-biotin method. In groups 1 and 2, extensive lesions were seen in the pons and mesencephalon, with astrocyte disappearance from the central area 24 h post-injection. Macrophagic infiltration and peripheral astrocytic reaction were noted after 3 days. Marginal astrocytes presented increased immunoreactivity to GFAP and DX-treated animals presented a greater number of GFAP-positive cells. In groups 3 and 4, discrete pontine lesions were observed, showing central astrocyte preservation and a peripheral GFAP staining less intense comparing to groups 1 and 2. No difference was found in GFAP immunostaining between groups 3 and 4 after saline injection. Astrocytes from the edges of the EB-induced lesions presented increased immunoreactivity to GFAP and DX seemed to increase this expression.
Changes in gene expression induced by activation of P2X7 receptors C. Santos-Berrios a , N. Chorna a , G. Weisman b and F. Gonzalez a a University of Puerto Rico, San Juan, Puerto Rico; b University of Missouri, Missouri, USA Activated lymphocytes, macrophages, microglia, and platelets, as well as cells undergoing necrosis or apoptosis, release high concentrations of nucleotide diphosphates and triphosphates into the extracellular space. Astrocytes respond to extracellular nucleotides that are released upon nerve stimulation or from damaged brain cells. P2 receptors mediate the responses to extracellular nucleotides. From this family of receptors, two subtypes are known-the G protein-coupled P2Y receptors and the ligand-gated ion channels P2X receptors. In this study, we tested the hypothesis that activation of P2X7 receptors with BzATP, an analog of ATP, leads to the modulation of the expression of genes related to inflammation. Our results show a time-and dose-dependent increase in the expression for cox and cfos mRNA. Studies made with cDNA microarrays before and after activation of P2X7 receptors revealed additional genes that were modulated by this receptor. Our findings will help us better understand the role of nucleotide receptors in inflammatory responses in the nervous system, information that is bound to have pharmacological applications and clinical significance. ADAMTS-1, -4 and -5 are members of the ADAMTS family of metalloproteinases. ADAMTS substrates include extracellular matrix (ECM) components, aggrecan, versican and brevican, which form part of CNS ECM, therefore these enzymes may have a role in CNS ECM turnover. ADAMTS-1 and -4 exhibit anti-angiogenic activity. Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a naturally occurring inhibitor of the ADAMTSs. The involvement of ADAMTSs in the pathology of multiple sclerosis (MS) and stroke may include a role in ECM breakdown, demyelination, and prevention of remyelination and in stroke, prevention of angiogenesis. The expression of ADAMTS-1, -4 and -5 was studied in primary human astrocytes at the mRNA and protein level using real time RT-PCR, western blotting and immunocytochemistry. Cells were untreated or treated with pro-inflammatory cytokines IL-1-beta, TNF-alpha and TGFbeta. Increased expression of ADAMTS-1 was seen following treatment with all three cytokines. ADAMTS-4 expression was increased by TGFbeta and TNF-alpha. TIMP-3 was increased by TGF-beta. This data indicates that during inflammation there is an imbalance in ADAMTS and TIMP3 expression, which could cause increased enzyme activity resulting in breakdown of the CNS ECM and prevention of angiogenesis. This work was funded by the Wellcome Trust.
Neuroinflammation and oxidative stress up-regulate and translocate multidrug resistance-associated protein 1 (Mrp1) in mouse astroglia F. Gennuso a , C. Tirolo a , N. Testa a , S. Caniglia a , F. L'Episcopo a , M.C. Morale a , C. Fernetti b , L. Pascolo b , D. Ostrow c , C. Tiribelli b and B. Marchetti a,d a Neuropharmacology, OASI, Troina, Italy; b Biochemistry, University of Trieste, Trieste, Italy; c Washington University, Seattle, USA; d Pharmacology, University of Sassari, Sassari, Italy Multidrug resistance associated proteins (MRPs) are ATP-driven export pumps that mediate the export of organic anions from cells. Various MRP family members including Mrp1 are expressed in enriched astrocytes, microglia, oligodendrocytes and neuronal rodent cultures. Mrp1 is also expressed in two CNS barriers, the endothelial cells of brain microvessels and the epithelial cells of the choroid plexus and its importance in preventing CNS accumulation of various xenobiotics has been demonstrated in knockout mice. The Golgi apparatus has been recently shown to represent a key station involved in the intracellular trafficking of Mrp1 in mouse astrocytes. Here, we report that Mrp1 is differentially expressed in resting vs. activated astrocytes and microglia and that exposure to oxidative/nitrosative stress, such as serum deprivation and lipopolysaccharide (LPS), time-and dose-dependently up-regulate Mrp1 expression and induce extensive membrane targeting. At a functional level, this effect is accompanied by enhanced Fluo-3-extruding activity and reduction of intracellular reactive oxygen species accumulation, whereas exposure to Mrp1 inhibitor MK571 sharply impaired such phenomena, implicating glial Mrp1 as a key redox sensor during brain inflammation.
Receptor machinery involved in MBP-induced astrocyte activation R. Businaro a , L. Fumagalli a , S. Leone b and G.M. Lauro b a University La Sapienza, Rome, Italy; b University Roma TRE, Rome, Italy
The object of the present study is to identify the receptor(s) involved in myelin basic protein (MBP) uptake and to evaluate their role in the subsequent MBP processing and presentation by human astroglial cells: central events in the demyelinating process. Our study deals with cultured astroglial cells, stimulated with MBP or its peptide fragments in order to evaluate heme oxygenase-1 (HO-1) and HLA class II antigen expression by cytometric analysis, RT-PCR and Western blots. Our attention was focused on the role of bFGF receptors, GM1 ganglioside and low-density lipoprotein receptor-related protein (LRP), detected into astroglial membranes. Our experiments, performed in the presence or in the absence of specific inhibitors of the above-mentioned putative receptors, suggest that MBP, carried by alpha2-macroglobulin, binds to LRP, undergoing subsequent receptor-mediated endocytosis. This mechanism may represent the first step of antigen processing and presentation function in astrocytic cells.
Monocarboxylates like lactate are provided by astrocytes and can be used as fuel by neurons and oligodendrocytes. In primary Lewis rat astrocytes, coculture with MHC class II-restricted myelin basic protein (MBP)-specific T cells resulted in a marked upregulation of the astrocytic lactate transporter MCT1 that is to export lactate into the extracellular space. It was evident that the increase in MCT1 was triggered by T cells in an antigen-dependent manner. The glial isoform of the glucose transporter GLUT1 was not regulated under these conditions. T cell blasts that had been pre-activated by antigen and splenic antigen presenting cells (APCs) beforehand also led to an increase in astrocytic MCT1. Even resting T cells stimulated the expression of MCT1 when anti-MHC class II-, but not when anti-MHC class I-antibodies were added to the co-culture. Therefore, complexation of MHC class II molecules on astrocytes might be mandatory for the regulation of MCT1. Consistent with the in vitro-experiments, an upregulation of MCT1 was observed in the spinal cord of autoimmune encephalitic rats while GLUT1 expression appeared to be unchanged. This T cell-mediated regulation of MCT1 might contribute to a compensatory or protective mechanism in order to guarantee substrate pools for neurons and oligodendrocytes under inflammatory conditions. While we have recently described that contact with astrocytes induces development of regulatory T cells, in the present study we investigated the mechanisms responsible for their suppressive properties. Pre-incubation with astrocytes rendered rat T cells completely unresponsive to mitogenic stimuli, which coincided with the block in G0/G1 phase of the cell cycle and the increase in number of subdiploid apoptotic cells. These anergic cells completely prevented mitogen-or antigen-induced growth of T lymphocytes, with the suppressive activity residing in both CD4+ and CD8+ T cell compartments. The observed inhibitory effect was not mediated by the immunosuppressive factors released from apoptotic cells, but apparently required metabolically active T cells. Heat-sensitive and protein synthesis inhibition-sensitive soluble T cell factors, not including TGF-beta or IL-10, were solely responsible for the observed suppression, which did not depend either on nitric oxide production, prostaglandin release, or tryptophan consumption. Interestingly, the soluble T cell products induced by contact with astrocytes were also able to transfer the suppressive activity to bnormalQ T cells, which could explain the extremely high potency of astrocyte-induced suppressor T cells. Finally, the cells with suppressive properties were readily generated upon contact of astrocytes and human T lymphocytes, indicating their potential therapeutic value in the treatment of CNS autoimmunity.
Inhibition of calpain activity and expression by interferon-beta in rat astrocytes: implications for MS pathogenesis and treatment T. Latronico a , V. Piscopo a , A. Fasano a , P. Riccio b and M.G. Liuzzi a a Department of Biochemistry and Molecular Biology, University of Bari, Bari, Italy; b University of Basilicata, Potenza, Italy
Calpains are a family of calcium-activated proteases that have been recently implicated in multiple sclerosis (MS) pathogenesis for both their ability to degrade myelin proteins and for their presence, at increased levels, in activated glial/inflammatory cells of MS plaques. Aim of this study was to investigate whether interferon-beta (IFN-beta), could modulate the activity and/or the expression of m-calpain in astrocytes. Rat astrocyte cultures were activated with LPS and simultaneously treated with different doses of IFNbeta. Culture supernatants and cellular extracts collected from astrocytes were subjected to casein-zymography and the presence of m-calpain activity was determined, after scanning densitometry, by the extent of casein degradation compared to control. Total RNA was extracted from glial cells and subjected to RT-PCR using primers specific for m-calpain. Increased activity of m-calpain was observed in cellular extracts from LPStreated astrocytes in comparison with cellular extracts from non-treated cells (control cells). On the contrary, dose-dependent inhibition of mcalpain activity was observed in cellular extracts from IFN-beta-treated astrocytes. RT-PCR revealed that the expression of m-calpain was increased in LPS-activated astrocytes and was dose-dependently inhibited by IFNbeta treatment. These results suggest that IFN-beta could modulate the expression of calpains in astrocytes and this effect could represent an additional mechanism by which IFN-beta decreases the development of new CNS lesions in course of MS. Within the central nervous system (CNS) in multiple sclerosis (MS), increased expression of two key chemokines, CXCL10 and CCL2, has been demonstrated. These chemoattractants are involved in the recruitment into the CNS of T cells and monocytes, which express the corresponding receptors, CXCR3 and CCR2. In the cerebrospinal fluid (CSF) of MS patients at times of relapse, there is an increase in CXCL10 and a decrease in CCL2. To further understand these in vivo findings, we have investigated the expression of CXCL10 and CCL2 by primary adult human astrocytes in vitro following treatment with pro-and anti-inflammatory cytokines. Secreted chemokine was measured by ELISA, cell-associated chemokine was visualised by immunofluorescent confocal microscopy and mRNA was measured by real-time PCR. Interferon gamma (10 ng/ml) induced the secretion of CXCL10 and CCL2 at 24 h with levels of 205 and 1853 pg/ml, respectively. TNF alpha (10 ng/ml) had no effect on CXCL10 expression but increased release of CCL2 to 1313 pg/ml. Increases in mRNA were also observed. TGF beta had no effect on chemokine expression. The fine control of chemokine expression by cytokines may explain the different chemokine expression profiles in the CSF in people with MS.
Expression and functional role of Toll-like receptors (TLR) on human astrocytes M. Bsibsi a , C. Persoon-Deen a , R. Ravid b and H. van Noort a a TNO Prevention and Health, Leiden, The Netherlands; b Netherlands Brain Bank, Amsterdam, The Netherlands
Background: Toll-like receptors play key roles in the recognition of invariant pathogen-associated molecules by the innate immune system and generally mediate pro-inflammatory responses to these agonists. We previously showed expression of various TLR family members on glial cells in the human central nervous system (CNS) and elevated levels of these TLR during neuroinflammation. Yet the functional significance of TLR in the CNS is still largely unexplored. Objective: To investigate regulation of TLR expression on cultured human astrocytes, and to evaluate the functional response of astrocytes to appropriate TLR agonists. Methods: Human astrocytes were isolated from post-mortem brains of healthy donors and treated with cytokines or TLR agonists. To monitor TLR expression, TLR-encoding mRNA was quantified by real-time PCR. To monitor functional responses, levels of mRNA encoding 268 genes for cytokines, chemokines, growth factors and their receptors were monitored using cDNA arrays. Results and conclusions: Selective TLR induction was observed in astrocytes by various stimuli. Surprisingly, activation of inducible TLR on astrocytes led to induction of several chemotactic factors along with a variety of mediators with known functions in repair processes. TLR activation in general did not activate a polarized pro-inflammatory response. Together, these data suggest that astroglial TLR play a role in repair functions rather than in promoting inflammation. MoMuLV-ts1-mediated neuronal degeneration in mice is a model for human immunodeficiency virus (HIV)-induced neurodegenerative disease in humans. In this study, we show increased expression of cyclooxygenase-2 (COX-2), an enzyme known to be associated with inflammatory and neurodegenerative diseases, in the brainstem tissues of ts1-infected mice. In sections of the same tissues, we find that astrocytes in areas of spongiform lesions contain increased amounts of immunoreactive COX-2. COX-2 is also upregulated in ts1-infected cultured astrocytes, but this effect is significantly decreased by treatment of the cells with curcumin and by SP600125, both of which specifically inhibit the c-Jun N-terminal kinases (JNKs)/c-Jun pathway. By contrast, PD98059, MG-132, ALLN, and lactacystin, which inhibit extracellular signal-regulated kinase (ERK) activation, and proteasome activity, have no such effect. These data point to the JNK/c-Jun pathway as the mechanism by which COX-2 expression is induced in ts1-infected astrocytes. Notably, curcumin also decreases levels of (a) CCAAT/en-hancer-binding protein (CHOP), (b) glucose-related protein 78 (GRP78) and (c) phosphorylated eukaryotic initiation factor 2a (eIF2a), all of which are markers of endoplasmic reticulum (ER) stress signaling, and are upregulated in untreated C1 cells after ts1 infection. These findings link ER stress signaling in ts1-infected astrocytes to events that result in COX-2 induction.
Tuberculous meningitis and astrocyte derived matrix metalloproteinases J.E. Harris, J.S. Friedland Imperial College, London, UK Tuberculous meningitis (TBM) is the most deadly form of tuberculous infection, its pathology is characterised by extensive destruction of central nervous system (CNS) tissues. Matrix metalloproteinase-9 (MMP-9) has been implicated in tissue destruction in a range of CNS diseases including TBM. Astrocytes are the most populous cell-type in the CNS, therefore astrocyte-derived matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) maybe key mediators of CNS tissue destruction. In the present study, the regulation of gene expression and secretion of MMP-9 and key TIMPs are analysed using zymography, ELISAs, western blots and northern blots. Over 120 hours astrocytes, stimulated with conditioned media from infected monocytes secreted 6-fold more MMP-9 than controls with concentrations rising to a maximum of 1273796 units. TIMP-1 and TIMP-2 concentrations were not significantly different from controls over the study period. The upregulation of MMP-9 was inhibited by a combination of anti-TNF-alpha, IL-1 receptor antagonist and pertussis toxin which blocks G protein-linked processes; such inhibition was not seen in response to a single inhibitors. TNF-alpha induced MMP-9 expression in astrocytes, while IL-1 and cholera toxin did not. These data suggest that networks active between astrocytes and monocytes play a key role in creating a matrix degrading environment during TBM and that a combination of mediators are involved with TNFalpha, having a critical role. Astrocyte-expressed tissue inhibitor of metalloproteinase (TIMP)-1 is an important molecule in CNS homeostasis and inflammatory diseases including HIV-1-associated dementia (HAD). Recently, TIMP-1 has emerged as a multifunctional molecule promoting cell survival in addition to MMP regulation. Previously, we uniquely demonstrated that HAD brains have reduced levels of TIMP-1. Astrocyte-TIMP-1 expression is differentially regulated with elevated levels in acute and diminished levels in chronic inflammation. These observations indicate that TIMP-1 replenishment in HAD may have therapeutic value. Laboratory and animal model systems were used to examine mechanisms of astrocyte-TIMP-1 regulation and neuroprotection. Human astrocytes/neurons, neurotoxins, virus/viral proteins, TIMP-1 promoter-luciferase reporter constructs and RNAi gene silencing were used. Preliminary data confirm that acute versus chronic TIMP-1 responses in astrocytes are regulated predominantly at the promoter level. Neurotoxicity and/or neuroprotection were measured by MTT activity, TUNEL, and DNA fragmentation ELISA. TIMP-1 siRNA-transfected astrocytes had enhanced neurotoxicity that correlated with diminished TIMP-1 levels. TIMP-1 treatment partially reversed neurotoxicity in challenged neurons. In addition, TIMP-1 supported neuronal differentiation during early development suggesting that TIMP-1 may function as a neurotrophin. TIMP-1-neuroprotection may be mediated by activation of anti-apoptotic proteins such as Bcl-2/Bcl-xL and/or through neurotrophic activities. Our results indicate that the complex astrocyte-TIMP-1 regulation in HAD may influence neuroprotection. These studies are relevant to glial inflammation in HAD and may unravel novel mechanisms of glialneuronal interactions. Vagus nerve prevents systemic inflammation by inhibiting TNF transcription in the spleen via alpha7 nAChR L. Ulloa, H. Liao, V. Pavlov, M. Ochani and K.J. Tracey North Shore University Hospital, Long Island Jewish Research Institute, Manhasset, NY, USA Endotoxin, a constituent of Gram-negative bacteria, stimulates macrophages to release pro-inflammatory cytokines including tumor necrosis factor (TNF), which can cause lethal shock and tissue injury. We have recently discovered that vagus nerve stimulation attenuates circulating TNF levels during endotoxemia. This mechanism was named bthe cholinergic anti-inflammatory pathwayQ because acetylcholine, the principle neurotransmitter of the vagus nerve, inhibits TNF production in macrophages. The object of this study is to determine how vagus nerve regulates TNF production in different organs. Here we report that stimulation of the vagus nerve prevents lethal systemic inflammation by inhibiting TNF transcription in the spleen. Splenectomy attenuates circulating TNF levels during endotoxemia (sham=181.3F31 pg TNF/ ml serum vs. splenectomy=41.2F13.4 pg TNF/ml serum; pb0.05). Vagus nerve stimulation fails to further attenuate circulating TNF levels in splenectomized mice. Electrical stimulation of the vagus nerve activates a splenic neuronal network, and sectioning of the common celiac trunk of the vagus nerve, proximal to the spleen, ablates vagus nerve regulation of TNF in both spleen and serum. Vagus nerve stimulation fails to modulate splenic or circulating TNF levels in animals deficient in the alpha7 subunit of the acetylcholine receptor. Thus, the vagus nerve prevents endotoxin-induced lethal systemic inflammation through cholinergic signaling in the spleen. Neuropeptide Y (NPY), is a 36aa peptide distributed throughout the body which has been implicated in the regulation of several physiological processes such as energy balance, feeding, reproduction and anxiety. NPY actions are mediated by five G-protein coupled receptors, designated as Y1, Y2, Y4, Y5 and Y6. We assessed the effects of NPY and its receptors in regulating immune responses. NPY was found to inhibit the migration of B cells to chemokine CXCL12 (SDF1-alpha). We have also shown expression of Y receptors, Y1, Y2, Y4 and Y6 in murine spleen. Y1-deficient mice (Y1À/À) have smaller spleens, less mature B cells and more naive T cells in their secondary lymphoid organs. Furthermore, T cell differentiation to Th1 T cells in Y1À/À mice appears to be defective as lower IgG2a and IL-12 levels are detected in their serum. When challenged with a T-dependent or T-independent antigen, Y1À/À mice still produce significantly lower levels of IgG2a antibodies. Response of these mice was tested in a Th1 model of delayed-type hypersensitivity (DTH). Interestingly, Y1À/À mice had significantly reduced footpad swelling compared with wildtype mice. Finally, macrophages lacking Y1 receptors produce less TNF-alpha following stimulation with LPS in vitro. This data strongly suggests that the Y1 receptor is a key molecule controlling fundamental immune functions, confirming the interplay between the neuroendocrine and immune systems.
Neuropeptides generate tolerogenic dendritic cells D. Ganea a and M. Delgado b a Rutgers University, Newark, USA; b Institute of Parasitology and Biomedicine, Granada, Spain Dendritic cells (DC) act as potent antigen presenting cells initiating T cell activation, or as tolerance-inducing agents. The cells/factors and molecular mechanisms controlling these opposite functions of DC are not elucidated yet. In this study, we show that bone marrow DCs generated in the presence of the neuropeptides VIP and PACAP (VIP/PACAP-DC) induce or activate regulatory T cells (Treg). In contrast to DCs generated in the absence of VIP or PACAP, VIP/PACAP-DC secrete high levels of IL-10, and no detectable IL-12, IL-6, or TNF upon stimulation with LPS. LPS-stimulated VIP/ PACAP-DC induce unresponsiveness in co-cultured CD4+T cells in terms of proliferation and Th1 cytokine release (IFNgamma and IL-2). The cocultured Ag-specific T cells secrete however high levels of IL-10. The Treg induced by co-culture with VIP-DC inhibit the proliferation of responder T cells, and this inhibition is completely reversed by the addition of a mix of neutralizing Abs (anti-IL10+anti-TGFbeta+anti-CTLA-4) suggesting that the suppressive activity is mediated by both cellular contact and cytokines. The VIP-DC induce Treg generation in vivo, and the in vivo-induced Treg are capable to transfer tolerance to naRve hosts. The generation of antigenspecific Treg in vitro in a controlled environment is of high therapeutic potential for autoimmune diseases and transplantation (AI47325 & AI052306).
The endogenous cannabinoid 2-arachidonoyl glycerol as chemoattractant for dendritic cells and adjuvant for T helper cell type 1 response to soluble antigens G. Maestroni Istituto Cantonale di Patologia, Locarno, Switzerland Toll-like receptors (TLRs) expressed in dendritic cells (DCs) alert the innate immune system to the presence of pathogens so that the appropriate response can be mounted to contain the infection. However, the decision-making mechanisms that determine the type of effector response are poorly understood. In the preceding work, we showed that the sympathetic nervous system may influence the antigen-presenting and Th priming ability of DCs. Here we report that murine epidermal Langherans cells as well as lymphoid and the myeloid DCs express the mRNA coding for the cannabinoid CB2 receptor. Intradermal injection of 2-arachydonoylglycerol (2-AG), the physiological ligand for the CB2 receptor, shifted the immune response to a soluble protein and a TLR2 agonist from the mixed Th1/Th2 type to a sheer Th1 type. This effect evidenced by enhanced hypersensitivity response, Ig2a production and Th1 pattern of cytokine production in the draining lymph nodes was abolished by the specific CB2 antagonist SR 144528. The mechanism of this interesting effect might be related to a CB2-mediated recruitment of lymphoid DCs precursors. Consistently, 2-AG showed a potent chemotactic activity in vitro toward DCs. As 2-AG production may by induced by inflammatory agents, we have identified an endogenous lipid mediator which might influence the choice of the appropriate immune response to a pathogen. In the evolutionary scale, Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) involved in the innate and adaptative immune responses. Stimulation of TLR2 and TLR4 mediates a signalling cascade that finally leads to the expression of inflammation-related genes. Vasoactive intestinal peptide (VIP) is an immunopeptide present in the lymphoid microenvironment that exerts a therapeutic effect in several animal models of inflammatory and autoimmune disorders. We have demonstrated that treatment with VIP reduced the clinical and histopathological severity of TNBS-induced colitis by decreasing the inflammatory and Th1-driven autoimmune components of the disease, but nothing is known about the putative involvement of TLRs both in the disease and healing. Our results show the constitutive expression of TLR2 and TLR4, as well as the up-regulation after TNBS-treatment at protein and mRNA levels in colon that was reduced after VIP treatment. This behaviour is also exhibited in the expression of both receptors in macrophages, dendritic cells and lymphocytes from mesenteric lymph nodes. The present study demonstrates that the modulation of TLR2 and TLR4 receptors is involved in the beneficial effects of VIP in an animal model of Crohn's disease, representing a new step in the design of potential therapeutic strategies. Ghrelin is a recently identified gastric hormone with a variety of functions. This neuroendocrine peptide is involved in the release of growth hormone (GH) and neuropeptide Y (NPY), following activation of the GH secretagogue receptor (GHS-R). Prior studies have demonstrated that NPY would play a regulatory role in EAE by causing an immune bias towards Th2. Here we explored if Ghrelin might also exert an immunomodulatory effect similar to NPY and protect against development of EAE. We induced EAE in C57BL/6 mice with myelin oligodendrocyte glycoprotein (MOG)35-55 and treated the mice with subcutaneous injections of Ghrelin (0.5, 5 or 50 Ag/kg) every other day. Our results revealed that the continuous treatment with 5 Ag/kg of Ghrelin after EAE induction (up to day 35 or to day 10) could significantly ameliorate the clinical severity of EAE, whereas treatment with an acyl-modified Ghrelin showed no effects. Unexpectedly, IFN-gamma production of the draining lymph node (LN) cells to MOG35-55 was not affected by Ghrelin treatment, indicating a mechanism of EAE suppression that is independent of NPY. These results indicate a novel role for Ghrelin to modulate autoimmune pathology in EAE. Endogenous catecholamines (CA) in human peripheral blood mononuclear cells (PBMCs) modulate activation-induced apoptosis. In multiple sclerosis (MS), PBMCs have dysregulated CA production and are less susceptible to apoptosis. Since interferon (IFN)-beta is beneficial in MS, we investigated its effects on CA production and apoptosis in PBMCs obtained from healthy donors. Stimulation with phytohaemagglutinin (PHA) 10 Ag/ml triggered production of CA. Incubation with IFN-beta (500-1000 U/ml) increased CA production and efflux, an effect which was prevented by antibodies against IFN-beta or its receptor. Percentage of apoptotic PBMCs increased after PHA, and was reduced by alpha-methyl-p-tyrosine 10 AM (which prevents PHA-induced CA production). IFN-beta per se did not affect apoptosis but completely reverted the antiapoptotic effect of alpha-methyl-p-tyrosine. Incubation of PBMCs with IFN-gamma (0.1-2.0 pg/ml) per se completely inhibited CA production, an effect which was reverted by coincubation with IFN-beta 1000 U/ml. Under such experimental conditions however, IFNbeta failed to induce CA efflux. In conclusion, in human PBMCs IFN-beta (1) induces CA production and efflux; (2) restores impaired apoptosis secondary to inhibition of CA production; (3) counteracts IFN-gammainduced effects on CA. These findings shed new light on the mechanisms of action of IFN-beta as an immunomodulatory drug in MS. Pregnancy represents a physiological transitory state of immune tolerance to avoid the rejection of the foetus. Multiple sclerosis (MS) ameliorates during pregnancy, with a rebound of activity after delivery. Alterations in CD4+CD25+ regulatory T-cells (TR) are known to cause organ-specific autoimmune disease in experimental models. Our goal was to quantify TR during pregnancy and puerperium in MS patients. We conducted a prospective study of 10 patients with definite MS during pregnancy, and compared with two control groups: 12 healthy pregnant and 14 nonpregnant women. We quantified the TR and activated (CD4+HLA-DR+CD38+) cells by flow cytometry. During pregnancy, we observed a progressive increase in CD4+CD25+ T-lymphocytes in healthy as well as MS patients, whereas a decrease in activated T-cells was noted. The proportion of TR cells was significantly higher in pregnant than in nonpregnant women ( p=0.01), whereas no differences were observed neither in the percentages of total nor activated CD4+ T-cells. The CD4+CD25+ and CD4+CD25high T-cells significantly decreased when comparing the third trimester and puerperal proportions in MS patients ( P=0.01, and P=0.002, respectively). No significant differences were noted between healthy and MS pregnant in neither activated, nor TR cells, during any of the gestational trimesters and at postpartum. Our findings are consistent with the expansion of circulating regulatory CD4+CD25+T-cells pool during pregnancy, which may represent relevant factors in the activity course of MS. In a post-mortem study, we investigated the relationship between multiple sclerosis (MS) lesions in the hypothalamus and the state of activity of corticotropin releasing hormone (CRH)-producing neurons that control the hypothalamus-pituitary-adrenal (HPA) axis. A high incidence (15/16) of MS lesions was found in the hypothalamus, of which more than 50% was active, i.e. contained activated macrophages. MS patients have increased numbers of CRH-immunoreactive neurons co-expressing vasopressin (CRH/VP neurons), a sign of chronic activation of CRH neurons, and increased CRH mRNA expression. Active MS lesions correlated with a low number of hyperactive CRH/VP neurons. High HLA -DR, -DP, -DQ expression, a measure for macrophage and microglial activation, correlated with low CRH mRNA expression. The nearer the HLA expression was situated to the CRH neurons, the stronger the inhibiting effect, suggesting that activated microglial cells or macrophages suppress these neurons. The more active MS lesions were present in the hypothalamus, the shorter was the disease duration until the moment of death, indicating an unfavorable course of the disease. Thus, MS patients have a chronically activated CRH system, but in the subgroup of patients with active MS lesions in the hypothalamus, this activation is impaired and the disease course is worse. (Tissue provided by the Netherlands Brain Bank). Recently, it has been found to be active in neurons, and mice lacking NF-kappaB subunits p50 or p65 show deficits in specific cognitive tasks. Here we demonstrate a strikingly low level of anxiety-like behavior in the p50 knockout mouse. In an open field, the mutant mice showed significantly less defecation, more rearing, and more time spent in the center compartment relative to wild type control mice. The p50 knockout mice also spent more time investigating a novel object placed in the open field. On the elevated plus maze, p50 knockout mice spent more time on the open arms and had increased numbers of open arm entries relative to wild type. In tests of general health, sensorimotor function, and daily activity on a circadian rhythm, p50 knockout mice were normal. Thus, absence of the p50 subunit of the NF-kappaB transcription factor, which results in altered NF-kappaB transcriptional activity in cells throughout the body and brain, alters neuronal circuitry underlying manifestation of emotional behavior. The p50 subunit appears to play a role in normal expression of certain forms of anxiety. There is evidence of an innervation of lymphoid organs by sympathetic fibers releasing noradrenaline (NA) and neuropeptide Y (NPY). In previous work, we have observed the modulatory in vitro effect of NA and NPY on several immune functions. The aim of the present study was to evaluate the in vitro effect of these neurotransmitters, individually and jointly, on several inflammatory parameters of peritoneal leucocytes, such as adherence to fibronectin and collagen type I, PGE2 and NO release as well as susceptibility to H 2 O 2 -induced apoptosis. Adult female BALB/c mice have been used to obtain peritoneal suspensions containing both macrophages and lymphocytes. The concentrations of NA and NPY were 10 À10 M for NPY and 10 À8 M for NA. The results show an inhibitory effect of both neurotransmitters, when used jointly, on PGE2 and NO release (from 137 to 104 ng/ml, pb0.001, and from 47 to 34 AM, pb0.001, respectively). The susceptibility to apoptosis was decreased by NPY and NE, used individually or jointly, in basal conditions as well as in the presence of H 2 O 2 (50 mM). The adherence to fibronectin and collagen was decreased only by NPY, used individually or with NA. These results suggest an banti-inflammatoryQ effect of the sympathetic neurotransmitters studied (NPY and NA), at physiological concentrations, on murine peritoneal leucocytes. Work supported by FIS and CM grants. Crohn's disease (CD) is a chronic inflammatory pathology of the colon characterized by diarrhoea and weight loss. Vasoactive intestinal peptide (VIP), an immunoregulatory neuropeptide, has proved beneficial in a mice model of human CD: trinitrobenzene sulfonic acid (TNBS)-colitis. The aim of the present study was to determine the molecular mechanisms of action of VIP on this model of colitis studying the time-course expression of proinflammatory and regulatory mediators implicated in the pathophysiology of the disease. Gene array and real-time PCR analysis were performed to measure mRNA levels of several proinflammatory mediators, such as cyclooxygenase-2 (COX-2), CCR5, IL-1 beta, IL-6, IL-12, IL-18, IFN gamma and IL-4. TNBS-mice showed an increased expression of all proinflammatory mediators studied, with a specific time course pattern for each molecule. Besides, TNBS-treated animals showed a Th1 lymphocytic response with high levels of IFN gamma mRNA but low IL-4, correlated with a high expression of IL-12 and IL-18, two Th1 differentiating factors. VIP downregulates the expression of all these mediators, exerting an antiinflammatory function that explains the remission of the disease in the treated animals. VIP also favours the Th2 versus Th1 response, contributing to the remission of the disease. The results support a potential therapeutic action of VIP for the therapy of inflammatory diseases.
Role of substance P/NK-1 receptor system in the vanilloid VR1 receptor-mediated capsaicin-induced apoptosis of rat thymocytes C. Amantini, R. Lucciarini, M. Mosca, D. Martarelli, M. Perfumi, P. Ballarini and G. Santoni University of Camerino, Camerino, Italy
We investigated the involvement of the substance P/NK-1 receptor system in the regulation of the vanilloid receptor (VR1)-mediated capsaicin (CPS)-induced apoptosis in rat CD5+ thymocytes. We evaluated by confocal laser scanner microscopy analysis the expression of substance P (SP) and its NK-1 receptor in CD5+VR1+ rat thymocytes. We found that the NK-1 and VR1 receptors completely colocalize in the plasma membrane of CD5+ rat thymocytes, while only a moderate colocalization was detected by comparing the expression of SP in CD5+VR1+ rat thymocytes. Evidence of the involvement of the SP/NK-1 receptor in the CPS-induced apoptosis have been provided by analyzing the capability of SP to protect CD5+ rat thymocytes from CPS-mediated apoptotic cell death. SP inhibits in a dose-and time-dependent manner VR1-mediated CPS-induced apoptosis of rat CD5+ thymocytes, as evaluated by its ability to completely reverse the CPS-induced caspase-3 activation and oligonucleosomal DNA fragmentation. The specific NK-1 receptor antagonist, SR140333 completely reverses the anti-apoptotic effect of SP on the VR1-mediated CPS-induced apoptosis of CD5+ rat thymocytes. CPS markedly inhibits in a time-dependent manner the expression of SP in CD5+ rat thymocytes and a specific VR1 antagonist, capsazepine markedly blocks in a time-dependent manner the CPS-induced SP synthesis/release in CD5+ rat thymocytes. Neuropeptide Y (NPY) is involved in the modulation of functions related to immune and inflammatory reactions such as lymphocytes proliferation, NK cell activity, immune cell trafficking, cytokine secretion and release of oxidative agents. NPY exerts biological effects through several receptor subtypes (named from Y1 to Y5, in the rat). We have investigated the effect of NPY and NPY-related peptides with different pharmacological profiles on NO and H 2 O 2 production in rat granulocytes obtained from rat air pouch. Carrageenan (20 Ag/rat) was injected directly into the air pouch. Granulocytes obtained 24 h later by lavage were treated in vitro with NPY, peptide YY (PYY), pancreatic polypeptide (PP, Y4 receptor agonist), LeuPro-NPY (Y1,5 receptor agonist), NPY13-36 (Y2,5 receptor agonist) and hAlaAib-NPY (Y5 receptor agonist) in concentrations 10 À6 -10 À12 M, and stimulated with LPS (1 Ag/ml, 48 h) for NO or zymosan (125 Ag/ml, 30 min) for H 2 O 2 production. Both NPY and PYY stimulated basal and LPS-induced NO production in granulocytes, and LeuPro-NPY potentiated NO release from LPSstimulated cells. NPY13-36 and hAlaAib-NPY decreased NO production in cells cultured in medium. NPY and LeuPro-NPY decreased peroxide production in zymosan-stimulated granulocytes. It is concluded that NPY and NPY-related peptides differentially modulated NO and H 2 O 2 production in rat granulocytes. (Supported by MSTD, Republic of Serbia, Grant 1239).
The induction of interleukin-1 (IL-1B) beta and interleukin-12 (IL-12) by substance P (SP) in human peripheral blood mononuclear cells (PBMC) M. Braitch, M. Chopra, A. Fahey and C. Constantinescu University of Nottingham, Nottingham, UK Substance P (SP), neuropeptide stored in the termini of sensory neurons, induces pro-inflammatory cytokines and mediates neurogenic inflammation. Aim: To examine the effects of SP on the production of interleukin-1 beta (IL-1h) and interleukin-12 (IL-12) from peripheral blood mononuclear cells (PBMCs). Method: PBMCs were stimulated with SP alone (10 À6 -10 À12 M) and in combination with Lipopolysaccharide (LPS) or Phytohaemagglutinin (PHA). Supernatants were analysed for IL-1h, IL-12 p40 and IL-12 p40/p70 by ELISA. Results: 10 À6 M SP alone and in combination with LPS and PHA induced a significant increase in IL-1h production by PBMCs. PHA was a strong inducer but in concert with other stimuli LPS proved more effective. After 24-h, 10 À6 M SP induced the production of IL-12 p40 and IL-12p40/p70 secretion alone and in combination with LPS and PHA. As the concentration of SP decreased, the amount of IL-12 produced declined. This effect was more profound after 48-h stimulation. Conclusion: These studies confirm the ability of SP to enhance IL-1h production by human PBMC cells and show for the first time the production of IL-12 from human PBMCs by SP. The ability of SP to augment secretion of pro-inflammatory cytokines supports the role of SP in the inflammatory response. SP antagonists may represent future tools for down-regulating inflammation in environment rich in SP including the peripheral nerves, gut, inflamed joints and brain.
Interactions between neuro-endocrine and immune systems in ageing: trade-off analysis by means of mathematical modeling T. Sannikova a , A. Romanyukha a and A. Yashin b a Institute of Numerical Mathematics, RAS, Moscow, Russia; b Max Planck Institute for Demographic Research, Rostock, Germany
The aim of this work is to quantify the trade-offs between human immune and hormonal systems in the aging process. For that purpose, we construct mathematical model that describes mutual influences arising during agerelated decline in functioning of immune and endocrine systems. We suppose that this closely coordinated systems change under the allostatic and antigen load actions. These two types of environmental pressure affect the rate of resource exhausting for each of the systems. Neuro-endocrine and immune systems are responsible for maintenance of homeostasis of the internal milieu and intensity of the energy metabolism. On the other hand, parameters of the machine of the energy metabolism determine the activity of immune and endocrine systems. Dynamics of the cell concentrations, the rate of the hormonal synthesis and intensity of the energy transformation and consumption processes take place in mitochondria are modeled by the ODE system. The results of the simulation reveal that there are explicit trade-offs between immune space and power of hormonal reactions. Existence of the trade-offs should be taken in consideration when hormonal therapy is used in elderly people. Maintenance of body weight around a set point requires regulatory processes as energy homeostasis and depends on tightly regulated interactions between nervous, immune and endocrine systems. Thus, obesity may result from energy homeostasis deregulation of multiple causes; including viral infection, as association of human obesity and virus has been reported. Our mouse model of obesity induced by brain CDV infection may give insights in how virus-induced brain damages may lead to obesity. This negative-stranded RNA virus predominantly targets neurons located in hypothalamus, a key structure for integration of central and peripheral signals regulating energy homeostasis. Obesity syndrome, associated with persistence of virus in hypothalamus, could be due to specific neuronal loss and/or epigenetic alteration of hypothalamic genes involved in food intake, energetic control and basal metabolism. To identify these genes, we used differential display PCR (DDRT-PCR) and subtractive libraries and compared hypothalamic mRNA profiles from infected-lean and -obese mice. Among the identified deregulated genes, UROP 11 was selected according to the magnitude of increase and novelty of sequence. Its full-length sequence and cellular central and peripheral localization were determined. The functional relevance of UROP11 in obesity was demonstrated in other obesity models (leptin deficient mice ob/ob and db/db; high fat diet). Our data pointed out Urop11 as a new gene, implicated in emerging or maintenance of obesity status, likely regulated under a leptin control.
IL-18 modulates CRH and prostaglandin production from the rat hypothalamus in vitro G. Tringali, M. Vairano, G. Pozzoli and P. Navarra Institute of Pharmacology, Catholic University School Medicine, Rome, Italy
Interleukin-18 (IL-18) is a pro-inflammatory cytokine showing similarities with IL-1beta in structure, receptor and signalling mechanisms, as well as biological functions. We have previously shown that IL-1beta directly stimulates corticotrophin-releasing hormone (CRH) secretion from the rat hypothalamus via a mechanism requiring an increased prostaglandins production. Here we investigated whether: (a) IL-18 shares with IL-1beta the ability to stimulate CRH gene expression and peptide release from rat hypothalamic explants in short-term (1 and 3 h) in vitro incubations; (b) IL-18 modulates basal and IL-1beta stimulated prostaglandins production in hypothalamic explants as well as in primary cultures of rat cortical microglia and astrocytes in 24-h experiments. CRH released in the incubation medium was evaluated by radioimmunoassay (RIA), whereas CRH mRNA levels were analysed using RNase protection assay. PGE2 production was assessed by RIA. We found that IL-18 decreases in a concentration-dependent manner both basal and KCl-stimulated CRH release. These findings were paralleled by a significant decrease in CRH gene expression. IL-18 had no effect on basal PGE2 production by hypothalamic explants but completely abolished production stimulated by IL-1beta. Moreover, IL-18 produced a reduction in both basal and IL-1betastimulated PG2 production and release from microglia and astrocytes. These data clearly demonstrate that IL-18 shows a profile of neuroendocrine activities in vitro that are opposed to, and even antagonizing, those of IL-1beta in the control of stress axis.
Endothelin-1 increases the release of prostaglandin (PG) E2 from the preoptic area of rat anterior hypothalamus POA in vitro without altering cytokine gene expression A.S.C. Fabricio a , G. Tringali a , G. Pozzoli a , G.E.P. Souza b and P. Navarra a a Institute of Pharmacology, Catholic University Medical School, Rome, Italy; b University of São Paulo Faculty of Pharmaceutical Sciences, Ribeirão Preto, Brazil Aims: We have previously shown that fever is induced in rats by injection of endothelin-1 (ET-1) into the preoptic area of rat anterior hypothalamus (POA) while i.c.v. ET-1 induces an increase in cerebrospinal fluid prostaglandin (PG) levels. Here we investigated if ET-1 increases the release of PGE 2 from the POA or posterior hypothalamus (PHyp) explants and if this effect is related to changes in cytokine gene expression. Methods and results: Male Wistar rats (250 g) had the whole hypothalamic region explanted and divided at the optic chiasm to obtain isolated POA and Phyp areas. In 1-h incubation experiments, ET-1 induced a concentrationdependent increase in immunoreactive PGE 2 release in the incubation medium from the whole hypothalamus, with statistical significance at 10 and 100 nM. ET-1 (100 nM) also induced an increase in PGE 2 release from the POA but not from the PHyp. RNase protection analyses of total RNA extracted from the explants showed that 100 nM ET-1 produced no effect on interleukin (IL)-1alpha, IL-1beta and IL-6 gene expression. Conclusion: ET-1 induces an increase of the PGE 2 release at the major thermoregulatory area in the brain, i.e. the POA. The acute stimulatory effect of ET-1 on PGE 2 release from the POA does not appear to be related to an action on cytokine mRNA expression.
Orchidectomy affects the monoamine content in rat thymocytes B. Vidic-Dankovic a , K. Radojevic a and G. Leposavic b a Institute of Immunology and Virology bTorlakQ, Belgrade, Yugoslavia; b Faculty of Pharmacy, Belgrade, Yugoslavia Object of study: It has been shown that: (i) orchidectomy influences the intrathymic concentration of monoamines and (ii) some T-cell lines produce catecholamines. Therefore, this study was designed to investigate: (i) whether thymocytes contain detectable level of monoamines and (ii) if so, whether orchidectomy affects the intrathymocyte monoamine content. Methods: The concentrations of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) were estimated in the whole thymus and in thymocyte suspensions from AO rats one month post bilateral orchidectomy performed at age of either 30 or 60 days, by HPLC with electrochemical detection. Results: In spite of age at surgery, orchidectomy diminished the intrathymic concentration of NA and 5-HT, while increased that of DA. The thymocytes from both orchidectomized (ORX) and control (sham-ORX) rats showed detectable levels of NA and 5-HT, but not that of DA. Orchidectomy, in both groups of rats, increased the intrathymocyte concentration of NA. The intrathymocyte concentration of 5-HT was increased in rats ORX at age of 60 days, but it was reduced in those ORX at age of 30 days. Conclusions: The results revealed detectable levels of NA and 5-HT in rat thymocytes, and showed that the intrathymocyte levels of these monoamines are influenced by gonadal hormone milieu.
Regulation of dipeptidyl peptidase IV (DPPIV/CD26) on human dermal fibroblasts by endomorphin-2 J. Gabrilovac a , B. Cupic a and J. Jakic-Razumovic b a Ruder Boškovic Institute, Zagreb, Croatia; b Clinical Hospital Centre Rebro, Zagreb, Croatia DPPIV is a membrane-bound peptidase that cleaves dipeptides from Nterminal of the polypeptides if proline is at the penultimate position. Besides the enzyme activity, DPPIV can act as an adhesive and as a costimulatory molecule transducing the growth signal. Its expression is regulated by cytokines (IL-1-alpha, IL-12, TNF-alpha). Neuropeptides can also regulate the expression of membrane peptidases that in turn cleave the neuropeptides. We examined whether opioid peptide endomorphin-2 (Tyr-Pro-Phe-Phe-NH2; E-2), which is a physiological DPPIV substrate, could affect the expression of DPPIV on human dermal fibroblasts. The role of the cleavage site in the interaction was examined by using a synthetic tetrapeptideTyr-D-Pro-Phe-Phe-NH2 (DE-2), which is resistant to cleavage by DPPIV. The role of opioid receptors was examined by using an opioid receptor antagonist, naloxone. DPPIV enzyme activity was measured by means of hydrolysis of fluorochrome-labeled substrate and by immunohistochemical staining. Time-and concentration-dependent up-regulation of DPPIV with both endogenous E-2 (DPPIV sensitive) and synthetic DE-2 (DPPIV resistant) was observed. However, naloxone abrogated or diminished only the effects of DPPIV-resistant DE-2, suggesting that E-2 and DE-2 may use different mechanisms in regulating DPPIV. Collectively, the data show that endomorphin-2 can up-regulate DPPIV expression on dermal fibroblasts either via an interaction with the enzyme itself or via the opioid receptor. Increased DPPIV expression on fibroblasts may affect their functional activity and growth. ET [Br. J. Pharmacol. 136 (2002) 947-955] . The aim of the present study is to investigate the effects of ICV injections of PAT on the increased intracerebral levels of cytokines induced by ICV injection of ET. Methods: Several groups of rats received ICV injections of either ET only (1.5 Ag in 2 Al) or ET preceded by PAT (0.2, 1, or 5 Ag in 2 Al). After 4 h, the brain tissues were sampled from the brainstem, diencephalon and hippocampus for the determination of the levels of tumor necrosis factor (TNF) alpha, interleukins (IL) 1 beta and 6 by ELISA. Results: Pretreatment with PAT produced a dose-related alteration of the increased levels of all cytokines by ET. Injection of PAT only did not produce significant alteration of the basal concentration of cytokines in the brain. Conclusion: The observed results provide evidence on the potential protective role of PAT against brain tissue damage during inflammatory conditions.
Melatonin receptors in goat membranes lymphocytes: light and stress regulation G. Carrillo and C.I. Gutierrez Universidad Nacional Experimental bFrancisco de MirandaQ (UNEFM), Coro, Estado Falcón, Venezuela
Neuroendocrine and immunological systems are connected structurally and functionally. There are anatomical, physiological and pharmacological evidences that supported this close interaction. The melatonin neurohormone plays an important role in the immune system, which is also supported by the existence of melatonin-specific binding sites in lymphoid cells, central nervous system and peripheral organs. Goats are domestic animals, widespread in Venezuela, especially in arid zones like Falcó n state; they have lower incidence of neoplasic disease and lower susceptibility to infections. The aim of this work was to characterize melatonin receptors in membranes lymphocytes of goat's peripherical blood through binding studies with [ 3 H]iodomelatonin. The binding was dependent of time, temperature and cellular concentration, also stable, reversible and saturable. Saturation assays demonstrated a high-affinity binding site (K d =3.6F0.21 nM) and low capacity (B max =335.2F50.2 fmol/mg). The affinity suggests that caprines receptors may recognize serum melatonin level physiological. When the animals were stressed by immersion and immobilization in water, we find an increased affinity without capacity modification, but under light constant for 7 days only the capacity increased. The results indicate that adult goats have high-affinity receptors to melatonin in lymphocytes and under different conditions vary and show the interaction and communication between pineal gland and immune system.
Regulation of secretion of nociceptin/orphanin FQ from rat splenocytes A. Fulford University of Bristol, Bristol, UK N/OFQ mRNA is expressed by various immunocyte populations although the significance of endogenous N/OFQ in the immune system is poorly understood. In this study, the regulation of N/OFQ secretion in rat splenocytes was investigated. Splenocytes (5Â10 6 cells/ml) were cultured with varying concentrations of mitogens or inflammatory mediators. Production of N/OFQ after stimulation by LPS (1 or 5 Ag/ml), Con A (1 or 5 Ag/ml) and IL-1 beta (5-20 ng/ml) was measured after 24-or 48-h incubation (37 8C in 95% O 2 /5% CO 2 ). Additionally, splenocytes were activated with Con A (5 Ag/ml) for 24 h prior to the addition of CRF (10 À12 -10 À8 M) for 24 h. Supernatants were harvested and retained for measurement of N/OFQ content by EIA. LPS and Con A caused dosedependent stimulation of N/OFQ secretion confirming that B and T cells have the capacity to secrete N/OFQ (basal secretion in ng/ml) at 48 h 0.06F0.009; LPS 5 Ag/ml 0.13F0.03; Con A 5 Ag/ml 0.16F0.04, n=4/5, pb0.05). IL-1 beta stimulation of splenocytes for 24 h also stimulated N/ OFQ secretion (basal 0.093F0.006; IL-1 beta (20 ng/ml) 0.175F0.02, n=4/ 5) although lower doses of IL-1 beta were without effect. N/OFQ secretion from Con A-activated splenocytes was enhanced by CRF in vitro (30% increase by CRF 10 À12 M vs. Con A alone and reversible with alpha-helical CRF 10 À7 M, n=4/5). These observations are consistent with the findings for other opioids and identify possible sources of immune-derived N/OFQ that may contribute to the inflammatory process. Rationale: To characterize beta2-adrenoceptor (b2AR)-mediated modulation of growth and function of human intestinal mast cells (MC). Methods: MC were isolated from intestinal surgery specimens, purified, and subsequently cultured for 14-21 days in the presence of stem cell factor (50 ng/ml) with our without addition of adrenaline or salbutamol. MC adhesion assays were performed using fibronectin-coated plates as well as human umbilical vein endothelial cells (HUVEC). Cellular F-actin content was measured by flow cytometry after labeling with fluorescent phallacidin. Results: The b2AR agonists dose-dependently reduced the MC growth to 23.4% (salbutamol) and 25.3% (adrenaline) of control conditions (at 10 À6 M, n=5) which was mainly attributable to a strong inhibition of MC proliferation. Moreover, the b2AR agonists (at 10 À6 M) abolished adhesion of MC to HUVEC monolayers as well as adhesion to fibronectin-coated plates following MC activation (n=5). The cellular F-actin content decreased to 70% of basal levels following challenge of the MC with the b2AR agonists for 5 min, whereas, stimulation of the MC by IgE receptor crosslinking lead to a rapid doubling in F-actin content. This activationinduced increase in F-actin content, however, was abrogated in the presence of the b2AR agonists (n=3). Conclusions: Our data show that b2AR agonists regulate human intestinal MC proliferation and interaction with HUVEC and fibronectin by mechanisms that possibly involve an alteration of cellular F-actin homeostasis.
Electrical stimulation of the mesencephalic periaqueductal gray matter in rats alters NK cell function A. Pert a and R. Weber b a National Institutes of Health, Bethesda, USA; b University of Illinois College of Medicine, Peoria, USA
We have previously reported [Science 245 (1989) 188-190 ) that microinjections of morphine into the periaqueductal gray matter (PAG) of the rat midbrain suppresses natural killer (NK) cell activity, thus implicating this brain region in the regulation of immune function by opioids. The purpose of this study was to examine the effects of electrical stimulation of the PAG on natural killer cell (NK) activity. Bipolar electrodes were introduced into various aspects of the periventricular-periaqueductal axis from its rostral to caudal extent. One week following surgery, animals were stimulated with intermittent trains of biphasic pulses for 20 min and splenic NK cell activity was determined 3 h following termination of stimulation. It was found that stimulation of sites rostral to the dorsal raphe nucleus was relatively ineffective in decreasing NK cell activity while 11 of the 14 sites in the caudal PAG caused significant suppression of NK cell activity. The sites most active were found in the ventral PAG and its caudal aspect at the level of the dorsal raphe nucleus. Studies are underway to determine the neural and peripheral circuitry through which electrical stimulation of the PAG gains access to the immune system.
Exogenous dehydroepiandrosterone replacement in humans induces regulatory T cells S.A.J. Thompson a , S. Curran b , A. Cox a , K. Chatterjee b and A. Coles a a Department of Clinical Neurosciences, University of Cambridge, UK; b Department of Endocrinology, University of Cambridge, UK DHEA, an adrenal steroid, declines with age and has been implicated in neurodegeneration. We took advantage of an unusual cohort of patients to study the immunological effect of exogenous DHEA replacement. Patients with Addison's disease, autoimmune gland destruction, report persistent fatigue with reduced quality of life and cognitive abilities, despite standard corticosteroid replacement therapy. It has been previously shown that restoration of serum DHEA levels by oral administration enhances cognition and reduces fatigue. PBMCs were extracted from patients before, during and after treatment with DHEA. NK and NKT cells were defined by FACS of surface cell markers CD3/CD56 and regulatory T cells identified as CD4 + CD25 hi CD45RO + . Cytokine protein and mRNA were measured by ELISA and real-time quantification. We found that DHEA treatment of patients with Addison's disease reduced peripheral NK and NKT cell numbers. It also induced lymphocyte cytokine mRNA and protein expression, without a particular bias to any T helper phenotype. CD4 + CD25 hi cell numbers and foxP3 expression was also increased, suggesting that DHEA had induced regulatory T lymphocytes. We conclude that oral DHEA treatment has significant effects on the immune system.
Modulation of immune responses in mice exposed to toluene by noseonly inhalation H. Fujimaki a , S. Yamamoto a , M. Kakeyama a , Y. Kurokawa a , N. Kunugita b , H. Hori b and K. Arashidani b a National Institute for Environmental Studies, Ibaraki, Japan; b University of Occupational and Environmental Health, Fukuoka, Japan
In this study, we examined the effect of low level toluene inhalation on immune and brain regions. Male C3H/He mice were exposed to filtered air (control), 9 and 90 ppm toluene for 30 min by nose-only inhalation on day 1, 2, 3, 7, 14, 21, and 28. One day after the last inhalation, mice were anesthetized by injection with Nembutal sodium, and blood, thymus, spleen, olfactory bulb and hippocampus were collected. In spleen cells, the percentage of CD3 + and CD4 + T cells was significantly decreased in toluene-exposed mice. However, the percentage of CD19 + cells in tolueneexposed mice was increased compared with air-exposed mice. Mitogenic stimulation by PHA and LPS induced the production of IL-4 and IFNgamma from spleen cells of control mice, but exposure to toluene gave no significant increase in the cytokine production. Total IgE production in plasma from toluene-exposed mice significantly decreased. It is possible to speculate that toluene inhalation may affect immune functions in brain regions via olfactory system. In olfactory bulb and hippocampus of tolueneexposed mice, the production of nerve growth factor, TNF-alpha, and IL-1beta was equivalent to the control mice. These things suggest that exposure of mice to low dose toluene via intranasal route may induce immune abnormalities. Having in mind that the neuroendocrine system products might modulate intrathymic maturation of T cells, the aim of this study was to investigate whether somatostatin-14, centrally applied, alters relations between thymocyte subpopulations. Adult male AO rats were cannulated intracerebroventriculary and treated with three doses of SRIH-14, in the concentration of 0.6Â10 À10 M per day, or saline, every other day. Animals were sacrificed 24 h after the last treatment. Thymuses were processed for analysis of thymocyte subpopulations and apoptosis by flow cytometry, and for evaluation of morphometric parameters by stereological analysis. Our results showed that SRIH-14 significantly decreased volume of the thymus cortex accompanied with decrease of cellularity and the number of thymocytes, per volume unit, in it and increased the percentage of apoptotic cells. The percentages of CD4 À CD8 À and the both single positive thymocyte subpopulations were significantly increased, while the percentage of CD4 + CD8 + cells was diminished. The analyses of cell subsets in the relative proportion to expression of TCRah revealed increase in the percentages of the least mature CD4 À CD8 À TCRah À/low and CD4 À CD8 + TCRah À cells and the most mature CD4 À CD8 À TCRah hi , CD4 + CD8 À TCRah hi and CD4 À CD8 + TCRah hi cells, while the percentage of positively selected CD4 + CD8 + TCRah hi cells was decreased. This increase in the percentages of the least mature and the most mature thymocyte subsets, suggest the involvement of somatostatin-14 applied intracerebroventriculary in the modulation of T cell maturation, whose consequence could be appearance of CD4 À CD8 À TCRah + regulatory T cell subset. Thyroid hormone regulation of immune responses was demonstrated. In addition, differential expression of protein kinase C (PKC) isoenzymes and high nitric oxide synthase (NOS) activity were described on tumor respect to normal T lymphocytes. l-Thyroxine (T4)-mediated actions on both enzymatic activities, related to T lymphocyte and BW5147 T lymphoma cell proliferation were studied. Thyroid hormones increased tumor, but not normal, cell proliferation and potentiated mitogen-stimulation of T lymphocytes as measured by tritiated-thymidine incorporation. Twentyfour-hour incubation with T4 induced a rise in total and membraneassociated PKC activities, as measured by [gamma-32 P]-ATP phosphorylation of a PKC specific substrate, on both cell types. In addition, T4 potentiated mitogen-induced PKC translocation in normal T lymphocytes and lead to a rapid and transient effect on tumor cells. T4 increased atypical PKC zeta expression on BW5147, but classical PKC isoenzymes on mitogen-stimulated normal T cells by immunoblotting analysis. Additionally, T4 augmented NOS activity, measured by production of [U-14 C]citrulline from [U-14 C]-arginine, only on tumor cells. These results show, for the first time, differential intracellular signals involved in T4 modulation of lymphocyte biology.
Substance P involvement in chemotactic activity of human brain endothelium C. Cioni and P. Annunziata
In the last few years, the mechanism underlying the recruitment of T lymphocytes through the blood-brain barrier has been receiving increasing attention. Recently, substance P (SP), a neuropeptide with a number of proinflammatory properties, has been found to be produced and released by cytokine-stimulated brain endothelium in rodents and to mediate leakage and activation of brain endothelium cultures exposed to proinflammatory cytokines. To test whether SP could exert chemotactic activity at the human blood-brain barrier, we analysed SP production by human brain endothelium (HBE) cultures exposed to proinflammatory cytokines (TNF-alpha, IFN-gamma) and tested whether SP may mediate the chemotactic activity of cytokine-stimulated HBE for human T lymphocytes. Biotin-labelled T lymphocytes were seeded on the membrane and HBE cells on the bottom of a Boyden chamber. Biotin-labelled T cells that migrated to the chamber bottom were assayed by ELISA. SP immunoreactivity was found on the surface of cytokine-stimulated human brain endothelial cells and in the culture supernatants. Low rate (11%) of T cells migrated through chamber membrane in the presence of unstimulated HBE, increasing up more than 50% when HBE was exposed to proinflammatory cytokines. This percentage returned to baseline values when spantide, a SP antagonist or anti-SP polyclonal antibody was used. These findings demonstrate that SP is involved in chemotactic activity of in vitro cytokine-stimulated HBE. Objective: To analyze beta adrenergic receptor (BAR) regulation of T lymphocyte proliferation in mice according to different thyroid status. Methods: Specific radioligand binding assays were performed on T cells from eu-, hypothyroid (by propylthiouracil treatment) and hyperthyroid (by thyroxine-T4-administration) mice. Beta-agonist isoproterenol effects on intracellular levels of cyclic AMP (cAMP) were determined. Mitogeninduced T-cell proliferation was measured by tritiated-thymidine incorporation. Protein kinase C (PKC) activity in cytosol and membrane were determined using radiolabelled enzymatic substrates. Results: A decrease or a not significant increase in BAR number was found, respectively, on T cells from hypo-and hyperthyroid and ISO-stimulation of cAMP was lower in hypothyroid and higher in hyperthyroid T lymphocytes, respect to controls. T-selective mitogen-induced proliferation was increased in hyperthyroidism but decreased in hypothyroidism. BAR was downregulated during the proliferation peak in all animals. A higher or a lower decrease was observed respectively in hyper-and hypothyroid T cells. A higher translocation of PKC activity in hyperthyroid cells, and a lower one in hypothyroid lymphocytes was observed. Conclusions: Intracellular signals triggered by mitogen activation would be related to differential BAR down-regulation in T lymphocytes depending on the thyroid status, contributing to the distinct proliferative responses found in hypo-or hyperthyroidism. Endogenous catecholamines (CA) in peripheral blood mononuclear cells (PBMCs) contribute to activation-induced apoptosis. PBMCs from multiple sclerosis (MS) patients are less susceptible to apoptosis and we previously showed dysregulated CA production under phytohaemagglutinin (PHA)stimulation. Since interferon (IFN)-beta positively modifies MS course, we investigated CA production, expression of mRNA for tyrosine hydroxylase (TH, the limiting enzyme in CA synthesis), and apoptosis in PBMCs from patients undergoing treatment. So far, 38 relapsing-remitting MS patients were included: data are presently available from 27, 23, 13, 6 patients with 1, 3, 6, 12 months follow-up, respectively. TH mRNA increased during the first 3 months, then decreased to baseline levels. CA production and efflux increased similarly in the first quarter, then maintained high plateau levels. The capacity of IFN-gamma added in vitro to inhibit CA production decreased progressively and the antagonistic effect of IFN-beta in vitro increased consistently with treatment. Pharmacological inhibition of CA production significantly reduces apoptosis in healthy subjects: this effect was not observed in MS patients, even during IFN-beta therapy. Our results suggest that IFN-beta treatment antagonises IFN-gamma on TH mRNA expression and CA production, but its relevance on activation-induced apoptosis remains unclear. A longer follow-up is warranted to assess possible differences between responders and non-responders to IFN-beta. EAE is a animal chronic inflammatory demyelinating disease of the central nervous system (CNS) serving as a useful model for human multiple sclerosis (MS). Previous studies have shown that the severity of MS and EAE is reduced by increased level of sex hormones during pregnancy. In this study, we demonstrate that estrogen treatment led to the induction of a novel subpopulation of regulatory cells, which also occurs naturally in pregnant mice. These previously uncharacterized cells display a low level expression of CD45 (CD45 dim ), and no detectable expression of many cell surface markers related to TCR signaling, including CD3 and TCR. However, these cells retained expression of VLA-4. Several lines of evidence suggest that these novel cells defined as CD45 dim VLA-4 + may play a role in the protective effects of estrogen on EAE. Injection of purified CD45 dim VLA-4 + cells conferred protection from spontaneous EAE (Sp-EAE). CD45 dim VLA-4 + cells also suppressed antigen-specific proliferation of primed lymphocytes in co-culture. A better understanding of how CD45 dim VLA-4 + cells suppress harmful immune response of EAE may explain the induction of immune tolerance during pregnancy and lead to novel therapeutic approaches to combat MS and other autoimmune diseases.
Effects of exposure to formaldehyde and NO 2 on neurotransmitterrelated mRNAs expression in the mouse brain M. Kakeyama a , Y. Kurokawa a , S. Yamamoto a , R. Hojo a , N. Kunigita b , H. Hori b , K. Arashidani b and H. Fujimaki a a Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba, Japan; b University of Occupational and Environmental Health, Kita-Kyushu, Japan
Effects of low-dose exposure to two types of air pollutants, formaldehyde (FA) and nitrogen dioxide (NO 2 ) on the brain neural transmitter-related mRNAs were examined in mice. Female C3H/He mice (10 weeks old) were exposed to 400 ppb of FA for 12 weeks (16-h daily, 5 days/week), or 1 ppm of NO 2 for 3 weeks and levels of expression of glutamate receptor subunits (GluR1, GluR2, epsilon1 and epsilon2), dopamine receptors (D1) and serotonin receptors (5-HT1A) mRNAs were semi-quantified by RT-PCR in the neocortex (NC), the hippocampus (HIP), the amygdala (AMG), and the hypothalamus (HT). Exposure to FA resulted in the increase in epsilon1 mRNA levels in the NC and HIP, epsilon1, epsilon2 and D1 mRNAs in the AMG, and 5-HT1A mRNA in the HT, and reduced epsilon 2 mRNA levels in the NC and HIP. On the other hand, exposure to NO 2 caused a reduction of these all mRNAs in NC, HIP and HT. These results indicate that the brain neurotransmitter-related mRNAs can be a sensitive marker for effects of exposure to air pollutants. In addition, there arise a possibility that difference between patterns of changes in neurotransmitter-related mRNAs after exposure to FA or NO 2 reflects the difference in neurotoxicity between FA and NO 2 . Rationale: Cyclosporin A (CsA) attenuates antinociceptory effects of morphine, development and expression of morphine-induced tolerance and dependency via nitric oxide pathway. Objectives: In our study, the effect of systemic CsA on the morphine-induced conditioned place preference (CPP) and the probable involvement of nitric oxide were assessed in mice. Methods: The effect of morphine (0.5-10 mg/kg), CsA (5, 10, 20 mg/kg) and N G -nitro-l-arginine methyl ester, l-NAME, (2.5, 5, 10 mg/kg) administration on the induction of CPP was assessed. The effects of CsA, l-NAME and also co-administration of CsA (1, 2.5, 5 mg/ kg) and l-NAME (2.5 mg/kg) on the acquisition of CPP induced by morphine (5 mg/kg) were studied. Results: Our data showed that administration of morphine significantly increased the time spent in the drug-paired compartment in a dose-dependent manner. CsA (5, 10 mg/kg) and l-NAME (2.5, 5, 10 mg/kg) did not induce either CPP or conditioned place aversion (CPA), while CsA (20 mg/kg) induced CPA. Both CsA (10, 20 mg/kg) and l-NAME (5, 10 mg/kg), in combination with morphine (5 mg/kg) during conditioning, significantly suppressed acquisition of morphine-induced CPP. Lower and per se non-effective doses of CsA (1, 2.5, 5 mg/kg) and l-NAME (2.5 mg/kg), while coadministered, exerted a significant potentiating inhibitory effect on morphine-induced CPP. Conclusions: CsA attenuates the reinforcing effects of morphine. This effect is potentiated by inhibition of nitric oxide synthase.
Cortisol, cytokines and symptom severity in patients with schizophrenia during treatment with risperidone and haloperidol X.Y. Zhang Peking University, Beijing, P.R. China Backgrounds: The aim of this study was to investigate the inter-relationships among cortisol, cytokines and symptoms in schizophrenia, and to compare the effects of typical and atypical antipsychotic drugs on these variables. Methods: Seventy-eight schizophrenia were randomly assigned to 12 weeks of treatment with 6 mg/day of risperidone or 20 mg/day of haloperidol using a double-blind design. Clinical efficacy was determined using PANSS. Serum cortisol and IL-2 was assayed by RIA, and serum IL-6 by quantitative ELISA, as compared to 30 sex-and age-matched normal subjects. Results: Serum levels of cortisol, IL-2 and IL-6 were elevated in schizophrenia (all pb0.05). The elevated cortisol was associated with the increased IL-2 and IL-6 in schizophrenia, respectively (both pb0.01). Cortisol was associated with negative symptoms ( pb0.05) and IL-2 with positive symptoms ( pb0.05). As compared with haloperidol, risperidone decreased the elevated cortisol and improved negative symptoms significantly (both pb0.05), but had similar effects on IL-2 and IL-6 as well as on positive symptoms (all pN0.05). The improvement of negative symptoms of schizophrenia was related to the change in cortisol (r=0.34, df=69, pb0.01). Conclusions: Schizophrenia may have hypothalamic-pituitary-adrenal (HPA) axis and cytokines dysregulation, which may be implicated in clinical symptoms of schizophrenia, and further in the response to antipsychotic treatment. The aim of this study was to investigate perceived stress, neuroimmune function, Th1/Th2 cytokine balance and allergy symptoms in medical students, with and without atopy, during a calm and stressful (exam) period of academic studies. Among several studied parameters, results from questionnaires, phenotyping of white blood cells, cytokine assays, urine cortisol measurements are presented. Participants perceived elevated stress in response to examination, which was accompanied with an increase in urine cortisol. The CD4/CD8 ratio was increased in both groups during stress, while the total number of T cells was unaltered. The number of NK cells decreased in atopic subjects during stress. Interestingly, regulatory T cells increased during stress in both groups. The cytokine profiles in atopic subjects reflected a Th2-like predominance in response to stress, which was not seen in healthy subjects. We conclude that perceived stress, Th1/Th2 cytokine profiles and important immunoregulatory lymphocyte subsets were observed in response to examination. Some immune parameters, such as NK cell numbers and cytokine profiles were differentially regulated in atopic and healthy individuals in response to stress. However, other parameters, such as the CD4/CD8 ratio and the number of regulatory T cells, were similarly regulated in both groups. Our results indicate that healthy and atopic subjects share certain features of a stress response but differ in others. The autoimmune sialadenitis developed by NOD mice is a suitable model to study the ethiopathogenic mechanisms leading to sicca symptoms in Sjfgren's syndrome. As both nitric oxide and vasoactive intestinal peptide (VIP) are common messengers to nervous and immune systems mediating secretory and inflammatory responses, we examined nitric oxide synthase activity with special focus on VIP-mediated effects in salivary glands of NOD mice and Th1/Th2 cytokine balance in sera and glands. We measured NOS by the conversion of l-[U-14 C]arginine to citruline in submandibular and parotid glands of NOD and BALB/c mice and the levels of cytokines in sera, supernatants of splenocytes and homogenates of salivary glands. We found a decreased NOS activity and NO-mediated signaling in salivary glands of NOD mice that starts at 12 weeks coincident with the onset of salivary dysfunction. The increase of IL-12, TNF-alpha and IFN-gamma levels in sera was detected 3 weeks later and at this time no changes in cytokine levels could be stated in salivary glands. Infiltrates in the glands appeared at 20 weeks as well as histological lesions. Our results support the hypothesis of an impaired NO production and signaling in salivary glands as early events to take place in NOD glands and these events precede the autoimmune response. Autoantibodies to the brain opiate and glutamate receptors as putative biomarkers of morphine addiction O. Granstrem a , W. Adriani b , D. Giannakopoulou b , S. Dambinova c , G. Izykenova c and G. Laviola b a IP Pavlov's State Medical University, St. Petersburg, Russia; b Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità , Rome, Italy; c Emory University, Atlanta, USA Knowledge on the pathophysiological changes, underlying loss of control over drug intake, is still rather limited. We have recently developed a novel approach based on the hypothesis that autoimmune responses, which are well documented for several nervous-system disorders, may also occur in the drug-addicted organism. Human heroin addicts present elevated levels of autoantibodies (aAbs) to opioid receptors. In the present study, SD rats were administered morphine (5 mg/kg s.c.) once daily for 2 weeks. The levels of aAbs to mu-delta opioid (MDOR) and glutamate (GluR1 AMPA and NR2A/B NMDA) receptors in the rat serums were determined with synthetic peptides as antigens (0.5 ng per ELISA well). Confirming previous reports, morphine treatment increased significantly serum titers of autoantibodies against opioid and AMPA glutamate receptors, but not NMDA glutamate receptors. It is possible to hypothesize that an autoimmune response to repeated drug exposure occurred. The relationship between presence in the plasma of autoantibodies to these neuroreceptors and pathological changes in brain function is so far unknown. Circulating autoantibodies may functionally affect the AMPA glutamatergic input within the brain. Noteworthy, AMPA receptors have been very recently implicated in drug-seeking behavior and addictive habits. Objective: To determine if neuropsychiatric manifestations in patients with systemic lupus erythematosus (SLE) are influenced by anti-DNA antibodies cross-reacting with the human N-methyl-d-aspartate (NMDA) receptor types NR2a or NR2b. Methods: A decapeptide was synthesized containing a sequence motif present in the extra-cellular ligand-binding domain of NMDA receptors NR2a and NR2b, bound by the monoclonal murine anti-DNA antibody R4A. In an ELISA-assay with the murine monoclonal R4v as positive control, plasma samples of 57 patients with SLE were examined for the anti-peptide (anti-NR2) antibody after the patients had been subjected to comprehensive psychological and cognitive testing. Results: Seven out of 31 neuropsychological variables were associated with elevated levels of anti-NR2 antibody in a correlation matrix. These candidate variables were entered into a MANOVA statistical model with anti-NR2 as the independent variable. Poor performance on the Visual Paired Associates Test (immediate), the Grooved Pegboard Test, as well as high scores on the Beck Depression Inventory, and scales D-2 (Depression), Pd-4 (Psychopathic deviate), Sc-8 (Schizophrenia), and Ma-9 (Hypomania) of the MMPI-2 were significantly associated with elevated levels of anti-NR2 antibodies. Conclusion: Findings in several domains indicate an association between anti-NR2 antibodies and depressed mood in addition to decreased short time memory and learning. Antibodies to NMDA-receptors may represent one of several mechanisms for cerebral dysfunction in patients with SLE. It has been suggested that schizophrenia and affected disorders may be associated with viral infections. However, search for viral nucleic acids in the brain of patients with schizophrenia revealed a paucity of virus. We hypothesized that T cells found in the CSF of patients with schizophrenia may contain populations of T cells that initially responded to the virus (although the virus is no longer present). Using the NPA-PCR/V betaspecific PCR, we demonstrated high proportions of identical beta-chain TCR transcripts in the CSF of seven out of seven patients with schizophrenia or affective disorders, suggesting the presence of antigen(s)-driven clonally expanded population of T cells. These T cell clonal expansions may be the only remaining trace of the initial viral infection. Alternatively, these T cells may be generated in response to reactivation of a viral infection, or molecular mimicry or epitope spreading. In contrast, TCR transcripts from the CSF of normal donors were polyclonal. It is unknown whether clonally expanded T cells in the CSF of patients with schizophrenia and affective disorders contribute to brain tissue loss and mental/cognitive impairment.
Psycho-immunological evaluation of stress preventive intervention programmes U. Sack, K. Meier, K. Bauer and M. Stqck University of Leipzig, Leipzig, Germany
We aimed to examine immunological and psychological parameters reflecting stress management capabilities, particularly the correlation between self-regulative therapeutic stimuli and immunoglobuline A in saliva. As part of the integrative stress management training-concept for schools, a 10-week stress management training as well as the intervention method Biodanza, based on motions, emotions, and body contact, were carried out with teachers. It was the aim of this intervention to enable teachers to better cope with their daily pressures. The effects of both intervention methods were to be determined in an accompanying processevaluation study. For this purpose, health-psychological (stress-relevant psychological variables; work-related behavioural and experience patterns) as well as psychological (blood pressure, skin resistance) and immunological variables (immunoglobulin A in saliva) were taken. Twenty-four teachers were investigated before and after the sessions of both intervention studies. Parallel to this, different emotional parameters were ascertained. For both methods, overwhelming effects in improving stress resistance could be verified. In saliva, increase of immunoglobulin A could be found in persons with a positive response to our training. Furthermore, a significant positive correlation between the subjective sensation of relaxation or other health parameters and IgA could be measured. Both procedures could be shown to be effective in improving stress management capabilities. Psychological and subjective parameters correlated well with saliva immunoglobulin A concentration. Therefore, measurement of this simple immunological parameter could be proved to be a reliable indicator for stress resistance as well as training effects.
Changes of immune and endocrine parameters in PTSD over time A. Vidovic a , K. Gotovac a , M. Vilibic b , A. Sabioncello a , S. Rabatic a , V. Folnegovic-Smalc b and D. Dekaris a a Institute of Immunology, Zagreb, Croatia; b Vrapce Psychiatry Hospital, Zagreb, Croatia
Our study performed on post traumatic stress disorder (PTSD) patients within 8 years from traumatic event revealed elevated plasma cortisol level and lower glucocorticoid receptors (GRs) expression in examined lymphocyte populations opposite to studies performed decades following exposure to trauma. We assumed that more time is needed for reversal of hormone and its receptor expression to take place due to hyperactivity of hypothalamic-pituitary-adrenocortical (HPA) axis. Participants were 18 Croatian war veterans with PTSD and 10 age-matched civilian controls retested 5 years later. Proportions of main lymphocyte populations and their activated subpopulations, as well as GRs expression in these populations were determined by flow cytometry. Cortisol levels were determined by RIA (radioimmunoassay). NK cell activity was measured by 51 Cr-release assays. Platelet activation status was determined by flow cytometry according to expression of CD63 activation marker and proportion of leukocyte-platelet aggregates in circulation. We found decreased percentages of Tc and total memory cells but did not reveal significant changes over time in GRs expression on total T, B and NK cells in this preliminary follow-up study. Brain atrophy and aberrant behaviour are common complications of systemic lupus erythematosus (SLE). Considering that their etiology is unknown, we use aberrant behaviour in lupus-prone MRL-lpr mice to reveal pathogenic mechanisms of autoimmunity-induced CNS damage. We previously documented profound damage in the dopaminergic system, both in human and animal forms of the disease. The present study explores the course of self-injurious behaviour (SIB) after sustained pharmacological challenge with quinpirole (QNP), a D2-receptor agonist. The MRL-lpr mice and congenic controls were injected with QNP (i.p., 0.5 mg/kg) daily for 15 days. This treatment gradually decreased grooming frequency, but increased duration of grooming episodes in both strains. However, these effects were less profound in the MRL-lpr group with 60% of these animals showing advanced SIB with repeated QNP administration. This was not seen in the age-matched, nonautoimmune CD1 strain of mice, suggesting that autoimmunity is required for emergence of the aberrant behavioural response. We presently examine whether SIB is associated with excessive damage of the dopaminergic system and whether SIB can be induced before autoimmune manifestations develop. Currently, obtained results support the hypothesis that chronic inflammation and autoimmunity damage dopaminergic neurons and increase the sensitivity of their cognate receptors thus underlying SIB. This work was supported by funds from the NIH to B. Sakic, who is a recipient of the FSORC career development award. S. Chun is a recipient of the NSERC Graduate Scholarship. There is evidence of a bidirectional communication between Central Nervous System (CNS) and Immune System (IS), and that opioids exert a modulatory role on the immune response. Our main goal was to study: (1) the influence of acute and chronic d-amphetamine treatment on ConAinduced lymphocyte proliferation in rats, (2) the participation of dopaminergic and opioidergic systems in these effects, (3) the amphetamineinduced effects on proenkephalin and derived peptides in immune organs and different CNS areas. Wistar rats received chronic (1 or 2 mg/kg/day for 5 days IP) or acute (2.5 or 5 mg/kg IP) amphetamine; 4 days after the last injection, the proliferative response was assessed. Since similar immunosuppressive effects were observed following acute and chronic treatments, Naloxone, SCH-23390 and Sulpiride were given only before 5 mg/kg amphetamine. Since all antagonists abolished the amphetamine-induced effects on IS, dopamine and opioid systems seem to be involved. The proenkephalin and derived peptides levels in the CNS and IS were studied following 5 mg/kg amphetamine. An increase in Met-enkephalin was observed in spleen, thymus, nucleus accumbens and prefrontal cortex. These changes in Met-enkephalin point out that it could be used as a common biological marker of pertinent stimuli (i.e. psychostimulant drug) acting at SI and SNC, and reflect the functional changes at both levels.
Morphofunctional characteristics of the rat thymus exposed to forced swim stress A. Rakin, I. Zivkovic, D. Petrovic-Djergovic, D. Kosec and M. Micic
Having in mind recent studies about the effects of stress on the immune system, we investigated whether chronic stress, induced by the forced swimming procedure, alters the morphofuncional parameters in the rat thymus. For this purpose, adult male rats were exposed to the forced swimming during 21 days, on the first day 15 min and in the following days 5 min. The experimental and control animals were sacrificed by decapitation, the thymuses were processed for morphometric and flow cytometry analysis and blood samples were taken for measurement of corticosterone level by RIA. Our results showed that every day treatment with the same stimulus leads to reduction of (i) the thymus mass and cellularity, (ii) the volume of thymus cortex and medulla, (iii) numerical density of thymocytes in the inner cortex and medulla and (iv) the number of thymocytes in the both thymus compartments. However, the percentage of apoptotic cells and the level of corticosterone were significantly increased. The phenotypic analysis of thymocyte subpopulations showed significantly decreased percentage of the least mature CD4 + CD8 À TCR À thymocytes. On the other hand, the percentages of CD4 À CD8 À TCR low/high and CD4 À CD8 + TCR-thymocytes were significantly increased. These results show that in adult rat recurred swimming procedure induces thymus hypotrophy, through change of the thymus compartments volume and cellularity. Additionally, elevated percentage of CD4 À CD8 À TCR + cells suggests that chronic stress is involved in the modulation of T cells maturation.
The influence of restraint stress and beta-endorphin on inflammatory edema and macrophage function in AO rats K. Mitic a , M. Dimitrijevic a , V. Vujic b , V. Kovacevic-Jovanovic a and S. Stanojevic a a Immunology Research Center bBranislav JankovicQ, Institute of Immunology and Virology bTorlakQ, Belgrade, Serbia; b Institute of Chemistry, University of Belgrade, Medical School, Belgrade, Serbia
The influence of i.pl. treatment with beta-Endorphin (beta-End, 0.01-10 Ag) and restraint stress (RS) on Concanavalin A (Con A)-induced paw edema were investigated in male Albino Oxford (AO) rats. Besides, peritoneal macrophages obtained from intact and stressed rats of this strain were additionally in vitro treated with beta-End (10 À12 -10 À8 M) and tested for capacity to produce reactive oxygen species and reduce nitro blue tetrazolium salts (NBT) to formazans. Results showed that Con-A-induced paw edema was suppressed by i.pl. treatment with 10 Ag of beta-End, which was antagonized by naltrindole and nor-binaltorphimine, suggesting involvement of delta and kappa opioid receptors. Quite the opposite, 2 h of RS applied after but not before inflammation induction significantly increased diameter of inflamed paws in AO rats, but did not influence the NBT reduction capacity of peritoneal macrophages. The NBT reduction was influenced by beta-End in such way that beta-End potentiated reactive oxygen radical production in unstimulated macrophages from animals exposed to RS, while dose-dependently increased and decreased NBT reduction in cells stimulated with phorbol myristate acetate in both intact and stressed animals. It could be concluded that RS and beta-End exerted opposite effects on in vivo and in vitro inflammatory responses in AO rats. The proinflammatory cytokine interleukin-1 beta (IL-1 beta) influences neuroendocrine activity and promotes central effects such as anxiety and anhedonia. The melanocortin neuropeptides, like alpha melanocyte stimulating hormone (alpha-MSH), antagonize many actions of IL-1, including fever, anorexia and HPA axis activation through specific melanocortin receptors in central nervous system. However, it is unknown if melanocortins can modulate IL-1 beta-induced anxiety. The objective of the present study was to establish the effect of MSH peptides on IL-1 beta-induced anxiety-like behavior and the type of melanocortin receptors involved. The present study evaluated the effects of intracerebroventricular (i.c.v.) administration of IL-1 beta (30 ng) and melanocortin receptor agonists: alpha-MSH, an MC3/MC4-R agonist (0,2 Ag) and gamma-MSH an MC3-R agonist (2 Ag) or HS014, an MC4-R antagonist (2 Ag), on elevated plus-maze test. Injection of IL-1 beta induced an axiogenic-like response, as indicated by reduced open arms entries and time spent on open arms. The administration of alpha-MSH reversed IL-1 beta-induced anxiety. Coadministration of HS014 inhibited the effect of alpha-MSH. The associated treatment with gamma-MSH did not affect the anxiety response to IL-1beta. These data suggest that melanocortins, through central MC4-R can modulate the anxiety-like behavior induced by IL-1 beta. Expression of Toll-like receptors in MG thymus P. Bernasconi a , M. Barberis b , M. Cannone b , F. Baggi a , E. Arnoldi a , C. Cappelletti a , F. Cornelio a and R. Mantegazza a a National Neurological Institute bCarlo BestaQ, Milan, Italy; b MultiMedica, Milan, Italy Myasthenia gravis (MG) is often associated with thymic alterations (hyperplasia, thymoma and thymitis); however, the aetiopathological basis of this association is still unknown. Intercellular signals as well as surface molecules may represent key factors characterizing the thymic microenvironment. Elements of innate immunity are emerging as contributors to the development of autoimmunity. We studied, by semi-quantitative RT-PCR, the expression of Toll-like receptors (TLR) 2-5 in 38 myasthenic thymuses and in 4 non-pathological young thymuses. TLR transcripts were found not differently expressed among groups, except for TLR4. By realtime PCR, TLR4 mRNA expression was significantly higher in thymitis than in hyperplastic ( p=0.002), non-pathological young thymuses ( p=0.03) and thymoma ( p=0.048). By immunohistochemistry, the protein was detected in all thymic subgroups analysed, but the extent of positivity was greatly variable. In thymitis and hyperplastic thymuses, a strong TLR4 positivity was detected in correspondence of cytokeratin-positive cells within the thymic medulla, surrounding cortex and at the borders between cortical and medullary regions. In thymoma and in young non-pathological thymuses, TLR4 protein was rarely detected. These findings suggest a possible involvement of the innate immunity in thymus alterations characterized by an abnormal cell proliferation, and evoke the possibility that antigen mimicry might play a crucial role in MG pathogenesis. In early-onset MG (EOMG), thymic infiltration is well established. However, the thymus and its muscle-like myoid cells have not been well studied in patients without anti-AChR autoantibodies (seronegative MG)i.e. those with (MuSK Ab + ) or without (MuSK Ab À ) anti-MuSK antibodies. The histopathology is usually reported as normal or involuted, and these patients are rarely thymectomised, unlike those with EOMG. We compared thymic histology in patients with EOMG, MuSK Ab + and MuSK Ab À SNMG, labelling paraffin sections for mature T cells and germinal centres (GC), and for markers of inflammation and complement activation. In most MuSK Ab + and 70% MuSK Ab À SNMG cases, the thymus appears normalfor-age. B cells express only CD20 and are mainly in the medullary epithelial areas (MEA), near the apparently normal myoid cells. Interestingly, in about 30% of MuSK Ab À cases, we see mild infiltration, very similar to that in EOMG. B cells and follicular dendritic cells in GC express receptors for activated complement components. Some of myoid cells are very close to, or within, the infiltrates where they appear to be under immune attack, as in EOMG. We conclude that the thymus is normal or bburnt-outQ in MuSK Ab + SNMG. By contrast, MuSK Ab À SNMG is heterogeneous; in one form, the target antigen may be expressed by thymic myoid cells, which seem to be involved in pathogenesis. The target organ of myasthenia gravis (MG) is the postsynaptic membrane of the neuromuscular junction (NMJ). The primary autoantigen, the acetylcholine receptor (AChR), is clustered and anchored in the postsynaptic membrane of the NMJ by rapsyn. Previously, we found that a naturally increased rapsyn concentration in aged rats correlates with a resistance to experimental autoimmune MG (EAMG). Here, we report that the overexpression of rapsyn by in vivo electroporation confers resistance to EAMG in treated legs of susceptible rats. The rapsyn overexpression increased the level of AChR in muscles 2 weeks after electroporation. In EAMG rats, rapsyn-overexpressing muscles showed no loss of AChR, in contrast to control muscles. Repetitive nerve stimulation showed no decrement of the compound muscle action potential of the rapsynoverexpressing muscles, whereas sham-treated muscles of the same EAMG animals did. Electron microscopic examination showed that endplates of rapsyn-overexpressing muscles of EAMG rats had a normal morphology of the postsynaptic folds, while endplates of sham-treated muscles of the same animals had damaged folds. Rapsyn overexpression was effective to increase AChR levels in ongoing disease of chronic EAMG in the presence of high titers of anti-AChR antibodies. These results suggest an important role for rapsyn in the susceptibility to EAMG and show a new promising therapeutic for treating myasthenia gravis in humans.
Induction of experimental autoimmune myasthenia gravis by an acetylcholine receptor peptide conjugated to lipopolysaccharide I. Wirguin a , C. Sicsic b , M. Bersudsky a and T. Brenner b a Ben-Gurion University, Beer-Sheva, Israel; b Hadassah University Hospital, Jerusalem, Israel Background: Molecular mimicry is implicated in causing autoimmune disorders, such as Guillain-Barré syndrome which is attributed to crossreactivity between carbohydrate epitopes in Campylobacter jejuni lipopolysaccharides (LPS) and nerve gangliosides. We showed that covalent bonding of LPS to gangliosides enhances immunity to self-gangliosides. Objective: To test the hypothesis that LPS-bound antigens could overcome tolerance to protein antigens. Methods: The decapeptide WNPDDYGGVK, [the main immunogenic region (MIR) of the chain of acetylcholine receptor (AChR)], was conjugated to Salmonella Re-LPS by heterodimeric crosslinking. Lewis rats subcutaneously presented by Torpedo AChR or by MIR (days 1 and 21), received intraperitoneally 10 Ag of the MIR-LPS conjugate (day 28). Control rats received MIR or Re-LPS only. Animals were tested by repetitive nerve stimulation, by treadmill exercise tolerance and by immunoassays for AChR antibodies. Results: 15/25 LPS-MIR injected rats showed pathological decrements to repetitive nerve stimulation at 3 Hz compared to 2/25 of controls. 4/12 MIR-LPS injected rats failed to complete 10 min of treadmill exercise compared to 1/13 of controls. Rat AChR antibodies were detected by RIA in 3 of the MIR-LPS injected rats and in none of the controls. Conclusion: MIR-LPS conjugates can induce EAMG without additional adjuvants. The molecular bond of the LPS to the antigen appears to play an important role in overcoming tolerance to selfantigens such as AchR. The expression level of interleukin-10 is related to the polymorphisms À1082 (G/A), À819 (T/C) and À592 (A/C) in the promotor region of the IL-10 gene constituting three haplotypes (GCC, ATA, and ACC). The haplotype combination GCC/GCC is associated with high IL-10 expression, whereas GCC/ATA and GCC/ACC are associated with medium and ATA/ATA, ATA/ACC and ACC/ACC with low expression. The distribution of these polymorphisms was analysed in 64 myasthenia gravis patients and 87 controls to determine whether they could influence disease susceptibility. We found a different genotype distribution in MG patients compared with controls. ACC/ACC and ATA/ATA were more frequent in MG patients ( p=0.02). This was reflected in a higher frequency of ACC/ACC (21.4% vs. 3.4%) in thymoma MG patients ( p=0.05) and titin antibody positive MG patients (regardless of thymus pathology). Early-onset MG patients and titin antibody negative MG patients (regardless of age of onset or thymus pathology) had a higher frequency of ATA/ATA (19.2% vs. 3.4%), ( p=0.05). There were no differences between MG patients and controls for high, medium or low IL-10 expression. Conclusions: MG patients with thymoma and thymus hyperplasia are associated with different IL-10 genotypes. MG patients with titin antibodies are similar to thymoma patients while titin antibody negative patients are similar to early onset MG patients in regard to IL-10 genotypes. The functional plasticity of dendritic cells (DC) makes them ideal targets for immuno-therapies. From cancer immunology, we learn how the plasticity of DC function is ensured by capability of DC to both produce and respond to certain cytokines. For example, melanomas or breast cancers are able to produce large amounts of IL-10, which modulate tumor-associated DC. Such IL-10-modulated immature DC (IL-10-DC) induce anergic T cells. However, cytokine-matured DC can induce effector T cells against tumor. Here, we investigated therapeutic potentials of DC from EAMG rats modulated with IL-10. Effects of IL-10-DC and LPS-matured DC on autologous T cell responses in MG compared to healthy controls (HC) were also investigated. DC derived from EAMG rats, modulated in vitro with IL-10, ameliorate ongoing EAMG. This was associated with suppression of both Th1 and Th2 cytokines, and of B cell responses. Surprisingly, IL-10-DC from human MG, like mature DC, increased CD4 + CD25 + T cells expressing CD69, i.e. activated T cells, compared to IL-10-DC from HC. IL-10-DC induced IL-10 and IL-4 production by T cells from MG patients, but only IL-10 production from HC, without affecting Th1 cytokines. Conclusions: (a) IL-10-DC differently affect EAMG vs. MG in terms of T cell activation, and Th1/Th2 cytokine production; (b) IL-10-DC in MG have pronounced costimulatory capacity for Th2 cells, reflected by increased CD4 + CD25 + T cells expressing CD69 and increased production of IL-10 and IL-4, compared to IL-10-DC from HC.
The Lambert-Eaton myasthenic syndrome has a more progressive course in patients with small cell lung carcinoma P. Wirtz, A. Wintzen and J. Verschuuren Leiden University Medical Centre, Leiden, The Netherlands HLA studies suggest a major difference in immunopathogenesis between HLA-B8-associated LEMS with small cell lung cancer (SCLC-LEMS) and non-HLA-associated LEMS without associated tumour (non-tumour, NT-LEMS). In contrast, there is no known difference in the neurological signs and symptoms between SCLC-LEMS and NT-LEMS patients, but some reports suggest a more progressive course of disease for SCLC-LEMS. We assessed in detail the chronology in which symptoms developed in 38 LEMS patients, of which 13 with SCLC. Patients were interviewed using a structured checklist, backed up by a review of their clinical records. We compared the frequency and time scale of symptoms during the course of LEMS. In SCLC-LEMS patients, proximal leg weakness, dry mouth and impotence (in males) were the first symptoms, followed by proximal arm weakness, double vision, hand weakness and slurred speech. In at least 50% of the SCLC-LEMS patients, all these symptoms occurred within 6 months. All NT-LEMS patients started with proximal leg weakness. A dry mouth was the only symptom that occurred in at least 50% of patients within 6 months after start of the leg weakness. Symptoms in SCLC-LEMS patients developed within a shorter timeframe than in NT-LEMS patients, indicating a more progressive course of disease in SCLC-LEMS. Therefore, in a patient with an aggressive course of LEMS one should be extra alerted for an underlying lung carcinoma. Experimental autoimmune neuritis (EAN) is an inflammatory autoimmune demyelinating disease of peripheral nervous system (PNS) and represents an animal model of Guillain-Barré syndrome (GBS) in man. The inflammatory cell infiltrating into the PNS is a prerequisite for developing EAN. To explore the role of CC chemokine receptor 5 (CCR5) in the inflammatory process of EAN, we induced EAN in CCR5 deficient (CCR5 À/À ) mice with P0 protein peptide 180-199. We found that CCR5 À/À mice showed a similar EAN clinical course and severity as well as profile of infiltrating macrophage and T cells in cauda equina (CE) of EAN and the same levels of spleen mononuclear cell (MNC) response to antigen and mitogen when compared with CCR5 +/+ control mice. However, increased IP-10 and MIP-1alpha production in sciatic nerves were seen in CCR5 À/À mice. These results suggest that CCR5 deficiency does not prevent P0 peptide 180-199 immunized mice from EAN. Increased MIP-1alpha and IP-10 in sciatic nerves may compensate the CCR5 deficiency and contribute to inflammatory cells infiltrating to the PNS. Guillain-Barré syndrome (GBS) is a post infectious polyradiculoneuropathy characterized by demyelination and axonal degeneration of peripheral nerves. Inflammatory cells, predominantly macrophages and T cells, and humoral factors, including autoantibodies, have been implicated in disease pathogenesis. We recently documented the capacity of GBS-associated anti-GM1 IgG to induce IgG receptor (FcgammaR)-mediated leukocyte activation, as measured by leukocyte degranulation and phagocytosis. To investigate the association between antibody functionality and disease development, we used a recently described GBS rabbit model. Immunization of rabbits with ganglioside mixtures results in the formation of high anti-GM1 IgG antibody titers. Nevertheless, only GM1 specific antibodies from rabbits with clinical overt GBS induced leukocyte activation ( p=0.002). Next, the phagocyte activating capacity of sequentially drawn sera from GBS-affected rabbits was tested. Maximal phagocyte activation was detected 1 week before or at the occurrence of clinical symptoms. Increased functionality may be explained by both the increase in specific antibody titer and increased antibody avidity. Taken together, these data suggest that functionality of anti-ganglioside antibodies, as measured by in vitro assays, is associated with the presence of GBS clinical symptoms in rabbits, thus providing additional evidence for their pathogenic role in vivo. Gangliosides are implicated as target antigens in various autoimmune neurological disorders. IgM antibodies against b-series gangliosides including GD1b are associated with chronic sensory ataxic neuropathy. Animal model of sensory ataxic neuropathy can be induced by sensitization with GD1b. However, it has yet to be clarified whether GD1b is expressed on proprioceptive neurons of dorsal root ganglia (DRG). Genetically engineered mice lacking in b-series gangliosides, immunologically naRve to GD1b, were immunized with GD1b to generate anti-GD1b monoclonal antibody (mAb). Generation of anti-ganglioside antibodies upon immunization with GD1b was greatly enhanced, exhibited class switching to IgG isotypes and immunological memory, indicating that tolerance to self-gangliosides is a major regulatory factor. Six clones with anti-GD1b IgG activity were obtained, three of which reacted exclusively with GD1b. One of the monospecific anti-GD1b mAb preferentially stained the large cell bodies in rat DRG. Immunohistochemical studies showed that GD1b co-localized with parvalbumin, but not with calcitonin gene-related peptide, substance P, and somatostatin. This indicates that GD1b is expressed on proprioceptive neurons in rat DRG. These results support the concept that anti-GD1b antibodies may cause selective injury to proprioceptive sensation of primary afferent neurons, and development of sensory ataxic neuropathies.
Effective prevention and treatment of experimental autoimmune neuritis with rapamycin T.Y.-H. Lin and J. Spies The University of Sydney, Sydney, Australia Objective: To determine whether rapamycin, a relatively new immunosuppressive agent effective in preventing transplant rejection, is effective treatment for EAN, an animal model for human inflammatory demyelinating neuropathy. Background: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) responds to immunomodulatory therapy but long term immunosuppressive therapy is often required, with significant associated toxicity. Rapamycin has a more favorable side effect profile than agents such as cyclosporine which are currently used to treat CIDP. Methods: EAN was induced in Lewis rats by immunization with peripheral nerve myelin or passive transfer of P2-specific T cells. Rapamycin or placebo was administered daily by intraperitoneal injection commencing just prior to the expected onset of paralysis (prevention) or at the first signs of EAN (treatment). Clinical grade and weight were recorded daily. Neurophysiology and histological examination were performed at predetermined intervals. Results: Rapamycin given prior to the onset of disease prevented the development of significant paralysis in both EAN (mean clinical grade 0.1 vs. 3.4; pb0.00001) and adoptive transfer EAN (mean clinical grade 0 vs. 2.2; pb0.0002). Rapamycin given at the onset of clinical weakness effectively reduced the severity (mean clinical grade 0.5 vs. 3.7; pb0.00001) and hastened recovery of clinical weakness and electrophysiological abnormalities. Conclusion: Rapamycin is effective in prevention and treatment of EAN and adoptive transfer EAN in Lewis rats and may be an effective and less toxic alternative treatment for refractory CIDP. Based on the evidence that ONO-2506 suppresses S-100 protein production, preventive and therapeutic effects of this agent on immunemediated neuropathy were studied in EAN. As to the preventive effect, neurological symptoms were remarkably lessened and the duration of disease was clearly shortened in the group receiving intraperitoneally ONO-2506 (10 mg/kg/day) every day after inoculation, as compared with the non-treated control group. As to the therapeutic effect, a remarkable suppressive effects on the neurological symptoms and a clear shortening of the duration of disease were also noted in both the early phase-treated group receiving ONO-2506 for 7 days from the onset of limp tail and active phase-treated group receiving the agent for 7 days from the onset of paraparesis, as compared with non-treated group. In the pathological observation at 30 days after antigen inoculation, remarkable demyelination and fibrosis were found in the non-treated group, whereas only a mild perivascular cell infiltration was noted in the preventively treated group, mild cellular infiltration and demyelination in the early phase-treated group and moderate cellular infiltration, demyelination and fibrosis in the active phase-treated group. The above results suggested ONO-2506 to exert preventive and therapeutic effects on EAN.
Campylobacter jejuni-derived LPS abrogates oral tolerance to experimental autoimmune neuritis S. Jung a , J. Voss a , M. Frosch b , H. Karch b , K. Toyka c and S. Sch7fer c a Department of Neurology, University of the Saarland, Homburg, Germany; b Institute of Hygiene and Microbiology, University of Würzburg, Würzburg, Germany; c Department of Neurology, University of Würzburg, Würzburg, Germany
The inflammatory form of the Guillain-Barré syndrome (GBS) is an acute polyradiculoneuritis mediated by T lymphocytes, macrophages, and antibodies and is associated with a recent infection by Campylobacter jejuni in about 30%. The animal model of GBS, experimental autoimmune neuritis (EAN) of Lewis rats can be induced by immunization with PNS myelin. Immunization with antigen preparations of various C. jejuni strains derived from GBS patients did not induce clinical or histopathological signs of neuritis. Nevertheless, animals generated high titers of C. jejuni-specific antibodies. Subsequent injection of P2-specific T cells caused typical neuritis and opened the blood-nerve barrier, but clinical disease course or nerve histopathology were not aggravated in C. jejuniimmunized rats compared to CFA-sensitized animals. Signs of active EAN induced by immunization with PNS-myelin could be mitigated by precedent feeding of myelin (oral tolerance). By adding pronase-digested LPS prepared from C. jejuni to the enterally applied myelin, tolerance induction was completely prevented and rats developed an accelerated course of neuritis. Feeding of LPS alone did not modulate EAN. The findings demonstrate that C. jejuni-derived LPS can disturb natural immunoregulation by the gut-associated lymphoid tissue. Objective: To determine whether human immunoglobulin (Ig) is effective and its mechanism of action in the treatment of experimental allergic neuritis (EAN), an animal model for human inflammatory demyelinating neuropathy. Background: Administration of high-dose intravenous immunoglobulins (IVIg) has become one of the most successful therapies for inflammatory demyelinating neuropathies, yet the mechanism of action is not fully understood. Methods: We induced EAN in Lewis rats and administered human Ig or albumin intravenously commencing at the onset of neurological deficit. Clinical grade and weight were recorded daily. Neurophysiological and histological examinations were performed at predetermined intervals. Results: IgG treatment effectively reduced the severity and fastened recovery of clinical weakness. This improvement was associated with an accelerated rate of recovery from electrophysiological abnormalities. Conclusion: Human Ig is an effective treatment for EAN in Lewis rats. Further investigations are underway to determine whether the Fc or Fab component of IVIg is responsible for the observed therapeutic efficacy.
Anti-ganglioside antibodies and intrathecal IgG synthesis in Guillain-Barré syndrome S. Matà, E. Galli, A. Amantini, F. Pinto, S. Sorbi and F. Lolli University of Florence, Florence, Italy Objective: To investigate the relationship between clinical, electrophysiological, immunological findings and CSF changes in the Guillain-Barré syndrome (GBS). Background: Increased anti-ganglioside antibody titers are a frequent finding in GBS patients and, to a lesser extent, in patients with chronic inflammatory demyelinating polyneuropathy (CIDP). Another frequent feature of these diseases is a dysfunction of blood-brain barrier (BBB), whereas the intrathecal synthesis of immunoglobulins is still a matter of debate. The relationship between these findings is unknown. Methods: The authors studied a cohort of GBS patients examined by standard EMG, by ELISA for anti-ganglioside antibodies, and by standard laboratory procedure for CSF examination. We used four formulas for the calculation of intrathecal synthesis of IgG: IgG Index, Reiber and Felgenhauer's formula, Blennow et al.'s formula and IgG/total protein ratio. Controls consisted of patients with non-dysimmune polyneuropathy, inflammatory and non-inflammatory CNS disorders and blood donors. Results: Thirty-eight percent (28/73) of GBS patients and 28% of CIDP patients had detectable serum titers of anti-ganglioside antibodies. In both patient groups, the anti-ganglioside reactivity was associated with high incidence of motor conduction block and prolonged F wave latencies. In GBS patients, there was a clear association between anti-ganglioside antibody and high value of intrathecal IgG production indexes. Conclusion:
The association between anti-ganglioside antibodies and high CSF IgG production indexes value in GBS patients suggests a pathogenetic relationship between these findings. Objective: Campylobacter jejuni (Cj) is the leading cause of Guillain-Barré syndrome (GBS) in Japanese. Recently, we cloned and characterized the iron-binding protein Dps from Cj (C-Dps). The aim of this study was to determine whether or not C-Dps contributes to pathogenesis of GBS. Methods: Anti-C-Dps antibody in sera was determined by ELISA in 40 patients with GBS, 11 patients with Cj enteritis, 139 patients with other inflammatory neurological diseases and 61 healthy controls. Binding of C-Dps to various glycolipids and rat neural tissues was studied. Furthermore, PC12 cells were exposed to various concentrations of C-Dps and LDH release was measured at 1 h later. Results: We found that (a) anti-C-Dps antibody was only present in GBS patients with preceding Cj infection (38.9%) and those with Cj enteritis (18.2%), (b) C-Dps bound to neurons in rat spinal cord and myelin sheath in rat peripheral nerves, (c) C-Dps bound specifically to sulfatide, (d) in vitro, C-Dps bound to PC12 cell surface and induced the LDH release from the cells. This LDH release was inhibited by heart-inactivation and co-incubation with anti-C-Dps monoclonal antibody. Conclusions: C-Dps is considered to bind to neural cell body and myelin sheath through sulfatide. After binding, C-Dps exerts direct neuro-toxicity, which may contribute to axonal damage in GBS with preceding Cj infection. This paper updates the Cochrane systematic reviews of immunotherapy for Guillain-Barré syndrome. The primary outcome was the change in a sevenpoint disability grade scale after 4 weeks. Plasma exchange was tested against no exchange in five trials with 606 participants. The weighted mean difference was 0.89 (95% CI 0.64 to 1.15, pb0.00001) of a grade more improvement with plasma exchange than without. Intravenous immunoglobulin was compared with plasma exchange in five trials involving 536 participants. There was 0.04 of a grade more improvement with intravenous immunoglobulin than with plasma exchange (95% CI 0.26 more improvement to 0.19 less improvement, p=0.74) so that these treatments have been considered equivalent. One trial involving 249 participants compared plasma exchange followed by intravenous immunoglobulin with plasma exchange alone: the sequentially treated group had 0.20 (95% CI: À0.14 to 0.54, p=0.24) of a grade more improvement than the other. Five trials with 567 participants compared corticosteroids with placebo. Despite marginally significant results in one trial seeking a synergistic effect with intravenous immunoglobulin, the synthesis of all the trials showed no significant difference (weighted mean difference only 0.05, 95% CI À0.16 to 0.25 more in the corticosteroid treated participants, p=0.66). This synthesis shows that intravenous immunoglobulin and plasma exchange have equivalent efficacy in Guillain-Barré syndrome but the effect of corticosteroids alone is neutral. Objective: To determine if there is a neuropathic process selectively affecting small-diameter epidermal nerve fibers (ENFs) in patients with systemic lupus erythematosus (SLE). Methods: Sixty patients, age 43.2F13.5 years, were subjected to a clinical examination. Neuropathy Impairment Score (NIS) and Neuropathy Symptom and Change Score (NSC) with emphasis on sensory symptoms were obtained as quantitative estimations of neurological deficits and symptoms of both small and large-diameter nerve fiber function. In two punch biopsies taken from the distal part of the leg, ENFs were visualized staining with a rabbit polyclonal antibody to the panaxonal marker anti protein gene product 9.5. The density of ENFs was considered abnormal when the number of ENFs was less than the 2.5th percentile for controls (3.4 fibers/mm). Nerve conduction velocity studies (NCV) were performed for an evaluation of the presence of large-diameter nerve fiber neuropathy. Results: The density of ENFs was 7.5F3.8, and 12.4F4.6/mm in controls, P=0.0001. Eight patients (13%) had less than 3.4 fibers/mm. Thirty-seven patients (62%) had an abnormal NIS score, 53 patients (88%) an abnormal NSC subscore for sensory symptoms, and 7 patients (12%) an abnormal NCV. No associations were found between reduced number of ENFs and symptoms, clinical findings, or NCV. Conclusion: A selective pathological process affecting only ENFs is prevalent in patients with SLE.
Motor nerve fibers blocking dynamic variations along the first month after intravenous immunoglobulin treatment in multifocal motor neuropathy A. Villa a , G. Sandoval b , R. Saizar a , M. Di Egidio a , A. Morillo b , O. Garcea a , G. Nores c and R. Sica a a Ramos Mejia Hospital, Buenos Aires, Argentina; b Hospital Aleman, Buenos Aires, Argentina; c Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Cordoba, Argentina Objective: To examine the effects on nerve conduction studies (NCS) after the administration of 1 g/kg of IVIg in a patient suffering MMN. Material and methods: NCS were performed in a patient affected by MMN, with high titers of IgM anti-GM1, before the treatment and at days 7th,14th, 21st and 28th after the IVIg administration. Multisegmental motor nerve conduction studies were performed by measuring the compound muscle action potential (CMAP) and the conduction velocity (CV). Results: The neurophysiological changes were first seen at the 7th day post-IVIg. Proximal conduction block (CB) sites CAMP amplitudes and areas increased more than 50%; simultaneously, motor nerve conduction velocities values across the CB sites became faster. After day 21st post-IgIV, there was a progressive decline in CMAP amplitudes and areas, while CVs slowed down. The M wave amplitude increased 50% of its original value at the 7th day after treatment, but declined after day 21st. Discussion: Our findings showed reversible changes of the CMAP characteristics at the CB sites. This fact suggest that the axons ion channels, modify, somehow, their permeability properties due to the activity of a pathogenic factor, possible IgM anti-GM1 antibodies, signaling a dynamic modification rather than a structural change.
Rituximab in the treatment of multifocal motor neuropathy with anti-GM1 antibodies: a case report R.F. Bunyan a and M.R. Swenson b a King Faisal University, Al-Khobar, Saudi Arabia; b University of Louisville, Louisville, KY, USA Objectives: Rituxamab is a chimeric anti-CD20 monoclonal antibody used in the treatment of B cell lymphoma. There is new evidence of its efficacy in antibody-mediated autoimmune disorders. Multifocal motor neuropathy is an uncommon progressive autoimmune neuropathy often associated with anti-GM1 antibodies and poorly responds to conventional immunosuppressive therapy. We describe a case of multifocal motor neuropathy with anti-GM1 antibody responsive to Rituximab. Methods: A 45-year-old Caucasian gentleman developed generalized progressive motor weakness of 2-year duration predominantly involving the hands. He complained of a cold sensation of the distal extremities. He had neither bulbar symptoms nor sphincter disturbance. Clinical examination revealed generalized loss of muscle bulk, weakness of the appendicular and axial muscles, areflexia, and distal loss of sensation. Electrodiagnostic studies revealed a motor neuropathy. Laboratory studies showed anti-GM1 IgM antibody titers of 1:6400 (Normal b1:800). Other laboratory and CSF analyses were normal. Results: Initial treatment with intravenous immunoglobulin was not of benefit. Rituximab was initiated with four weekly doses of 375 mg/m 2 followed by a 375 mg/m 2 monthly maintenance dose. This resulted in objective clinical improvement of motor power and hand function. He did not suffer any adverse effects from the treatment. Conclusions: (1) Rituximab was useful in this patient with multifocal motor neuropathy associated with anti-GM1 antibodies. (2) Maintenance therapy resulted in progressive improvement and no adverse effects. Detection of auto-antibodies to gangliosides is routinely carried out in our laboratory by immunostaining on thin-layer chromatography (ITLC) using gangliosides purified by partition of total lipids extracted from human peripheral nerves and spinal cord. A significant number of sera taken from patients with peripheral neuropathy showed staining of lipids that were identified as phospholipids (PL) recovered in the upper phase of partition along with gangliosides. Such sera were further tested by ITLC on a panel of standard phospholipids. The results show that the antiphospholipid antibodies reacted mostly with phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG) and less frequently with cardiolipin (CL). For the patients that also showed autoantibodies to glycosphingolipids, the presence of antibodies to PL was most often associated with reactivity to SGPG, and in rare cases to GM1 and GD1a. The antibodies binding SGPG and PL were always of the same isotype, mostly IgM, but IgG antibodies were sometimes prevailing. The presence of antiphospholipid antibodies in the antiphospholipid syndrome is generally associated with prominent clinical manifestations of neurologic disorders that are predominantly related to focal central nervous system thrombo-occlusive events. However, none of the patients with peripheral neuropathy tested in our study had any history of thrombo-occlusive problems. There is no comprehensive study about antecedent infection in Fisher syndrome (FS) and therefore mechanism of anti-GQ1b IgG antibody production is still unclear. We conducted a prospective case-control serological study about five antecedent infections (Campylobacter jejuni, cytomegalovirus, Epstein-Barr virus, Mycoplasma pneumoniae, and Haemophilus influenzae) in 73 FS patients and 73 sex-and age-matched hospital controls (HC). In FS patients, serological evidences of recent infection with C. jejuni (21%) and H. influenzae (8%) were found significantly more often than in HC. Anti-GQ1b IgG antibody was detected in all 15 FS patients with C. jejuni infection and most patients with H. influenzae infection. TLC with immunostaining showed that FS-related C. jejuni strains (n=20) had the GQ1b-like lipo-oligosaccharide (LOS) more often (50%) than did Guillain-Barré syndrome (GBS)-and enteritis-related strains (7% and 20%). Some FS-related strains also had GM1 or GD1a epitopes (both 20%), but the frequencies were lower than GBS-related strains (74% and 57%). GQ1b epitope was detected in 40% of the 10 FSrelated H. influenzae strains, but in none of the strains from patients with GBS or uncomplicated respiratory infection. These findings provide further evidence that C. jejuni and H. influenzae are causal agents in FS and that production of anti-GQ1b autoantibody is mediated by bacterial GQ1bmimicking LOS.
Synchronized immunomodulatory combination therapy as an initial treatment for chronic inflammatory demyelinating polyradiculoneuropathy J. Ikeda a , A. Harada a , T. Kohriyama b , M. Higaki b , S. Katayama b and M. Matsumoto b a Hiroshima Prefectural Hospital, Hiroshima, Japan; b Hiroshima University Hospital, Hiroshima, Japan Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) responds to treatment with immunomodulatory therapies. However, plasma exchange and intravenous immunoglobulin therapy (IVIg) have short-term clinical benefits and some patients may need these therapies repeatedly. Many patients relapse when the dose of steroids is reduced and fewer patients had complete remission with no treatment. This study was performed to investigate whether synchronized immunomodulatory combination therapy (SICT) in CIDP lead to short-and long-term remission. Seven patients at Hiroshima Prefectural Hospital from 2000 to present were included in this study. SICT was designed by immuno-adsorption therapy (IAT), methylprednisolone pulse therapy and IVIg. Maintaining 30 mg of prednisolone every other day was given orally after SICT. Clinical symptoms were evaluated by neurological disability score (NDS) and modified Rankin scale (mRS). Serum tumor necrosis factor (TNF)-alpha, interferon-gamma, interleukin (IL)-4, IL-10 and macrophage-colony stimulating factor (M-CSF) were measured by enzyme-linked immunosorbent assay (ELISA). Six patients showed improvement by decreased NDS and mRS after one course of SICT. Oral prednisolone was gradually reduced and discontinued in four patients. Remission has been observed in these cases. TNF-alpha, M-CSF and IL-10 were changed greatly during SICT. No response to all therapies was observed in one patient. SICT is useful for initial treatment of CIDP. Fluctuation of cytokines may reflect favorable response to immunotherapies.
A case of chronic inflammatory demyelinating polyradiculoneuropathy presenting external ophthalmoplegia M. Sugawara a , T. Imota b , K. Obara a , S. Watanabe a and I. Toyoshima a a Akita University School of Medicine, Akita, Japan; b Akita City Hospital, Akita, Japan Patient: We are presenting a 33-year-old man who developed an external ophthalmoplegia during a course of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). He reported right ankle weakness after running when he was 7 years old. He became slow at running, and easy to fall down. He had been diagnosed as CIDP in age of 20. Nerve biopsy revealed segmental demyelination. Corticosteroid pulse therapy followed by oral corticosteroid and cyclosporin A resulted in only a mild improvement of muscle power. He was admitted to our hospital due to the aggravation of the weakness of legs and the external ophthalmoplegia. Bilateral blepharoptosis, complete palsy in horizontal gaze and vertical gaze limitation were present. Cranial nerves V, VII, VIII, IX, X, XI and XII were intact. Antibodies: Anti-GM1, GM2, GM3, GD1a, GD1b, and GQ1b antibodies were not detected. Anti-AchR antibody was positive. Thymoma was not detected. Management: Four times of double-filtration plasmapheresis resulted in complete disappearance of external ophthalmoplegia but no improvement of the weakness of extremities. High dose injection of immunoglobulin (IVIg) improved muscle power of the patient remarkably and promptly, but the effect did not continue for more than 2 months. Muscle power of his limbs was maintained and the external ophthalmoplegia never occurred after the intermittent IVIg every 3 month. Conclusion: This case suggests that the patient with CIDP produced a wide diversity of antibodies, causing muscle weakness and external ophthalmoplegia, and maintained recovery by IVIg.
CIDP associated with lung cancer: a paraneoplastic disease? R. Fazio S. Raffaele Hospital, Milan, Italy
We described a 65-year-old smoker male followed for 5 years for a pure motor demyelinating peripheral neuropathy. The patient had a monthly motor relapse with severe weakness that restricted him to wheelchair, so he monthly needed high dose Ig ev. On EMG, the MCV were very slow (30 m/ s) without evidence of conduction blocks while SCV were in the normal range. CSF disclosed a high protein level. Laboratory findings did not reveal any other abnormality except for the presence of monoclonal gammopathy IgMk and high-titer anti-GD1a serum IgM antibodies (1:5000). On March 2003, he had the most severe relapse with flaccid tetraplegia and so severe respiratory failure that required ventilatory support. A total body CT scan revealed a nodular lung lesion with diffuse lymphangiitis. Biopsy disclosed a lung adenocarcinoma with a severe infiltration of CD8 cells. Surgical eradication of the tumor caused the last severe relapse. At the moment, the patient is relapse-free and no more treatment was administered. The clinical course of the motor demyelinating relapsing neuropathy suggests a possible paraneoplastic pathogenesis of the neurological illness also supported by the severe inflammatory infiltration of the tumor.
Immunomodulation of TGF-beta1 as possible antifibrotic therapy in muscular dystrophy: data from murine animal model F. Andreetta, P. Bernasconi, P. Ferro, E. Arnoldi, L. Oliva, F. Cornelio, R. Mantegazza and P. Confalonieri National Neurological Institute bC. BestaQ, Milan, Italy
Irreversible connective tissue proliferation in muscle is a pathological hallmark of Duchenne muscular dystrophy (DMD), and focal release of TGF-beta1 is considered a key factor in fibrosis development. Murine muscular dystrophy (mdx) is genetically homologous to DMD and histopathological alterations comparable to DMD limb muscle have been reported in diaphragm of older mdx mice. To investigate the early development of fibrosis and TGF-beta1 involvement in murine disease, we evaluated diaphragms of 6-to 36-week-old mdx and C57BL6 mice by morphometric analysis, immunohistochemistry for collagens, and TGF-beta1 expression by real-time PCR and ELISA. A significant increase of extracellular matrix and TGF-beta1 expression was found in the 6-12week-old mdx mice, with deposition of type I and III collagens. On the basis of these data, we treated mdx mice with either a neutralizing monoclonal antibody against TGF-beta1 or nonimmune murine IgG for 6 weeks, with alternate-day intraperitoneal injections. Morphometric analysis showed at 12 weeks of age a significant decrease of fibrosis and TGFbeta-1 mRNA levels in the group treated with anti-TGFbeta antibody. Our study describes for the first time the early and progressive development of fibrosis with overexpression of TGF-beta1 in mdx diaphragm, and suggests the immunomodulation of this fibrogenic cytokine as a promising therapeutic approach for muscle fibrosis.
Granulysin expression in infiltrating cells in inflammatory myopathies K. Ikezoe a , S. Oshima a , M. Osoegawa a , K. Ogawa b , K. Nagata b and J. Kira a a Kyushu University, Fukuoka, Japan; b BML, Kawagoe, Japan Objective: Granulysin is an effector molecule present in cytolytic granules of NK cells and cytotoxic T cells. The aim of this study is to clarify the difference of expression of granulysin in infiltrating cells in various inflammatory myopathies and the correlation of its expression with steroid resistance. Method: We analyzed the expression of granulysin and perforin in muscles from 10 cases of polymyositis (PM), 3 dermatomyositis (DM), and 5 inclusion body myositis (IBM) by immunohistochemistry. We also carried out the double staining of granulysin with CD4, CD8, and CD56. Results: In PM and IBM, most infiltrating cells with granulysin were found in endomysium and CD8 or CD56 positive. These results were the same as those of perforin. The ratio of the number of granulysin positive cells to that of CD8-positive cells (G/CD8 ratio) in steroid responsive PM (0.30) was lower than that of steroid-resistant PM (0.50) (Mann-Whitney, p=0.081). In IBM, the G/CD8 ratio was 0.17. In DM, there were few infiltrating cells with granulysin. Conclusion: There is a possibility that granulysin is concerned in steroid resistance in PM. In IBM, steroid resistance is based on factors other than granulysin. In DM, granulysin is not participating in muscle fiber damage.
Sporadic inclusion body myositis: immunotherapy downregulates inflammation and beta-amyloid associated factors in repeated muscle biopsies J. Schmidt, J. G. Voss, R. Raju and M. C. Dalakas National Institutes of Health, Bethesda, MD, USA To investigate effects of high dose prednisone (PRED) and intravenous immune globulin (IVIG) on chemokines, cytokines and beta-amyloid associated proteins, crucial for sIBM-pathogenesis. IVIG and PRED are ineffective in sIBM, although proven beneficial in other inflammatory muscle disorders. mRNA was extracted from 10 sIBM muscle biopsies before and 4 months after treatment with IVIG+PRED or PRED. cDNA was analyzed by quantitative (real-time) PCR in relation to GAPDH-expression, using sequence specific primers for chemokine-ligand CXCL9, CCL3, CCL4, interferon (IFN)-gamma, tumor-necrosis-factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-10, transforming-growth-factor (TGF)-beta, inducible-costimulator (ICOS) and -ligand (ICOS-L), perforin, amyloidprecursor-protein (APP) and ubiquitin. After IVIG+PRED or PRED, we observed a 3-12-fold downregulated mRNA-expression: The strongest decrease was noted for CXCL9, CCL3, CCL4, followed by IFN-gamma, TGF-beta and IL-10. In most patients, IL-1beta, APP and ubiquitin were clearly diminished. TNF-alpha, IL-6, ICOS, ICOS-L and perforin were modestly reduced. Immunohistochemical greyscale-analysis confirmed downregulation at the protein-level. In sIBM, IVIG+PRED or PRED drastically reduced the expression of proinflammatory chemokines, cytokines, APP and ubiquitin. Our results suggest that a stronger downregulation of these molecules may be necessary to achieve clinical efficacy, or that other factors are more relevant for the immunological-degenerative interplay in sIBM-pathogenesis.
The RAGE pathway in inflammatory myopathies M. Haslbeck a , U. Fries b , E. Schleicher b , A. Bierhaus c , B. Neundfrfer a and D. Heuss a a Department of Neurology, University Erlangen-Nürnberg, Germany; b Department of Medicine IV, University Tübingen, Tübingen, Germany; c Department of Medicine I, University of Heidelberg, Heidelberg, Germany
Oxidative stress and NF-kB activation are linked to the pathogenesis of many metabolic, degenerative, and chronic inflammatory diseases. Activation of the receptor for advanced glycation end products (RAGE) by its specific ligand Ne-(Carboxymethyl)lysine (CML) results in activation of NF-kB and production of proinflammatory cytokines. In order to determine, whether an engagement of RAGE might contribute to the pathogenesis of inflammatory myopathies, we studied the presence of CML, RAGE, and activated NF-kB by immunohistochemistry in muscle biopsies of patients with polymyositis (PM, n=8), dermatomyositis (DM, n=8) and in eight healthy controls. In inflammatory myopathies CML, RAGE and NF-kB was detected in mononuclear cells, regenerating muscle fibers and in degenerating muscle fibers. Colocalisation of CML, RAGE and NF-kB was seen in infiltrating mononuclear cells and regenerating muscle fibers. Our data suggest that the CML-RAGE-NF-kB pathway is an evident proinflammatory pathomechanism in mononuclear effector cells in PM and DM. RAGE-mediated NF-kB expression may play a role in muscle fiber regeneration in inflammatory myopathies. Autoimmune T cells in myasthenia gravis (MG) recognize several regions of the human muscle acetylcholine receptor (AChR), most of them presented by HLA class II DR molecules. By TEPITOPE analysis, we have identified eight synthetic AChR sequences and tested T cell responses in PBMC of 74 MG patients and 51 healthy controls in proliferation studies. The overall reactivity observed among patients was significantly higher than healthy controls but the frequency of response varied with specific HLA-DR phenotypes. Sequences p6-24, p36-52 and p131-147 were poorly recognized by HLA-DR5 + MG patients whereas p35-53 was strongly recognized by HLA-DR1 + MG individuals. HLA binding assays of these peptides revealed varied capacity of binding to the eight tested HLA-DR class II molecules. Female MG subjects, mainly with severe disease, displayed significantly higher responses against p36-52, p131-147 and p133-145 than male MG. Patients between 25 and 40 years showed strong reactivity against p36-52, p131-147 and p195-212. Reactivity against p8-22 was significantly higher in non-thymectomized patients as well as in patients with short duration of disease. These findings may have important implications in designing future therapeutic strategies for the disease. Supported by FAPESP (00/11889-9).
Immunology of ryanodine receptor and FK506 in myasthenia gravis M. Takamori Neurological Center, Kanazawa-Nishi Hospital, Kanazawa, Japan
To study the implication of anti-striational antibodies in muscle contraction fatigue in myasthenia gravis (MG), antibodies to ryanodine receptor (RyR, essential for excitation-contraction coupling in muscle) were measured in sera from 33 MG patients, 18 with thymoma and 15 without thymoma, by use of three synthetic peptides deduced from the RyR1 amino acid sequence. One region, close to the N-terminus, implicates in inactivation at high cytoplasmic calcium concentration; the other two peptides, C-terminal transmembrane region and C-terminal tail, are from the region responsible for functional calcium release channel. Antibodies were more frequently positive against the two C-terminal peptides, particularly in thymomaassociated MG. The N-terminal peptide antibodies were uncommonly detected. In 7 patients selected from 33 MG patients based on the authorized criteria for therapeutic trail, the treatment was designed to give FK506 which is an immunosuppressant acting as calcineurin inhibitor and also affects RyR-related muscle contraction through an accelerated sarcoplasmic calcium release. The efficacy was estimated by the reductions in disease severity score, prednisone dosage and anti-acetylcholine receptor antibody titers at 1-week and 1-year medication. Results showed early and sustained benefit of FK506 in patients positive for antibodies to C-terminal peptides. An early effect of FK506 may reflect its action as an enhancer of RyR-related sarcoplasmic calcium release and its effects on the RyR Nterminal region.
Myasthenia gravis patient sera exhibit cytotoxic effects on human primary muscle cell cultures S.P. Luckman, G.O. Skeie, G. Helgeland and N.E. Gilhus Haukeland University Hospital, Bergen, Norway Myasthenia gravis (MG) is caused primarily by autoantibodies directed against the nicotinic acetylcholine receptor (AChR), with additional antimuscle autoantibodies being detected in some MG patients. The role of non-AChR autoantibodies remains to be elucidated. In order to investigate the role(s) of these autoantibodies in MG, primary human muscle cell cultures have been exposed to MG patient sera and the morphological effects observed. Cell death in these cultures was also examined. Cultured human muscle cells were exposed to MG patient or control sera (normal donor, systemic lupus erythematosus, or Sjogren's syndrome patient sera) for 1-4 days, and observed by crystal violet staining and light microscopy. Sera from the most severe MG patients (MG crisis patients), induced a dramatic change in cell morphology, leading to multinucleated cell clusters (as opposed to the elongated multinucleated myotubes observed in control cultures), with obvious inclusion body and vesicle formation. A dramatic reduction in total cell number was also observed, indicating a cytotoxic effect. These effects were dose-and time-dependent and were not complement-mediated. The cytotoxic effects of the MG patient sera do not appear to be due to the induction of apoptosis. These studies demonstrate a direct cytotoxic effect of MG patient sera against normal human muscle cells in culture, with sera from the most severely affected patients demonstrating the greatest cytotoxic effect. In order to know whether acetylcholinesterase (AChE) autoimmune plays a role in both myasthenia gravis (MG), which is associated with antibodies directed against the nicotinic acetylcholine receptor (AChR) in 85% of patients, and Graves' disease (GD), we analysed by ELISA the ability of sera from 22 patients with MG without antibodies against AChR, 116 with GD having the antibodies against thyreoglobulin (Tg), and 50 healthy controls (CR) to react with AChE from human brain and human Tg. Results showed that significantly increased anti-AChE activity was exhibited by a high proportion of MG (50%) and nearly unchanged anti-AchE activity was observed in GD (1%) sera, being similar with CR, which no anti-AChE activity detected. Anti-Tg activity was detected in both MG patients (25% positive) and CR (22% positive). It is suggested that AChE autoimmune should be another important reason resulting in MG but plays no role in GD. Tg autoimmune may be ignored in MG.
The in vivo role of complement activation in myasthenia gravis F. Romi, E.K. Kristoffersen, J.A. Aarli and N.E. Gilhus Haukeland University Hospital, Bergen, Norway Background: Antibodies to the acetylcholine receptor (AChR), titin, and the ryanodine receptor (RyR) occur in myasthenia gravis. These antibodies have been found capable of complement activation in vitro. The involvement of the complement system should theoretically cause consumption of complement components such as C3 and C4. Materials and methods: Complement components C3 and C4 were assayed in sera from 78 AChR antibody positive MG patients (48 titin antibody positive of whom 20 were also RyR antibody positive; 30 titin and RyR antibody negative of whom 15 had early-onset MG and 15 late-onset MG) and 52 healthy controls. Results: MG patients with AChR antibody concentrations above the median had significantly lower mean C3 and C4 concentrations compared to those with AChR antibody concentrations below the median. Titin antibody positive MG patients, titin antibody negative early-onset MG patients, titin antibody negative late-onset MG patients, and controls had similar C3 and C4 concentrations. Nor did mean C3 and C4 concentrations differ in MG patients with RyR antibodies. Patients with severe MG had similar C3 and similar C4 levels compared to those with mild MG. Conclusion: There is serological evidence of increased C3 and C4 consumption in MG in vivo in patients with high AChR antibody concentration, unrelated to MG severity and other muscle antibodies. This study does not exclude, however, the role of complement activation in the pathophysiology of other muscle antibodies in MG.
Modulation of antigen specific responses against AChR by recombinant erythropoietin F. Ubiali a , S. Nava a , C. Antozzi a , P. Ghezzi b , C. Cappelletti a , F. Cornelio a , R. Mantegazza a and F. Baggi a a National Neurological Institute C. Besta, Milan, Italy; b Mario Negri Institute for Pharmacological Research, Milan, Italy
Recombinant erythropoietin (rEPO) treatment alters the number of circulating T-cells and CD4/CD8 ratio; these effects might influence immune responses pathways such as antigen presentation and/or recognition by T cells. The aim of this study was to evaluate rEPO effect on a T-cell line specific for peptide p97-116 of rat-AChR alpha-subunit, a myasthenogenic T-cell epitope in Lewis rat. T-cells were challenged with ConA and p97-116 in the presence of rEPO (50, 100, 200 ng/ml), and specific responses were measured by 3 H-thymidine incorporation. Proliferative responses (ConA 20012F936 cpm; p97-116 16171F1176 cpm) were significantly reduced by spleen cells (conventional APC) preincubated with rEPO (100 ng/ml, 3 h): ConA 9232F812 cpm ( p=0.0012); p97-116 11366F670 cpm ( p=0.0069). Similar results were obtained by preincubation with 200 ng/ ml of rEPO (ConA 7114F1044 cpm; p97-116 8289F945 cpm). These experiments were repeated using as APC bone marrow-derived DCs, differentiated with GM-CSF and IL-4. p97-116-specific responses (8493F832 cpm) were reduced (5199F1819 cpm) when rEPO was directly added to wells; this result was enhanced by DCs pre-incubation (3 h) with rEPO (4846F933 cpm, p=0.043). rEPO was also tested on TAChR-specific responses from lymph node cells (LNC) derived from TAChR-immunized Lewis rats. LNC proliferations were reduced by rEPO (added to cultured LNC), confirming its modulatory properties. These results suggest that rEPO might be implicated in the regulation of the autoimmune response to AChR.
Use of ELISPOTS to monitor cloning of AChR-specific T-cells from myasthenia gravis patients W. Zhang, I. Leite, U. Kishore, P. Water and N. Willcox Neurosciences Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK Autoimmune CD4 T-cells that recognise peptides from the acetylcholine receptor (AChR) play an important role in the initiation and progression of Myasthenia Gravis (MG). Because of the complexity of the AChR molecule, difficulties in purifying its subunits and the low frequency of AChR-specific T-cells in the peripheral blood lymphocytes (PBL), cloning them is laborious and inefficient. We have modified a specific ELISPOTS assay and used it to monitor our recent T-cell cloning. This assay has enabled us to determine precisely whether patient's' PBL have responded specifically to one round of in vitro stimulation with AChR subunits or peptides. It also makes it quicker and easier to define the epitope(s) being recognised and to estimate frequencies of the responding T-cells. We have now identified two promising lines for cloning.
MuSK antibodies in myasthenia gravis N. Desai, M.E. Farrugia, J. McConville and A. Vincent Oxford University, Oxford, UK Antibodies to the receptor tyrosine kinase, MuSK, are found in up to 70% of patients with generalised myasthenia gravis without acetylcholine receptor antibodies (bseronegativeQ, SNMG). Here we describe the characteristics of the antibodies, and their frequency in myasthenia gravis patients kindly provided by neurologists from 17 countries. The antibodies are very high affinity (kDs around 100 pM), are predominantly of IgG subclass 4, with some IgG1 and IgG2, and titres range from 1 to 200 nM. Patients with MuSK-antibody positive MG are most frequently female and tend to have marked bulbar weakness. The age at onset is mainly under 40 years, but older patients are relatively common in some countries. Altogether, antibodies to MuSK have been detected in a total of 128 out of 358 SNMG patients, representing 0-46% of SNMG patients from different centres, including nine in Europe, three in North America and five in the Far East. The highest prevalence was in patients originating from countries between 358 and 508N of the equator. The high affinities of the antibodies, and their variable frequency in SNMG patients from different geographical locations, raise the possibility that the antibodies are induced by a specific environmental antigen. Myasthenia Gravis (MG) with autoantibodies against Muscle Specific Kinase (MuSK) is an autoimmune disorder of neuromuscular transmission characterised by fluctuating weakness of predominantly bulbar muscles. We studied the epidemiology of anti-MuSK(+) MG in a distinct area in the province of South Holland with 1.6 million inhabitants. We reviewed charts of all patients diagnosed with MG in nine hospitals. In two hospitals without registration of diagnosis, we examined charts of all patients tested for anti-AChR antibodies. Between January 1st 1990 and January 1st 2004, MG was diagnosed in 246 patients. Anti-AChR antibodies were negative in 57 of whom 40 had their first symptoms within this period, an incidence of 1.8 per million per year. Among them, two patients were anti-MuSK(+), an incidence of 0.1 per million per year. The prevalence of anti-MuSK(+) MG on January 1st 2004 was 1.9 per million, based on 3 patients in a group of 52 anti-AChR(À) patients being alive. Among the 27 patients with generalised anti-AChR(À) MG, 3 of 20 patients tested for anti-MuSK were positive (15%). When restricting this group to EMG confirmed MG, the proportion of anti-MuSK(+) MG increased to 25%. In the Netherlands until now, we have found 82 patients with generalised anti-AChR(À) MG, 13 being anti-MuSK(+) out of 55 tested (24%). In this population-based study, the prevalence of anti-MuSK(+) MG is lower than suggested by previous reports.
Analysis of LIF, IL-6, TGF-beta1 and oncostatin M in myasthenic thymuses E. Arnoldi, P. Bernasconi, F. Baggi, F. Cornelio and R. Mantegazza National Neurological Institute C. Besta, Milan, Italy
Thymus is a crucial tissue in the pathogenesis of myasthenia gravis (MG) and is thought to be the site of loss of tolerance in the disease. Thymic abnormalities are often found in MG patients, including abnormal presence of B cell germinal centers. We investigated the transcription levels of cytokines involved in thymopoiesis and B cell function: leukemia inhibitory factor (LIF), oncostatin M (OSM), IL-6 and TGF-beta1. By real time-PCR, we studied the expression of these cytokines in 29 myasthenic thymuses (10 involuted, 11 hyperplastic, 8 thymoma). IL-6 and OSM transcript levels were higher in 55% of cases with hyperplastic thymuses and in 50% of thymoma cases than in involuted thymuses. LIF and TGF-beta1 expression was higher in hyperplastic thymuses than in involuted thymuses, while in all thymoma cases we did not observe an increase of the expression levels. These data suggest that the expression of the analyzed cytokines might be related to the presence of intrathymic cellular proliferation. A correlation between cytokine levels and serum anti-AChR antibody titers was not evident. Objective: To describe development and treatment of myasthenic syndrome after local botulinum-toxin injections for cervical dystonia. Methods: A 62-year-old male suffering from cerebral palsy (tetraparesis and dystonia) presented with severe cervical pain secondary to spasmodic torticollis. He was treated with several local injections of botulinum toxin type A with good results. He reported long-lasting (12 months) bilateral proximal brachial paresis after the last treatment and injections were stopped for 2 years. Pain became severe and the patient asked for treatment. Botulinum-toxin type B was administered four times. A rapidly evolving progressive tetraparesis presented 3 days after the fourth injection. Results: Myasthenic syndrome (facilitation on EMG and increase titter of antibodies for Ca channels) was diagnosed. Other studies (including nerve conduction velocities and SF analysis) were normal and no neoplasia was found. Immunoglobulin was administered intravenously and the progression of paresis immediately stopped. The patient quickly improved and steroids were given later with good results. Conclusions: The causal relationship between administration of Botulinum toxin and development of myasthenic syndrome warrants consideration. It is impossible to know whether toxin causally participated in the immunologic response against the neural structures or simply both toxins A and B unmasked pre-existing myasthenic syndrome. The response to treatment with immunoglobulin was remarkable.
Prognostic significance of thymectomy in autoimmune myasthenia gravis (MG) A. Ariatti, P Faglioni and G. Galassi
Objective: To verify efficacy of thymectomy in MG. Methods: A retrospective study was conducted on 72 MG patients (32 males, mean age 65.7; 40 females, mean age 63.4; range 17-84 years, mean age 64.0 years). Mean duration of patient illness was 69.9 months. Severity was defined according to Osserman's classification. Asymptomatic patients were scored b0Q for statistical purposes. At time of diagnosis, 26 patients were graded I, 29 as grade IIa, 17 as grade IIb; no patients were classified in grade III and IV. According to actual severity, 27 patients were included in grade I, 30 in grade IIa, 8 in grade IIb; no patients were graded as III and IV; 7 patients were asymptomatic at time of clinical evaluation. Clinical outcome was evaluated in patients who underwent surgery either, for thymoma (N=14), for non-neoplastic thymus (N=11) and in those not treated surgically (N=47). Results: (1) Percentages of subjects that improved were 21% and 55% among thymectomized patients either for thymoma, or hyperplastic thymus, respectively; 17% of patients who improved did not undergo thymectomy.
(2) The three percentages differed significantly: Chi square (2)=7.00, Pb0.03; (3) decomposition of total Chi square revealed that thymectomy raised chances of improvement (17% vs. 36%; Chi square (1)=3.30 Pb0.04 one tailed. Furthermore, hyperplastic thymus benefited from thymectomy more often than neoplastic thymus (55% vs. 21%; Chi square (1)=3.75; Pb0.05 one tailed). Paraneoplastic Neurological Syndromes (PNS) are immune-mediated, nonmetastatic disorders associated with cancer. They start before tumor's evidence and involve central or peripheral nervous system. The role of antineuronal antibodies is uncertain but they are important for the diagnosis. Myasthenia gravis (MG) is an autoimmune neuromuscular disorder sometimes associated with thymoma. Combination of MG with Hodgkin's Disease (HD) is rare. We report a case of 52-year-old female with generalized MG and radiological suspect of thymoma. Anti-acetylcholinereceptor (AChR) antibodies resulted positive but anti-titine and antiryanodine antibodies were negative. After neurological therapy, the patient was submitted to thymectomy. A nodular-sclerosing syncizial HD plus thymic atrophy was diagnosed. Oncological screening confirmed a clinically II A HD with mediastinal bulk. Neurological treatment was stopped and chemo plus radiotherapy was started. Successive 9 years of follow up revealed complete remission of both MG and HD with progressive decrease of anti-AChR antibodies. Combination of seropositive MG, thymic atrophy and HD may suggest that myasthenic symptoms could be triggered by immunological alterations underlying HD. Stable and complete resolution of MG after HD remission as well as progressive decrease of anti-AChR antibodies may reinforce this hypothesis and suggests a pathogenetic role of these antibodies in paraneoplastic MG associated to HD. Autoimmune diseases (AID) are known to cluster in families. We studied the frequency and nature of additional AID in patients with the Lambert-Eaton myasthenic syndrome (LEMS) and their family members, in both small cell lung carcinoma (SCLC) related and non-tumour (NT)-related cases. Additional AID in patients with LEMS were assessed by interviewing the patient and studying the medical record. Family histories up to second-degree family members were established by interviewing patients, controls and family members. Forty-four patients with LEMS were assessed, of whom eighteen (41%) had SCLC. In the NT group, seven patients (27%) had an additional AID, in the SCLC group two (11%) ( p=0.20). Thyroid disorder (five patients) and insulin-dependent diabetes mellitus (two patients) were the most common AID. AID were significantly more frequent in families of patients with NT-LEMS (64%) than in control families (27%, p=0.002), or in families of SCLC-LEMS patients (36%). Affected family members were linked to the NT-LEMS patient through the maternal line in all cases. In conclusion, AID were more frequently found in NT-LEMS patients and their families, than in SCLC-LEMS patients and their families. This suggests that NT-LEMS shares immunological risk factors with other AID. This finding and the remarkable preponderance of maternal inheritance, which was reported previously in myasthenia gravis, might help to identify the responsible mechanism or genes involved.
Daily versus alternate day plasmapharesis for induction of remission in patients with severe myasthenia gravis a randomised trial S. Singh, I. Trikha, V. Goyal, G. Shukla, T. Srivastava, R. Bhasin and M. Behari All India Institute of Medical Sciences, New Delhi, India Objectives: To assess the comparative efficacy of daily versus alternate day Plasmapharesis for inducing remission in patients with severe myasthenia gravis. Methods: Thirty-three patients with myasthenia gravis, Osserman's stage IIb and III were randomly allocated to receive alternate day (n=17) or daily plasma exchange (n=16). Myasthenia gravis disease scale (MGDS) score was compared before and after the procedure and for subjective improvement in symptoms. Time taken for weaning off ventilator, removal of nasogastric tube and total duration of hospital stay were also assessed. Results: Four to five sessions of plasma exchange were carried on each patient and about 20-25 ml/kg plasma was removed during each session. No statistically significant difference was found in daily vs. alternate day group with regards to change in MGDS score and percentage change in MGDS score at discharge. The total duration of stay in hospital was less in the daily group as compared to the alternate day group, which almost reached statistical significance ( p=0.054). Complications in the two groups were also similar. Conclusion: We conclude that daily and alternate day plasma pharesis are similar in their efficacy and complication rates, the duration of hospital stay is less with the daily plasma pharesis. Hence, it can be the preferred method of performing plasma exchange in patients with myasthenia gravis to induce remission of symptoms. Various mutants of the human anti-human-acetylcholine receptor (AChR) antibody IgG1-637 were produced and characterised. These included two mutants with changes in the complement-binding site, two mutations for generating bispecific antibodies and a mutation deleting the affinity for the antigen. Moreover, a His6 and a Flag tag were added for analysis and purification of different heavy chains. A transient expression system using HEK293 cells was successfully used for production of up to 30 Ag/ml. The non-complement-binding antibody IgG1-637-C1q was tested in a passive transfer EAMG model with Rhesus monkeys for its ability to compete with the pathogenic parental antibody IgG1-637.
A drug candidate for treatment of myasthenia gravis: efficacy of a novel immunosuppressant FTY720 in experimental autoimmune myasthenia gravis T. Kohno a , K. Hirayama a , R. Iwatsuki a , M. Hirose a , K. Watabe a , H. Yoshikawa b , A. Matsumoto c , T. Kohno c , T. Fujita c and M. Hayashi d a Faculty of Phamaceutical Sciences, Setsunan University, Osaka, Japan; b Kanazawa University, Ishikawa, Japan; c Research Institute for Production and Development, Kyoto, Japan; d Uwajima City Hospital, Ehime, Japan FTY720 was discovered by chemical modification of ISP-I, a metabolite of the fungus Isaria sinclairii, by Fujita who is one of the authors. The efficacy of FTY720 had been well established in preclinical transplantation models and has recently proven to be effective in renal transplantation in humans. In this study, the efficacies of FTY720 were examined, in preventing experimental autoimmune myasthenia gravis (EAMG) development, and in treatment of EAMG. Eight mice out of 12, which had been immunized three times with torpedo californica acetylcholine receptor (AChR) and received water, were developed EAMG. In six mice, which had been immunized and received FTY720 continuously (1 mg/kg, orally, three times a week), starting before immunization, no EAMG symptom was observed. The eight EAMG mice were divided into a placebo group (n=4), which had been received water, and a treatment group (n=4), which had been received FTY720 described as above after development of EAMG. The holding time in inverted grid test was significantly improved in the treatment group. These findings show FTY720 is effective not only in the prevention but also in the treatment of EAMG. In addition, to clarify the mechanisms of pharmacological actions of FTY720, cytokine mRNA levels, anti-AChR IgG levels (ELISA using mouse AChR as an antigen), and AChR in muscle (fluorescence microscopic detection using FITC-labeled bungarotoxin) had been examined.
Pyridostigmine 2-4-6 induction regimen is effective in myasthenia gravis R.S. Nicholas a , M. Roberts b , J. Sussman b , J. Winer c and I.K. Hart a a The Walton Centre, Liverpool, UK; b Hope Hospital, Manchester, UK; c Queen Elizabeth Hospital, Birmingham, UK Introduction: Pyridostigmine is the first-line treatment for myasthenia gravis (MG). However, there are few studies of its optimum use and neurologists often see patients on subtherapeutic doses or with drug adverse effects. Aim: To study in 50 MG patients, the pyridostigmine 2-4-6 induction regimen developed from the drug's pharmacokinetic profile. Methods: Patients entered an open-label study of the 2-4-6 regimen and worked up to the most useful dose: 30 mg twice/day for 2 days; 30 mg five times/day for 4 days; 60/ 30/60/30/60 mg per day for 6 days; 60 mg five times/day maximum dose. Patients and their neurologists ranked the disease symptoms by severity (0-5) on entry, and at 1 month along with drug adverse effects. Results: These are preliminary results from 18 patients: 7 ocular MG (oMG; average 1.6 symptoms), 6 oculobulbar MG (obMG; average 3.7 symptoms), 5 generalised MG (gMG; average 3.4 symptoms). 16/18 (89%) patients improved and completed the trial. The severest oMG symptom (ptosis 4, diplopia 3) fell 1.1F1.2 grades (meanFS.D., pb0.05), the severest obMG symptom (dysphagia 3, ptosis 2, diplopia 1) fell 3.3F1 grades ( pb0.001), and the severest gMG symptom (weakness 3, dysarthria 2, diplopia 1) fell 1.6F1.1 grades ( pb0.04). 12/18 patients reported adverse effects but in only 2 was pyridostigmine stopped. Conclusion: The 2-4-6 pyridostigmine induction regimen is well tolerated and effective in MG. (This work was supported by an unrestricted research grant from Valeant Pharmaceuticals).
Cyclosporin: a treatment in myasthenia gravis patients with anti-MuSK antibodies D.M. Bonifati a , R. Padoan a , A. Vincent b , E. Pegoraro a and C. Angelini a a University of Padua, Padua, Italy; b University of Oxford, Oxford, UK Objective: To evaluate treatment of myasthenic patients presenting antireceptor tyrosine kinase (MuSK) antibodies. Background: Recently, antibodies to the MuSK have been identified in 40% of seronegative generalised myasthenia gravis (MG) patients. They often present with bulbar weakness and are difficult to treat. Design/methods: In a large population of MG patients, we screened seronegative patients for anti-MuSK antibodies. We retrospectively evaluated both the efficacy of thymectomy and choice of immunosuppressive treatment. Results: Four patients were positive for anti-MuSK antibodies. A 38-year-old man, affected by class IIB myasthenia, at 28 years an atrophic thymus was removed, surgery worsened his condition and he required plasmapheresis, he was refractory to steroid therapy and then he was treated with cyclosporin A 300 mg daily. A 44-year-old woman presented since age 42 a class IV myasthenia. She was treated with IVIg and then with cyclosporin A 250 mg daily, with marked improvement. A 39year-old woman had bulbar myasthenia, after thymectomy she needed several plasmapheresis cycles, steroids and azathioprine were used with benefit. A 25-year-old patient with ocular myasthenia at 23 underwent thymectomy, but after 2 months presented a relapse with upper limb weakness, she was treated with steroids with benefit. Conclusion: Two of our patients with anti-MuSK antibodies responded well to cyclosporin A that might be therefore an effective treatment in this group of myasthenic patients. Thymectomy did not seem to benefit all patients.
Dramatic response of ocular symptoms to IVIG treatment in myasthenia gravis A. Kurne, E.M. Arsava and T. Dalkara Hacettepe University, Ankara, Turkey Introduction: Myasthenia gravis is an autoimmune disorder characterized by ocular, bulbar and generalized weakness. Ninety percent of myasthenic patients suffer from ocular involvement sometime during the course of the disease. Ocular symptoms of patients with myasthenia gravis respond incompletely to anticholinesterase and immunosuppressive therapies. Objective: We report six myasthenic patients whose ocular symptoms dramatically responded to intravenous immunoglobulin (IvIg) therapy and were generally refractory to anticholinesterase and immunosuppressive treatment modalities. Case reports: Characteristics of patients and treatment features are summarized in the table.
We propose that the IvIg should be considered in myasthenic patients whose ocular symptoms do not respond to conventional therapies. Future studies may disclose whether all patients with ocular symptoms or only a subset of patients dramatically respond to the IvIg. Pyridostigmine, Corticosteroid NGF regulates CGRP expression in human monocytes: effects on B7 expression and IL-10 production L. Bracci-Laudiero a , L. Aloe a , C. Caroleo b , P. Buanne c , N. Costa d , G. Starace a and T. Lundeberg e a CNR Institute of Neurobiology and Molecular Medicine, Rome, Italy; b University of Calabria, Cosenza, Italy; c University of L'Aquila, L'Aquila, Italy; d University of bMagna GreciaQ, Catanzaro, Italy; e Karolinska Institutet, Stockholm, Sweden Administration of Nerve Growth Factor (NGF) in EAE models delays the onset and prevents the full development of EAE lesions. In line with this anti-inflammatory activity of NGF are our recent results on the autocrine NGF synthesis in B lymphocytes, which directly regulate in these cells the expression of calcitonin gene-related peptide (CGRP), a neuropeptide which is a potent inhibitor of T cell proliferation and antigen presentation by macrophages and monocytes. Using cultures of human monocytes we investigated by RT-PCR, immunofluorescence and flow cytometry analysis whether NGF can regulate the expression of CGRP also in these cells and influence the expression of B7.1 and B7.2 and IL-10. Our data indicate that monocytes synthesise basal levels of NGF but, after LPS stimulation, upregulate NGF expression in dose-dependent fashion. When the cultures are deprived of endogenous NGF, CGRP expression in resting cells decreases and the up-regulation of CGRP induced by LPS is prevented. In addition, we observed that endogenous NGF reduction affects co-stimulatory molecule expression and IL-10 synthesis, and these effects can be partially mimicked by using CGRP receptor I antagonist. Our findings indicate that endogenous synthesis of NGF in monocytes has a functional role and, by influencing B7.2 expression and IL-10 production, contributes to the downregulation of the immune response and strongly support the newly found anti-inflammatory activity of NGF. Background: Myasthenia gravis (MG) is caused by autoantibodies directed to the acetylcholine receptor (AChR) at neuromuscular junction leading to muscle weakness. Dendritic cells (DC), usually regarded as antigen-presenting cells involved in T cell activation, also affect B cell function. Immature DC may induce tolerance while mature DC induce immunity. The presence of IL-10 during maturation of DC inhibits their terminal differentiation. We explored potential of IL-10-modulated-DCbased immunotherapy in experimental autoimmune myasthenia gravis (EAMG) in Lewis rats. Method: Splenic DC were isolated from onset of EAMG on day 39 post immunization (p.i.), exposed in vitro to IL-10, and then injected intraperitoneally (i.p.) into ongoing EAMG rats at a dose of 1,000,000 cells/rat on day 5 p.i. with AChR+complete Freund's adjuvant. Results: Lower clinical scores, less body weight loss, lower numbers of anti-AChR IgG antibody secreting cells and lower affinity of anti-AChR antibodies were observed in rats receiving IL-10-modified DC, accompanied with lower expression of CD80 and CD86 and lower lymphocyte proliferation compared with control EAMG rats. Lower levels of IL-10 and IFN-gamma were also found in the supernatants of AChR-stimulated lymph node mononuclear cell cultures in rats receiving IL-10-modified DC. Conclusion: IL-10-modified DC induce hypo-responsiveness by down-regulating co-stimulatory molecules, and reducing the production of anti-AChR antibodies possibly by inhibiting IL-10 production in EAMG.
Immature and mature dendritic cells in multiple sclerosis lesions B. Serafini a , B. Rosicarelli a , R. Magliozzi a , E. Capello b , G.L. Mancardi b and F. Aloisi a a Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità , Rome, Italy; b University of Genova, Genova, Italy Dendritic cells (DC) accumulating in target organs during autoimmune disease are thought to have a key role in the stimulation of autoreactive T cells. To address the functional relevance of DC in multiple sclerosis (MS), we evaluated the degree of DC infiltration in MS brain using immunohistochemical techniques and a panel of antibodies recognizing DC subsets and maturation markers. All MS lesions examined were of the chronic active and inactive type. The C-type lectin DC-SIGN that is present on immature myeloid DC was expressed by cells accumulating around inflamed blood vessels, predominantly in chronic active lesions. No CD1a+ immature DC were detected. Markers of mature DC, like CD83 and DC-lysosomeassociated membrane protein (DC-LAMP) were expressed by a few, isolated cells in the perivascular space and parenchyma of chronic active lesions. CCR7, a chemokine receptor typical of mature DC, was detected on numerous cells at the edge of some chronic active lesions. Most of these cells were tentatively identified as microglia due to their ramified morphology, scattered intraparenchymal distribution and coexpression of Iba-1. In normal brain, only DC-SIGN+ cells were detected in the meninges. The presence of immature and mature DC in chronic active MS lesions suggests that abnormal presentation of myelin antigens by these potent antigen presenting cells could break immune tolerance toward CNS antigens and allow continuous stimulation of autoimmune responses. Upon stimulation by microbial products through Toll-like receptors (TLR), dendritic cells (DC) can prime naive T cells and initiate a pro-inflammatory immune response. Recently, we have shown that APC within the CNS of MS patients contain peptidoglycan (PGN), a major cell wall component of Gram-positive bacteria, which signals through TLR2. We report here that Staphylococcus aureus PGN as a single component can replace whole Mycobacterium tuberculosis in Freund's complete adjuvant in EAE. Mice immunized with and encephalitogenic MOG-peptide in incomplete Freund's adjuvant did not develop EAE. In contrast, addition of PGN to the emulsion was sufficient for priming of auto-reactive Th1 cells and development of EAE. In vitro studies demonstrated that PGN stimulates DC-mediated processes, reflected by increased antigen uptake, DC maturation, Th1 cell expansion, activation, and pro-inflammatory cytokine production. These data indicate that PGN-mediated interactions result in pro-inflammatory stimulation of antigen specific effector functions important for development of EAE. These PGN-mediated processes may occur both within the peripheral lymph nodes as well as in the CNS and likely involve recognition by TLR on DC. As such, PGN signaling pathways may serve as novel targets for the treatment of MS.
Dendritic cells (DC) are the key regulators of tolerance and immunity to the self-antigens that they sample and present to T cells. Depending on their maturation state, DC either induce regulatory T cells (immature DC) or autoreactive T helper 1 cells (mature DC). DC maturation is controlled by the balance of C-type lectin receptors (CLR), and Toll receptors (TLR). TLR recognize pathogen-associated molecular patters and induce DC maturation (Yang), while the CLR recognition of carbohydrate structures on self-antigens antagonizes DC maturation (Yin). As essentially all extracellular proteins are glycosylated, it can be envisaged that self glycoproteins can maintain tolerance to self. In a healthy individual, Yin and Yang are in balance. We have hypothesized that a lack of Yin by disturbance of the normal glycosylation of self glycoproteins affects the capacity of CLR to inhibit DC maturation. As a consequence, DCs will mature and induce autoreactive Th1 cells to the defective glycoprotein. We will discuss new data from nonhuman primate models of experimental autoimmune encephalomyelitis which support this hypothesis.
Modulation of experimental autoimmune encephalomyelitis with the infectious agent Trypanosoma brucei brucei M. Wållberg and R.A. Harris Karolinska Insitute, Stockholm, Sweden
The protozoan parasite Trypanosoma brucei brucei (Tbb) causes African sleeping sickness in cattle and also experimentally in mice, with human forms of the parasite causing disease throughout equatorial Africa. Mice harbouring a Tbb infection do not develop Experimental autoimmune encephalomyelitis (EAE), an animal model for human disease multiple sclerosis (MS), upon immunisation with recombinant myelin oligodendrocyte glycoprotein (rMOG) in complete Freund's adjuvant (CFA), and mice that are infected with the parasites at the time of immunisation or a week later develop less severe EAE. Protected mice display a markedly diminished rMOG-specific proliferation and IFN-gamma production in lymph node cells, and have correspondingly low titres of serum anti-rMOG IgG. Antigen presenting cells (APC) from spleens of Tbb infected mice present rMOG less efficiently to rMOG specific T cells in vitro than APC from control spleens, and can also inhibit antigen specific proliferations in control in vitro cultures. Transfer of splenic APC from Tbb infected mice into mice immunised with rMOG-CFA 7 days previously abrogated disease significantly.
Visualization of peracute antigen presentation/T cell activation in spleen following high-dose soluble antigen application F. Odoardi, Z. Li, N. Kawakami, W. Klinkert, J. Bauer, H. Lassmann, H.
We studied the mechanisms of intravenous high dose soluble MBP application (HSM) in adoptive transfer experimental autoimmune encephalomyelitis (tEAE) of the Lewis rat using gene engineered myelin basic protein specific T cells, which express the green fluorescent protein (TMBP-GFP cells) as marker gene. HSM prevented CNS inflammation and clinical disease if applied few hours before entry of encephalitogenic T cells into the brain by trapping of the effector T cell in the peripheral immune organs. In lymph nodes, live-imaging showed a dramatically reduced motility of TMBP-GFP cells, which formed stable aggregates with resident phagocytes. In the spleen, HSM treatment induced redistribution of TMBP-GFP cells from the red to the white pulp. The morphological changes were associated with fast T cell activation: increase in cytokines mRNA expression was observed within 1 h and membrane phenotype rearrangement 2 h after HSM application. T cell activation was blocked by anti-MHC II antibodies indicating rapid antigen processing and T cell recognition process underlying these HSM effects. Twenty-four hours after HSM application, the entire TMBP-GFP cell population was eliminated by activation induced cell death.
In conclusion, HSM treatment elucidates the crucial role of CNS-infiltration by autoreactive CD4+ T cells in CNS autoimmunity and serves as ideal model system to visualize peracute antigen processing/T cell recognition in vivo. Matrix metalloproteinase (MMP)-12 is involved in the immunopathogenesis of multiple sclerosis (MS) and implicated in the active process of demyelination. MMPs may contribute to demyelination by cleaving myelin proteins, and consequently propagate the immune response by generating new immunodominant epitopes. Little is known about the role of MMPs influencing MHC class II related peptide presentation of dominant myelin epitopes, thus influencing epitope spreading or limitation. Here we show that a principal human HLA-DR2-restricted epitope-amino acids 85-99 of myelin basic protein, MBP(85-99)-contains a cleavage site for MMP-12. Presentation of this epitope by antigen presenting cells was completely abolished in the presence of MMP-12 as demonstrated by using MBP(80-99) specific TCR-transfectants. In contrast, myelin oligodendrocyte glycoprotein (MOG) was not cleaved by MMP-12. By immunohistochemistry, MMP-12 could be detected in active MS lesions, predominantly expressed by CD68+ cells, indicative of macrophages and activated microglia cells. Moreover, proteolytic activity of MMP-12 was measurable in the CSF from patients with MS but not in controls. Our data demonstrate that MMP-12, found in MS patients, exhibits a novel function: by destroying an immunodominant MBP-epitope, this protease could directly influence the cascade of MBP-specific T-cell autoreactivity in MS. These findings are not only of considerable relevance for a deeper understanding of mechanisms amplifying or limiting local CNS autoreactivity but also for general considerations on central and peripheral tolerance.
The criteria for definition of driver T cell clones J.S. Menezes, A. Ametani, P. van den Elzen, E. Maverakis, V. Kumar and E.E. Sercarz Torrey Pines Institute for Molecular Studies, San Diego, CA, USA Driver T cells (DTC) can be defined as necessary and sufficient for the induction of EAE or other autoimmune diseases. We have recently reported that in the B10.PL mouse, a very small group of clones is responsible for the causation of EAE and when this clone is down-regulated no further disease occurs and the animal enters remission. Thus, the disease course follows the life history of the DTC. In fact, this becomes one of the criteria in defining bdriversQ. We have spent considerable effort in the B10.PL model to derive a set of criteria which might be used to define such clones: (a) a high affinity for antigen, which underlies the successful competitive performance of drivers in becoming dominant members of the repertoire; (b) DTC should appear very early in the lymph nodes, and by the end of the second week, they have migrated to the spinal cord and spleen; (c) a characteristic of drivers is their sequential appearance in these central lymphoid organs; (d) their publicity and (e) their proinflammatory nature. DTC are likely to be public clones, present in each individual, and they are also usually Th1 in cytokine pattern. We now call clones fitting all of these criteria driver T cell clones and they have been found in mouse EAE as well as in the NOD model of type I diabetes. Immunization with myelin antigens leads to the development of experimental autoimmune encephalomyelitis (EAE). Auto-aggressive T lymphocytes migrate into the CNS where they recognize their cognate antigen and initiate an inflammatory cascade leading to tissue damage. The T cells need to re-encounter their cognate antigen in the context of an MHCII bearing antigen presenting cell (APC) in order to recognize their target. Whether the antigen presenting cell (APC) that mediates T cell entry into the CNS is located within the CNS or the systemic immune compartment remains heavily debated. To determine the contribution of the systemic immune compartment in guiding auto-aggressive T cells into the CNS, splenectomized alymphoplasia (aly) mice, which do not have secondary lymphoid structures, were used. We demonstrate that these mice are fully susceptible to EAE indicating that encephalitogenic T cells do not need to home into secondary lymphoid tissues prior to CNS infiltration. In order to assess the capacity of CNS residents (microglia) to present Ag in vivo, mice with an MHCII-deficient CNS parenchyma were used as recipients of MOG-reactive T cells. Surprisingly, these mice are fully susceptible to develop EAE induced by adoptive transfer. Finally, we demonstrate that transgenic mice expressing MHCII molecules exclusively on dendritic cells develop EAE induced by adoptive transfer. Our data clearly show that CD11c+ dendritic cells are sufficient to present Ag to auto-reactive T cells thus mediating CNS inflammation and clinical disease development.
Multiple sclerosis: impaired phenotype and maturation of plasmacytoid dendritic cells M. Stasiolek a , A. Bayas b , N. Kruse c , K.V. Toyka b , R. Gold d and K. Selmaj a a Medical University, Lodz, Poland; b University of Würzburg, Würzburg, Germany; c University of Gfttingen, Göttingen, Germany Dendritic cell (DC) subtypes possess strong but diversified immunoregulatory properties. Plasmacytoid DCs (pDCs) demonstrate pronounced ability to prime regulatory T cells or the Th2 type of the immune response. Considering the putative immunoregulatory function of pDCs in multiple sclerosis (MS), we assessed the phenotype of pDCs isolated from the blood of MS patients and the maturation of pDCs in culture. Blood samples from 32 untreated RR-MS patients and 22 healthy controls were included in ex vivo phenotypic analysis performed by flow cytometry. pDCs were assessed for the expression of maturation antigens and co-stimulatory molecules. In maturation experiments, pDCs isolated magnetically from leucapheresis material (18 MS patients, 12 controls) were cultured for 96 h with IL3 and sCD40L. Every 24 h cultured pDCs were analysed for the expression of maturation markers and co-stimulatory molecules. pDCs in MS showed immature phenotype compared to pDCs of healthy subjects. The expression of co-stimulatory molecules CD86 and 4-1BBL on pDCs was significantly lower in MS samples than in controls. pDCs of MS patients also showed impaired maturation as demonstrated by significantly lower or delayed up-regulation of CD86, 4-1BBL, CD40, CD83. The observed abnormalities in ex vivo phenotype and maturation process may contribute to the immunoregulatory imbalance in MS.
Serum of patients with multiple sclerosis induce activation of antigen presenting cells A. Karni a , D. Azulai a , A. Korczyn a and H. Weiner b a Department of Neurology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; b Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MS, USA Objective: To study the effect of serum from patients with multiple sclerosis (MS) on activation of antigen presenting cells (APCs). Methods: Peripheral mononuclear cells (PBMCs) from healthy donors were incubated with 25% serum of healthy controls (HC, n=12) or MS patients during remission (n=25) or relapse (n=11). The production of IL-12 and TNF-alpha in dendritic cells (DCs) and monocytes and the expression of HLA-DR, CD80 and CD40 measured by flow cytometry. Results: An increased percentages of IL-12 producing DCs detected after incubation with serum of secondary progressive (SP) MS (3.5F2.4%) compared to relapsing-remitting (RR) MS (1.3F1.0%, p=0.04) and to HC (0.8F0.7%, p=0.02). We searched the serum of MS patients for cytokines known to activate DCs and only IFNgamma was consistently detected. PBMCs were incubated with eight serum from SP-MS in the presence of anti-IFN-gamma blocking antibody or isotype control. IFN-gamma blockade decreased the percentages of IL-12 producing DCs from 3.14F2.15% to 0.84F0.79%, p=0.008. In a comparison with serum of patients in remission, the serum of the same patients during relapse induced increased expression of HLA-DR on monocytes (mean florescence intensity 2732F409 vs. 1171F355, p=0.05), increased percentages of TNF-alpha producing cells (2.5F0.9% vs. 1.5F0.5%, p=0.04, respectively). Conclusions: Serum of MS patients include factors that activate APCs. Serum IFN-gamma appears to be one of these factors. This serum effect may provide a proinflammatory signal via APCs for myelin autoreactive T cells.
CNS microvascular pericyte polarization to alternatively activated phenotypes P. Dore-Duffy a , R. Balabanov b and J. Williams a a Wayne State University, School of Medicine, Detroit Medical Center, Detroit, USA; b University of Chicago, Chicago, USA CNS microvascular pericytes are thought to be highly complex regulatory cells important to tissue homeostasis and vascular hemostasis. We have previously shown that rat primary capillary pericytes have immune potential. They present antigen to sensitized leukocytes and drive naRve T cells to the TH1 or TH2 phenotype. Pericytes may therefore function as a CNS antigen presenting cell (APC) and undergo TH1 or TH2-like polarization associated with alternatively activated phenotypes. To test this hypothesis, we examined freshly isolated rat pericytes. Pericytes were found to be primed to the APC phenotype and produce high levels of IL-12 and low levels of PGE. Seven-day-old primary pericytes constitutively release IL-18. They adhere CD4+ cells of the TH1 cytokine secreting phenotype. In the presence of IFN gamma, they express class II antigen and process antigen. In the presence of stress stimuli such as low oxygen or TH2 cytokines (IL-4), IL-12 production is decreased, eicosanoid synthesis is increased and pericytes synthesize TGF beta. During this stage of differentiation, pericytes are poor APC and selectively adhere TH2 cells. They express vascular endothelial cell growth factor (VEGF) and stimulate angiogenesis. These data suggest that pericytes have multiple states of activation/differentiation comparable to traditional APC. It is likely that pericyte APC have an important role in CNS inflammation. Perivascular macrophages (PVM) are a subpopulation of macrophages in the central nervous system. Based on animal studies and on the strategic location of the PVM at the interface between circulation and brain parenchyma, it has been postulated that PVM play a role in antigen recognition and presentation. Here we show that the hemoglobinhaptoglobin scavenger receptor, CD163, can be used as a marker for PVM in the human brain. By means of double immunofluorescence stainings in normal control brain tissue and in sections of several multiple sclerosis (MS) lesion stages, it appeared that CD163+ PVM express DC-SIGN, MHC II and several costimulatory molecules. Furthermore, we observed an increased number of CD163+ DC-SIGN+ macrophages as well as expression of CD163 and DC-SIGN on foamy macrophages. These double stainings suggest that PVM have the necessary tools for antigen recognition and presentation and supposedly play a role as antigen-presenting cell of the brain. Further studies are required to establish which immune regulatory role these cells exert upon antigen recognition. Multiple sclerosis (MS) has a relapsing-remitting nature in 85-90% of the patients, in which periods of disease and recovery alternate. This implies that there is a balance between detrimental and protective mechanisms. Cervical lymph nodes (CLN) are a major site of drainage of the brain. It has been shown that brain-derived myelin components are present in antigen presenting cells (APC) within the CLN during MS. We hypothesize that these APC play an important role in regulating the peripheral immune system during MS. Immunohistochemistry revealed that APC in the cervical lymph nodes not only contain myelin components, but also other central nervous system (CNS) components such as astrocytic and neuronal proteins. Immunohistochemistry was also used to determine the immunophenotype of CNS antigen-containing cells. MOG-containing cells express the anti-inflammatory molecules IL-1ra and TGF-beta, but did not express the pro-inflammatory cytokine TNF-alpha. To determine the antigenpresenting capacity of myelin-laden cells in vitro, monocyte-derived macrophages were pulsed with myelin and co-incubated with T cells in a mixed leukocyte reaction. Preliminary data demonstrate that pulsed macrophages increased T cell proliferation. In addition, IFN-gamma production by T cells paralleled T cell proliferation, suggesting enhanced antigen presentation. Our data suggest that phagocytic APC in CLN may display regulatory functions in the periphery during MS. Accumulating evidence suggests that, apart from clonal deletion and anergy, T cell mediated control of self-reactive T cells contributes to the maintenance of natural immunological self-tolerance. For example, expansion of CD25+CD4+ T cells, which constitute 5-10% of peripheral CD4+ T cells in rodents and humans, prevents autoimmunity by suppressing the activation of self-reactive T cells. It has been observed that dendritic cells (DC) can prime CD25+CD4+ T cells to acquire suppressive properties. In this context, gene expression analysis in murine models of autoimmunity revealed a functional role for the glucocorticoid-induced TNF receptor (GITR) in CD25+CD4+ T cells and recently it was shown in humans that a forced expression of the Foxp3 gene, which encodes a transcription repressor gene, can convert CD4+ T cells to T suppressor cells. In a preliminary analysis using real time PCR, we could not observe any significant differences in FOXP3 and GITR mRNA expression levels between MS patients and healthy donors. Therefore, we now extend our study employing gene expression microarray technology to assess gene expression differences in CD25+CD4+ T cells and DC between Multiple Sclerosis (MS) patients and young healthy donors. Differentially expressed genes in these key cells of immunological tolerance may further our understanding of the molecular pathogenesis of MS and provide new disease markers.
Brain dendritic cells: origin, maturation, function G. Reichmann a , A. Mausberg a , S. Jander a , D. Schlqter b and H.G. Fischer a a Heinrich-Heine-University, Düsseldorf, Germany; b University Mannheim-Heidelberg, Germany
It is generally accepted that DC are lacking in the normal brain, however, DC appear at intracerebral sites under inflammatory conditions. In the absence of a brain-directed T cell response as in cerebral ischemia, these DC remain immature, whereas in pathogen-induced neuroinflammation brain DC functionally mature. Intracerebral DC maturation precedes histopathology as evident by the early induction of CD83 and ex vivo allostimulatory activity. A contribution of microglia to the intracerebral DC population was delineated from the amyloid-related phenotype brain DC exhibit, the microglia-like morphology of cells expressing DC markers at parenchymal sites where any cellular infiltrate is lacking features that microglia share with immature DC, and the finding that microglia purified from adult brain can generate functional DC. Final proof that microglia constitute a portion of the DC population was obtained by sorting of donor versus host-type DC from inflamed brains of bone marrow chimeras. Distinct stages in microgliato-DC differentiation were demonstrated in vitro: GM-CSF drives microglia to acquire an immature DC-like phenotype while CD40 ligation triggers terminal activation. Since an inflammatory neural environment is a likely source of both stimuli, microglia must be regarded as an organotypic reservoir of pre-DC. Antigen presentation by brain DC in situ was substantiated by the finding that DC from the brains of mice with toxoplasmosis spontaneously activate pathogenspecific T cells ex vivo. The object of this study was to evaluate the effect of simvastatin on microglia immune function. We treated primary mouse and human microglia with simvastatin and evaluated the membrane expression of several immune regulatory molecules by FACS analysis. Next, we subjected simvastatin treated primary human microglia to a chemotaxis assay towards various chemokines. Finally, we evaluated the impact of simvastatin treatment on the subcellular structure of human microglia by cytoskeleton staining and structural electron microscopy. We show that simvastatin is able to reduce in primary microglia the expression of MHC class II, the costimulatory molecule CD86, the adhesion molecule CD40 and the chemokine receptor CCR5. In addition, we demonstrate that simvastatin reduces the capacity of microglia to migrate towards the chemokines RANTES and MIP-1alpha. Finally, we show that simvastatin treatment results in an altered subcellular structure of microglia. Together, these data suggest that simvastatin is able to reduce the capacity of microglia to migrate and interact with their environment by disturbing the metabolism of several important membrane-bound molecules. Supported by the Dutch MS Research Foundation (Grant 00-407 MS). Inflammation of the CNS is usually locally limited to avoid otherwise devastating consequences. Critical players implicated in this process are microglial cells. The B7 family of ligands and their receptors are recognized as pathways for the regulation of immune responses. Human and murine microglial cells were analyzed for the expression of B7-H1 and other B7costimulatory molecules. Functional relevance and expression of B7-H1 during MOG-induced EAE were investigated. Human and mouse microglial cells constitutively expressed B7-H1 mRNA and protein. Under inflammatory conditions, a significant upregulation of B7-H1 mRNA and cell surface protein was notable. Microglial expression levels of B7-H1 protein were substantially higher as compared to astrocytes and monocytes. Coculture experiments of the different APC with T cells demonstrated the functional consequences of B7-H1 expression: the production of inflammatory cytokines (IFN-gamma and IL-2) by T cells was markedly enhanced in the presence of a neutralizing anti-B7-H1 antibody. This effect was clearly more pronounced when microglia cells were used as the APC, compared to monocytes or astrocytes. B7-H1 was highly upregulated during the course of MOG-induced EAE in vivo and expression was predominantly located to the areas of inflammation. Taken together, our data point to microglial B7-H1 as an important immune-inhibitory molecule that downregulates T cell activation in the CNS and would contribute to the regulation of local levels of inflammatory responses.
Regulation of immunoproteasome and MHC class I expression by human brain endothelial cells H. Lau, M. van Zwam, K. Liu and K. Dorovini-Zis Vancouver General Hospital and the University of British Columbia, Vancouver, Canada
Proteasome is a multi-subunit protease responsible for the breakdown of misfolded and regulatory proteins into small peptides which posses anchor sites for MHC class I molecules (MHC I). Completely folded MHC Ipeptide complexes are then delivered to the cell surface and presented to CD8+ T cells (TC). Recent studies suggest that proteasomes play an important role in leukocyte-endothelial interactions. In inflammatory CNS diseases, TC subsets cross the blood-brain barrier (BBB) to initiate immune responses by mechanisms presently poorly defined. In this study, regulation of proteasome expression and its role on MHC I expression by HBMEC was investigated in an in vitro BBB model. mRNA and protein expression of the constitutive subunit alpha5 and the inducible beta1i (LMP2) and beta5i (LMP7) subunits by HBEMC was investigated by RT-PCR and ELISA and MHC I expression by ELISA. Unstimulated HBMEC express alpha5 and low levels of beta1i and beta5i subunits. Both subunits were upregulated following treatment with IFN-gamma (200 U/ml) and TNF-alpha (100 U/ml). MHC I expression on HBMEC was significantly upregulated after treatment with IFN-gamma for 48 h and downregulated by the proteasome inhibitor lactacystin. These results indicate that cytokine-induced upregulation of immunoproteasome subunits is required for MHC I upregulation on HBMEC and suggest that the proteasome may influence antigen processing and presentation at the BBB in CNS inflammation.
Peptide motif for the rat MHC class II molecule RT1.Da H. Duyar, J. Dengjel, K.L. de Graaf, S. Stevanovic and R. Weissert University of Tübingen, Tübingen, Germany MOG-induced EAE in DA rats (RT1av1) is a model of multiple sclerosis (MS) that reproduces major aspects of this detrimental disease. So far, no MHC class II peptide binding motif for the DA rat RT1.Ba and RT1.Da molecules has been described. We were interested to obtain an RT1.Da peptide binding motif. This will allow subsequent functional studies in rat models of autoimmune and infectious diseases. Sequence alignment of the beta-chain of the rat MHC class II molecule RT1.Da with human HLA class II molecules revealed strong similarity in the residues of the peptide binding groove of RT1.Da and HLA-DRB1*1501. According to the putative peptide binding pockets of RT1.Da after comparison with the pockets of HLA-DRB1*1501, we predicted the peptide motif of RT1.Da. To verify the predicted motif, naturally processed peptides were eluted by acidic treatment from immunoaffinity-purified RT1.Da molecules in lymphoid tissue of MOG immunized DA rats and subsequently analyzed by ESI tandem mass spectrometry. The identified peptides were aligned to the peptide binding pockets according to the putative peptide motif. Additionally, we performed binding studies with nonameric combinatorial peptide libraries to purified RT1.Da molecules. Based on these studies, we could define a peptide binding motif for RT1.Da. The peptide motif of RT1.Da will allow epitope predictions for analysis of peptides, relevant for autoimmune diseases.
Searching for autoimmunizing cell types in thymomas Y. Kadota a , M. Jones b , T. Meager c and N. Willcox a a Neuroscience WIMM; b Cellular Science, University of Oxford, Oxford, UK; c National Institute Biological Standards and Control, London, UK Thymomas are neoplasms of thymic epithelial cells (TEC), often generating abundant developing thymocytes. Around 30% of thymoma patients develop myasthenia gravis (MG), with antibodies against the muscle acetylcholine receptor (anti-AChR). We recently also found high titre neutralising auto-antibodies against Interferon-alpha (IFN-alpha) in N70% of thymoma patients at diagnosis, regardless of histology or HLAtype; titres increase sharply when thymomas recur, unlike those against AChR. Cultured thymoma cells spontaneously produce anti-IFN-alpha but not anti-AchR, again suggesting more direct autoimmunization against IFN-alpha in the tumours. To identify autoimmunizing cells, we labelled paraffin sections from 21 MG patients with thymomas; 15 with WHO type-B1-2, 5 with type-AB, and 1 with type-A. Anti-IFN-alpha titres were high in 10, medium in 5, low/negative in 6. IFN-alpha expression appeared strong in 6 (mostly with high serum anti-IFN-alpha), intermediate in 5, and low or negative in 10. Most IFN-alpha+ cells appeared large with dendritic morphology. They were consistently positive for CD68, and negative for Cytokeratin, CD20 and CD3. Conversely, CD68+ cells were also seen in 8 of the 10 thymomas with weak/negative IFN-alpha labelling. Most IFN-alpha+ cells were also CD14+ and HLA-DRint. We are currently labelling for co-stimulatory molecules. In conclusion, the most obvious autoimmunizing cells appear to be IFN-alpha+ macrophages rather than TEC, but unusual dendritic subsets cannot be excluded. Objective: To investigate the expression and role of the novel B7 family members ICOSL and B7-H1 in cultured muscle cells and in inflammatory myopathies. Methods: Expression and regulation of B7-H1 and ICOSL was analyzed in cultured myoblasts and muscle biopsy specimens by QRT-PCR, flow cytometry and immunohistochemistry. Functional relevance was investigated by coculture experiments of myoblasts and T cells. Results: Cultured human muscle cells constitutively express ICOSL but not B7-H1. Stimulation with IFN-gamma strongly upregulates B7-H1 expression. Coculture experiments of MHC-class I/II-positive myoblasts with CD4 and CD8 T cells in the presence of antigen demonstrated the functional consequences of muscle-related B7-H1 and ICOSL expression: whereas ICOSL provided stimulated T cells, B7-H1 was a strong inhibitor of T cell cytokine production and activation. Muscle fibers showed constitutive expression of ICOSL, but not B7-H1, in vivo. Importantly, both molecules were upregulated in specimens from patients with polymyositis, dermatomyositis and inclusion body myositis and were predominantly located in the areas of strongest inflammation. Conclusion: Cultured muscle cells and muscle fibers in vivo express the B7-molecules ICOSL and B7-H1. Whereas ICOSL provides an augmentatory signal for muscle immune cell interactions, inducible expression of the immune inhibitory molecule B7-H1 provides evidence for the capability of muscle to promote immunoprotective mechanisms putatively involved in the regulation of local inflammation. The recently discovered inducible costimulator (ICOS), a CD28 like molecule interacting with its B-7 like ligand ICOS-L on antigen-presenting cells (APC), is expressed preferentially by activated T cells. Recent data indicate that ICOS and ICOS-L represent an important pathway in the regulation of autoimmune or infectious diseases. To define the expression of ICOS and ICOS-L in inflammatory demyelination of the PNS, we studied their expression and distribution patterns in sural nerve biopsies from patients with Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and, for comparison, various non-inflammatory neuropathies (NIN) by RT-PCR and immunohistochemistry. In patients diagnosed as GBS and CIDP but not in NIN, a significant upregulation of ICOS mRNA was found. T cells were identified as the primary cellular source expressing ICOS in the inflamed PNS thus corroborating their state of activation. Similarly, significantly increased mRNA expression levels of its ligand, ICOS-L, were found in GBS and CIDP samples, but not in the NIN controls. Macrophages were deciphered as the cellular source of ICOS-L within the endoneurium. Schwann cells were negative for ICOS-L. Our data show that ICOS and its ligand ICOS-L are expressed in immune-mediated peripheral neuropathies and might play an important role in the augmentation of cellular immune responses in inflammatory peripheral nerve disorders.
The surface unit of MSRV envelope protein activates innate immunity through TLR4/CD14 receptors and promotes Th1 like responses A. Rolland a , E. Jouvin-Marche a , C. Viret a , H. Perron b and P.N. Marche a a CEA/INSERM, Grenoble, France; b bioMerieux, Marcy l'étoile, France
Multiple Sclerosis (MS) is a pro-inflammatory autoimmune disease causing lesions in the white matter of the central nervous system (CNS). Its etiology is not understood but appears to involve both genetic and environmental factors including viral infections. MSRV is a retroviral element previously isolated in cell cultures from MS patients and bearing pro-inflammatory properties characterized by the induction of proinflammatory cytokines in human PBMC cultures. It is increasingly clear that pattern recognition receptors are of primary importance for innate immune responses to viral components and for the initiation of inflammation. CD14 and Toll like receptor 4 (TLR4) are two members of this receptor group with the ability to sense microbial products from both bacteria and viruses. We report here that the surface unit of MSRV envelope protein (ENV-SU) specifically induces human monocytes to produce major pro-inflammatory cytokines through a TLR4/CD14 dependent mechanism. ENV-SU also induced dendritic cell maturation and conferred them the capacity to support a T helper 1 type of T cell differentiation. Thus, it appears that MSRV through the pro-inflammatory properties of its surface envelope protein could constitute an upstream event in the immunopathological cascade leading to autoimmunity and/or neuroinflammation in diseases such as MS. Encephalitogenic and beneficial roles of amyloid beta-specific T cells in Alzheimer's disease A. Monsonego a , V. Zota a , S. Petrovic a , J. Imitola a , T. Owens b , D. Selkoe a and H.L. Weiner a a Harvard Institute of Medicine, Brigham and Women's Hospital, Center for Neurologic Diseases, Boston, MA, USA; b Montreal Neurological Institute, Montreal, Canada Amyloid beta (Ab) vaccination of patients with Alzheimer's disease (AD) resulted in encephalitis observed in about 5% of the patients. Recently, we have shown that a significantly higher portion of healthy elderly and AD subjects had strong Ab-reactive T-cell responses than occurred in middleaged adults, strongly dependent on certain HLA-DR class II alleles. We then examined in a mouse model of AD whether Ab-specific T cells can migrate to sites of Ab plaques and affect disease pathogenicity. We show that a single immunization with a dominant Ab T-cell epitope induced agedependent transient meningoencephalitis only if IFN-gamma was expressed in the CNS. Immune infiltrates were targeted to sites of Ab plaques in the brain associated with enhanced microglia activation and clearance of Ab. Overall, we demonstrate that accumulation of Ab in the CNS, MHC genetic background, and inflammatory signal such as IFN-gamma are required to induce Ab-specific T-cell responses in the brain of APP-Tg mice similar to that observed in Ab-vaccinated patients with AD. These T-cell responses in the CNS to Ab are dependent on the local milieu and may be beneficial or detrimental depending on the type of T-cell response. It is widely accepted that cytotoxic T cells play a dominant role in viral encephalitis. The aim of this study is to determine the role of the various cytotoxic pathways used by CD8+ T cells in acute and chronic viral encephalitis. We investigated the distribution of GrB+CD8+ T cells in virus infected brains, the patterns of activation, cytotoxicity mechanisms and induction of cell death in target cells. We used immunohistochemical and confocal fluorescence double labeling techniques on formalin-fixed, paraffin embedded sections from selected viral encephalitis brains (cytomegalovirus encephalitis (CMV, n=7), tick borne encephalitis (TBE, n=5) and subacute sclerosing panencephalitis (SSPE, n=2)). Results show a predominance of CD8+ T cells in the inflammatory infiltrate of all encephalitis cases. In CMV cases, the percentage of GrB+ cells remains high during the studied course of disease (4-20 weeks) whereas in TBE we found a gradual decrease in percentage of GrB+ cells during disease course (1-9 weeks). Preliminary studies performed with a confocal laser scanning microscope revealed a close contact between CD8+ cells and CMV infected astrocytes. In TBE and SSPE, we also found close apposition of GrB+ cells to neurons in the early course of disease. These results indicate that especially during the early stage of disease, GrB-mediated cytotoxicity is important in viral encephalitis. This work was supported by a grant from the Austrian FWF (P-16063-B02).
Autoimmune responses against axonal proteins during experimental autoimmune encephalomyelitis (EAE) H.G. Huizinga, A. Jagessa, P. Smith, N. Heijmans and S. Amor Biomedical Primate Research Centre, Rijswijk, The Netherlands Objective: Axonal pathology plays an important role in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). The mechanism of axonal degeneration, however, is not yet known. We hypothesise that immune responses against axonal proteins contribute to axonal pathology in EAE. Therefore, we investigated whether autoimmune responses against axonal proteins develop in EAE and correlate with clinical disease. In addition, we studied the encephalitogenic potential of neuronal antigens, derived from newborn spinal cord in which myelination is just starting. Methods: Biozzi ABH mice were immunised with newborn and adult spinal cord homogenate (SCH). T-cell and antibody responses against neurofilaments (heavy and light) were measured during various stages of disease. Cervical lymph nodes were isolated to study presentation of neuronal antigens. Results: T-cell and antibody responses to both neurofilament light and heavy were observed in adult SCH-induced EAE. Antibody levels, however, did not correlate necessarily with disease stage or severity. Neurofilament staining was observed in cervical lymph nodes of mice with EAE, indicating that neuronal antigens can be presented in the lymph nodes. Although newborn SCH-immunisation induced T-cell and antibody responses to SCH and neurofilaments, animals did not develop EAE. Conclusions: Our data indicate that both T-cell and antibody responses against neurofilaments develop during chronic relapsing EAE in mice. However neuronal antigens, as in newborn SCH, was not encephalitogenic in mice. The relevance of these findings will be discussed. Inflammation plays an important role in the pathophysiology of ischemic stroke. We mucosally administered myelin oligodendrocyte glycoprotein (MOG) (35-55) peptide to C57BL/6 mice prior to middle cerebral artery occlusion (MCAO) to induce an anti-inflammatory T-cell response directed at CNS myelin. We found that nasal and oral administration of MOG (35-55) peptide decreased ischemic infarct size at 24 and 72 h after MCAO surgery. Nasal MOG (35-55) peptide was most efficacious and reduced ischemic infarct size by 70% at 24 h and by 50% at 72 h ( pb0.0001 vs. control) as well as improving behavior score. Immunohistochemistry demonstrated increased IL-10 and reduced IFN-gamma in the area surrounding the ischemic infarct following nasal treatment. Nasal MOG did not reduce ischemic infarct size in IL-10 deficient mice. Adoptive transfer of CD4+ T-cells from nasally tolerized mice to untreated mice prior to MCAO surgery significantly decreased stroke size ( pb0.001 vs. control) whereas, CD4+ T-cells from nasally tolerized IL-10 deficient mice had no effect. Furthermore, our results demonstrate that IL-10 secreting CD4+ T cells induced by nasal MOG reduce ischemic injury following stroke. Modulation of cerebral inflammation by mucosal tolerance to myelin antigens may improve outcome after stroke and enhance mechanisms of self-recovery.
T helper cells type 2 stimulate axonal outgrowth and attract cortical axons S. Müller-Röver, S. Sallach, C. Brandt, D. Lqdecke, D. Schwanzar, E. Kwidzinski, B. Heimrich and R. Nitsch Charité University Hospital, Berlin, Germany After mechanical lesions of the nervous system, activated T cells have been suggested to exert neuroprotective effects. However, the structural basis of these effects is not clear. Stimulation of axonal outgrowth by T cells might be a key mechanism for regeneration. Therefore, we have investigated the effects of activated T helper cells type 1 (Th1) and type 2 (Th2) on neuronal outgrowth in murine organotypic brain explants in vitro. In these T cell/ explant co-cultures, Th2 cells stimulated axonal outgrowth and attracted axons of cortical explants significantly. Since T helper cell subtypes might exert neuroregenerative effects by secreting neurotrophins, we analyzed their neurotrophin expression patterns using RT-PCR and ELISA. We have compared the expression patterns of nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and brain-derived neurotrophic factor (BDNF) as well as interferon-gamma (IFNgamma), interleukin-1beta (IL-1beta), IL-2, IL-4, IL-10, and transforming growth factor-beta 1 (TGFbeta 1) between TH1 and TH2 cells. Both subtypes expressed mRNA of all investigated factors except IFNgamma mRNA expression, which was limited to TH1 cells. On the protein level, TH2 cells secreted significantly higher levels of IL-4, IL-10, NGF, NT-3 and NT-4 compared to TH1 cells. The application of functionally inhibiting antibodies against NGF, NT-3 and NT-4 substantially reduced axonal outgrowth in T cell/explant co-cultures. These data suggest that one important structural element of bprotective autoimmunityQ is the stimulation of neurotrophin-dependent axon outgrowth by Th2 cells. The etiology of neurodegenerative disorders is at present unknown. Although a small percentage of these disorders are familial cases linked to specific genetic defects, most are idiopathic. Thus environmental factors are thought to play an important role in the onset and progression of such disorders. We have determined the effect of an exposure (4 h, 5 days per week for 2 weeks) to concentrated airborne particulate matter on brain inflammatory indices in cortical tissue of BALB/c mice. Animals were divided into three exposure groups: filtered air (control), ultrafine particles, or fine and ultrafine particles. The levels of proinflammatory cytokines interleukin-1 alpha (IL-1alpha) and tumor necrosis factor alpha (TNFalpha) were increased in cortical tissue of mice exposed to particulate matter compared to that of control mice. Levels of the immune-related transcription factor NF-kB were also found to be substantially elevated in the brain of exposed groups compared with the control group. These data indicate that components of inhaled particulate matter may trigger a proinflammatory response in nervous tissue. Since inflammatory events may contribute to the pathogenesis of neurodegenerative disorders, results suggest that environmental exposure to particulate matter may enhance pathogenic processes within nervous tissue.
Induction of antiphospholipid syndrome exacerbates amyloid and plaque deposition as well as memory impairment in an animal model of Alzheimer's disease A. Katzav, C.G. Pick, Y. Shoenfeld and J. Chapman Tel Aviv University, Tel Aviv, Israel
Immune and inflammatory mechanisms may be involved in the pathogenesis of neurodegenerative diseases and immune mediated diseases of the central nervous system lead eventually to neurodegeneration. The antiphospholipid syndrome (APS) is associated with dementia and can be induced experimentally in mice by immunization with the autoantigen beta2-glycoprotein I (beta2-GPI). We investigated the effects of APS induction on the course of neurodegeneration in an animal model of Alzheimer's disease. Transgenic mice with the pathogenic betaAPP mutation were immunized with beta2-GPI at the age of 4-5 and 4.5 months later were tested for hyperactivity on a staircase apparatus and for cognitive function in a swim T-maze alternation test. Brains were examined for mature plaque formation. There were significant differences in behavior between the APP transgenic mice compared to the controls in both tests and in total h-amyloid staining. The induction of APS had significantly reduced hyperactivity in the APP mice. Cognitive impairment and mature plaques were found only in the APP-APS mice. Induction of APS results in more rapid onset of amyloid deposition and cognitive impairment in betaAPP mice. Similar mechanisms may be involved in patients with APS and dementia.
The LPS receptor CD14 links Alzheimer's disease with innate immunity K. Fassbender University of Göttingen, Göttingen, Germany A pathophysiological hallmark of Alzheimer's disease (AD) is chronic neuroinflammation that appears to contribute to progressive neuronal injury in AD. The lipopolysaccharide receptor, CD14, is the key cellular receptor for components of gram-negative microorganisms. Here, co-immunoprecipitation experiments and studies in CD14 deficient microglia and monocytes demonstrate that CD14 recognizes Alzheimer's amyloid peptide, thereby mediating microglial and monocytic activation and release of neurotoxins. Further, we show that expression of CD14 is increased in brains of AD patients in correlation with senile plaques, suggesting a clinical relevance of this receptor in AD. Thus, CD14 mediated microglial activation by amyloid peptide may explain chronic neuroinflammation and subsequent neurodegeneration in AD. We hypothesize that a structural mimicry between amyloid fibrils and microbial components may underlie the mechanism of chronic neuroinflammation mediated by CD14 ligation. The lipopolysaccharide receptor, CD14, may, thus represent a novel therapeutic target in prevention of progression of AD.
Axonal injury at the onset of murine experimental autoimmune encephalomyelitis (EAE): a role for astrocytes D. Wang a , M. Ayers b , D. Catmull a , L. Hazelwood a , C. Bernard a and J. Orian a a La Trobe University, Melbourne, Australia; b University of Melbourne, Melbourne, Australia
Recent data have changed the view of Multiple Sclerosis (MS) from a primary inflammatory demyelinating disease to one in which cumulative axonal damage drives disease progression. Concurrently, studies of axonglia and glia-glia communication have emphasized the importance of interactivity between central nervous system cells. From this perspective, we studied axonal damage in the context of inflammation and glial responses in preclinical stages of murine EAE, an established MS model. We report several major findings: (1) we provide evidence of axonal injury before significant parenchymal T-cell entry, (2) we demonstrate an association between axonal injury and astrocyte responses from presymptomatic stages and (3) differences in astrocytic responses in two forms of the disease. Our data clearly show that mechanisms additional to those mediated by T cells are operating in early stages and strongly implicate astrocytes in disease initiation. In common with data from a growing number of neurodegenerative conditions, we show that murine EAE is characterized by early active contribution from astrocytes. These observations mark a change in the understanding of the role of astrocytes in disease pathogenesis and have important implications for development of neuroprotective strategies. Neurodegeneration and inflammation are fundamental aspects of many neurological diseases. Numerous molecular pathways that regulate these processes have been identified in recent years, but knowledge of how aspects of neurodegeneration and CNS inflammation are regulated on a genomic level is still very limited. We are currently dissecting these features using a standardized nerve trauma model in adult rats, ventral root avulsion (VRA), in combination with experimental genetic protocols to unravel genetic influences. In an F2 intercross between DA and PVG rats four quantitative trait loci (QTLs) controlling nerve cell death, expression of MHC class II on microglia and/or T cell infiltration after VRA were identified. These findings have now been extended in an advanced intercross line (F8(PVG.1AV1xDA)) reproducing the QTLs regulating MHC class II and neurodegeneration. Further fine mapping of the genomic regions are currently performed in the F10 generation of the same AIL. In parallel, congenic strains are being bred to allow for mechanistic studies in other models. Preliminary experiments performed in the founder strains reproduce considerable phenotypic differences also in experimental spinal cord and cortical contusion injuries. The demonstration that polymorphic genes in different loci control neurodegeneration and CNS inflammation has implications for various experimental rodent nervous system paradigms and this approach may lead to the identification of evolutionary conserved genetic polymorphisms in key controlling genes, which can serve as prime candidates for association studies in several human CNS disease. Multiple Sclerosis is a chronic inflammatory disease of the CNS leading to focal destruction of myelin, still the earliest changes that lead to lesion formation are not known. We have studied the gene-expression pattern of 12 samples of normal-appearing white matter from 10 postmortem MS brains. Microarray analysis revealed upregulation of genes involved in maintenance of cellular homeostasis, and in neural protective mechanisms known to be induced upon ischemic preconditioning. This is best illustrated by the upregulation of transcription factors such as HIF-1alpha and associated PI3K/Akt signalling pathways, as well as the upregulation of their target genes such as VEGF receptor 1. In addition, a general neuroprotective reaction against oxidative stress is suggested. These molecular changes might reflect an adaptation of cells to the chronic progressive pathophysiology of MS. Alternatively, they might also indicate the activation of neural protective mechanisms allowing preservation of cellular and functional properties of the CNS. Our data introduce novel concepts of the molecular pathogenesis of MS with ischemic preconditioning as a major mechanism for neuroprotection. An increased understanding of the underlying mechanisms may lead to the development of new more specific treatment to protect resident cells and thus minimize progressive oligodendrocyte and axonal loss. Heavy metals and free radicals have been related to MS pathogenesis. Serum levels of 26 elements were compared within 10 couples of disease discordant monozygotic twins. We developed a reliable method for the simultaneous quantification of Al, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, Hg, Li, Mg, Mn, Mo, Ni, Pb, Sb, Si, Sn, Sr, Tl, V, W, Zn and Zr using Sector Field Inductively Coupled Plasma Mass Spectrometry (SF-ICP-MS) and ICP Atomic Emission Spectrometry (ICP-AES). We carried out a similar comparison by photometric assay for the serum oxidant status (SOS) and serum antioxidant capacity (SAC). No significant differences emerged-at a p=0.05, nonparametric test-for the elements under study as well as for parameters relating oxidative stress, with the exception of Mg (median values of 19,213 for patients and 17,881 ng/ml for controls at a p=0.03). In affected twins, we observed a decreasing trend for Al, Co and Sr, especially in the case of Co (medians, 0.15 vs. 0.37 ng/ml, at a p=0.06) and a tendency to higher concentrations for Zn (medians, 883 vs. 818 ng/ml, p=0.10) and Be (0.48 vs. 0.40 ng/ml, p=0.18). Results on a larger series of patients will be presented.
The role of plasminogen activators in experimental allergic encephalomyelitis: inflammation and axonal pathology E. East, D. Baker, G. Pryce, M.L. Cuzner and D. Gveric Institute of Neurology, University College London, London, UK Entry of fibrin, normally excluded from the CNS and upregulation of urokinase plasminogen activator (PA) receptor (uPAR) and PA inhibitor-1 (PAI-1) are one of the earliest signs of inflammatory demyelination. Concentrations of tissue PA (tPA), the key fibrinolytic enzyme, are decreased in the CNS due to higher amounts of its inhibitor PAI-1, thus ensuring that fibrin entering the CNS is not efficiently removed. In addition to its proteolytic activity with uPA, uPAR is involved in the migration and adhesion of inflammatory cells. The roles of tPA and uPAR were investigated in EAE using knockout mice. Both tPAÀ/À and wild type mice experienced an acute disease lasting 7 days; however, tPAÀ/À animals incurred a significant degree of neurological deficit. In contrast, uPARÀ/À mice had a prolonged acute phase of disease, the peak of which was significantly delayed. This was accompanied by a decrease in leucocyte infiltration. Loss of neurofilament, an indicator of axonal integrity, was observed early on in all groups of mice indicating that axonal loss is an early feature of EAE and multiple sclerosis. High levels of CNS fibrin persisted in tPAÀ/À animals during acute and chronic phases of disease and deposits of fibrin could be localised on damaged axons. In conclusion, these data enforce the concept of the involvement of the PA system in neuroinflammation.
Neuroinflammatory conditions may disrupt the normal function of adrenomedullin (ADM) at the blood-brain barrier (BBB) E. Liverani a , A. Woods a , C. Bolton b and C. Paul a a University of the West of England, Bristol, UK; b William Harvey Research Institute, London, UK ADM appears intimately involved in the maintenance of BBB function. In vitro studies have clearly shown ADM capable of regulating key features of the BBB including transendothelial electrical resistance (TEER), permeability and p-glycoprotein pump activation. The BBB forms a highly selective barrier between the peripheral circulation and the central nervous system, which is, in part, under the control of glucocorticoids. Loss of normal BBB integrity is an integral feature of the neuroinflammatory process during multiple sclerosis (MS). Neurovascular function is intimately linked to glutamate-mediated events and we have shown that restoration of normal BBB permeability is associated with an improved clinical outcome in models of MS. We have examined the effect of glutamate and glucocorticoids on ADM function in vitro using ECV304 human endothelial cells and C6 rat astroglioma cells alone and in coculture. Our preliminary immunocytochemistry studies indicate that receptor-activity modifying protein-2 (RAMP-2) is normally expressed on ECV304 cells. However, on exposure to 0.1 mM glutamate, RAMP-2 expression is significantly reduced ( pb0.05). Additionally, TEER measurements in the co-culture BBB model are significantly elevated in the presence of co-administered ADM and dexamethasone compared to control values ( pb0.05). The data suggests that glutamate affects endothelial expression of ADM receptor proteins. Furthermore, the results indicate that steroidal compounds can influence ADM control of neuroendothelial permeability. The role of inflammation in central nervous system (CNS) damage and repair is controversial. Inflammation is considered a major cause of neurodegeneration in most CNS disorders. In contrast, autoreactive immune responses induced by injury were found to be part of the physiological repair mechanism. Thus, controlled boosting of protective immunity is proposed as a means of attenuating neurodegeneration and facilitating healing. Implantation of educated macrophages, produced from monocytes co-incubated with peripheral tissue (skin or sciatic nerve) but not with central tissue (optic nerve), was shown to improve functional recovery following optic nerve or spinal cord injury in rats. Since incubation conditions influence the activity of the resulting macrophages, skin-coincubated macrophages were characterized and compared to macrophages activated by lipopolysaccharide (bclassical activationQ) or interleukin-4 (balternative activationQ). The incubated cells were analyzed for morphological characteristics, expression of membrane markers and cytokine secretion. Activation with skin or lipopolysaccharide increased significantly the number and size of cytoplasmic granules and secretion of proinflammatory cytokines, in contrast with interleukin-4 that had opposite effects. Only lipopolysaccharide activation induced secretion of tumor necrosis factor alpha, as well as elevated expression of a co-stimulatory molecule, CD80. Interleukin-12 secretion was unaffected by any of the activators. Skin-co-incubated macrophages thus represent a unique cellular phenotype, distinct from bclassically'Q or balternatively-activatedQ macrophages. We propose that this phenotype promotes an immune response that supports neuronal cell survival and repair, resulting in functional recovery.
Effect of inflammatory mediators on axonal transport of synaptic vesicle proteins M. Stagi, N. Frank and H. Neumann European Neuroscience Institute, Göttingen, Germany Axonal transport disturbance is an early sign of most inflammatory brain diseases, but the molecular mechanism of this dysfunction remains elusive. Activated microglial cells are found to be in close proximity to dystrophic neurites and release nitric oxide (NO) and inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Confocal microscopy of fluorescence recovery after photobleaching (FRAP) was performed to analyze the axonal transport of the synaptic vesicle precursor protein synaptophysin tagged with green fluorescent protein (synaptophysin-EGFP) in transfected cultured hippocampal neurons. Microglia, pre-stimulated by inflammatory cytokines to produce NO and TNF-alpha, focally suppressed the axonal motility of synaptophysin-EGFP at the contact point. Direct application of TNF-alpha or short-term NO donor to cultured hippocampal neurons inhibited axonal motility of synaptophysin-EGFP within 10 min. Inhibition of axonal transport by this inflammatory mediator significantly increased the immobile fraction of synaptophysin-EGFP and was dependent on phosphorylation of c-jun NH(2)-terminal kinase (JNK). In particular, TNFalpha stimulated phosphorylation of JNK in axons. Reduced motility of synaptophysin-EGFP after TNF-alpha treatment was dependent on phosphorylation of JNK as determined by the JNK inhibitor SP600125. Thus, overt production of TNF-alpha and reactive NO by activated microglial cells blocks the motility of synaptic vesicle precursor via phosphorylation of JNK and may cause axonal and synaptic dysfunction in inflammatory and degenerative brain diseases. Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegeneration disease involving upper and lower motor neurons. Although microglia activation is a prominent feature of ALS and the mechanisms by which microglia contribute to motor neuron degeneration remain unknown, the supernatant derived from LPS-activated microglial cells (BV-2) induce death of a motor neuron hybridoma cell line (NSC-34 cells) through a TNF alpha dependent pathway. In this study, we have used both a cDNA superarray and real time PCR and observed a significant increase in the mRNA levels of the TNF receptor superfamily, including TNFR1, DR3, DR6 and the death ligand LT-beta following exposure of NSC-34 cells to LPS-activated BV-2 supernatant. In the presence of cycloheximide, LPSactivated BV-2 supernatant induced NSC-34 apoptosis (caspase 3 activation and PARP cleavage) with caspase 9 activation, suggesting a role for mitochondria in microglial-mediated NSC-34 apoptosis. In order to determine whether this was due to TNF alpha, recombinant murine TNF alpha was applied to NSC-34 cells in the presence of cycloheximide. The results were identical to those obtained with LPS-activated BV-2 supernatant. These findings suggest that LPS activated microglial BV-2 supernatant can induce NSC-34 motor neuron cell apoptosis through a mitochondria-dependent signaling pathway and that TNF alpha is a major contributor to this process. Research supported by the Scottish Rite Charitable Foundation of Canada.
Glutamate stimulates peroxynitrite production in a brain-derived endothelial cell line via N-methyl-d-aspartate (NMDA) receptor activation S.R. Bowman, G.S. Scott and C. Bolton The William Harvey Research Institute, London, UK There is compelling evidence indicating a role for glutamate in the pathogenesis of multiple sclerosis (MS). Studies in experimental allergic encephalomyelitis (EAE), the model of MS, demonstrate that pharmacological inhibition of specific glutamate receptors suppresses symptoms and prevents blood-brain barrier (BBB) breakdown. The mechanisms through which glutamate influences BBB function during disease remain undefined. Glutamate is known to trigger the production of nitric oxide and superoxide, which can lead to the formation of peroxynitrite. Recent studies have implicated peroxynitrite in the loss of neurovascular integrity during EAE. We propose that glutamate contributes to BBB breakdown through the actions of peroxynitrite. The current study aims to determine whether glutamate induces peroxynitrite formation in a brain-derived endothelial cell line (b.End3). b.End3 cells were incubated with glutamate (1 AM to 20 mM) for 1 to 24 h. The production of peroxynitrite was assessed by quantitating the oxidation of dihydrorhodamine. Results showed a concentration-and timedependent increase in peroxynitrite levels in glutamate-treated cells. Furthermore, glutamate-induced changes were suppressed by the peroxynitrite decomposition catalyst FeTPPS and the inhibitor uric acid. In addition, glutamate-mediated generation of peroxynitrite from b.End3 cells was reduced by the NMDA receptor antagonist MK801. The study reconfirms the importance of glutamate in the loss of neuroendothelial integrity and highlights the deleterious effects of the molecule through the actions of peroxynitrite and NMDA receptor activation. Developmental morphological changes were found in the cerebellar cortex after a single injection of the cytostatic drug cisplatin (5 Ag/g b.w.) to 10-dayold rats. One day after treatment (PD11), the external granular layer in the vermis showed several apoptotic cells; recovery of proliferating activity and ectopic granule cells were found in the lobules VI-VIII on PD17 and PD30, respectively. Purkinje cells slowed their differentiation and at the end of histogenesis they had changed/reoriented dendrite branches (Calbindin immunoreaction). However, degeneration of Purkinje cells has also been described (Scherini and Bernocchi, 1994) . Autofluorescence, depending on nature, amount, distribution of endogenous fluorophores, and on optical properties can provide information on structure and metabolic activity of biological tissues. Autofluorescence potentials for diagnosis of cerebellum structural changes during development were investigated. The pattern profile was analysed by nonlinear regression to estimate the depth of tissue involved (mm at which the signal is 1/e of the incident light). In neocerebellar lobules VI-VIII, this was 0.75, 0.37, 0.23 mm for control rats of 10, 17 and 30 days, and 2.22, 1.63, 0.51 mm for the corresponding treated rats. Paleocerebellum showed neither structural alterations nor depth values changes. Microspectrofluorometry and spectral fitting analysis indicated an increase in oxidised state. Results show a great influence of structural organization, and thus of optical properties, on autofluorescence emission, in addition to biochemical composition (Research Grant: Cofin 2002 to G.B., MIUR-Italy) .
Junction between tubulin and Na,K-ATPase in calf brain stem neurons N.M. Vladimirova, E.N. Sautkina, T.V. Ovchinnikova and N.A. Potapenko Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia
The function of Na,K-ATPase in the brain is poorly studied and least understood. It was shown that in the brain, in addition to conventional functions, Na,K-ATPase is involved in intercellular interaction between the neurons and glial cells and the binding to cytoskeleton proteins. In brain cells, all known isoforms of both Na,K-ATPase subunits are expressed. The goal of this study was the isolation, structural characterization, and search for structural peculiarities of isozymes of Na,K-ATPase that really function in various brain cells. Glial cells were shown to contain mainly Na,K-ATPase of the alpha1beta1 type and minor amounts of isozymes of the alpha2 beta2 (beta1) and the alpha3beta1(beta2) type. The axolemma contains alpha2beta1 and alpha3beta1 isozymes. An enhanced sensitivity of the alpha3 catalytic subunit of Na,K-ATPase from neurons to endogenous proteolysis was found. The presence of the cytoskeleton protein tubulin (beta3 isoform) in the high-molecular-weight complex of Na,K-ATPase alpha3beta1 isozyme from brain stem axolemma and the junction between Na,K-ATPase alpha3 subunit and tubulin beta3 subunit are shown for the first time. It should be noted that dysfunction of Na,K-ATPase leads to set of neurodegenerative diseases. This enzyme is trigger of some apoptotic ways of neurons. The finding of connection between the Na,K-ATPase and cytoskeleton protein may lead to the discovery of the novel functions of the sodium pump in the brain and understanding of the reasons of some diseases. The inflammatory response contributes to the evolution of brain ischemia/ reperfusion injury. We studied the effect of helper-dependent adenoviral vector (Ad) engineered with the anti-inflammatory cytokine interleukin (IL)-4 gene and with the green fluorescent protein (GFP) as a reporter gene, in a model of transient focal ischemia in mice. Ischemia was induced in 129SV mice by occlusion of the middle cerebral artery (MCA) using a nylon monofilament introduced into the internal carotid artery and advanced so as to block the origin of the MCA. At the end of 10 min ischemia, the filament was removed and reperfusion was allowed. The vectors expressing IL-4 (Ad-IL-4-GFP) or the reporter gene alone (Ad-GFP) were infused intracerebroventricularly at the dose of 8Â10 6 PFU/5 Al at the end of ischemic period. Five days later, general and focal neurological deficits were significantly improved in mice receiving IL-4 gene compared to those receiving the control vector. Mice treated with IL-4 had also significantly reduced infarct volume (Ad-GFP: 18.8F5 mm 3 ; Ad-IL-4-GFP: 5.2F1.3 mm 3 ). Thus IL-4 gene delivery using Ad vectors induces protection in ischemia/reperfusion injury.
Identification of signaling pathways associated with rat strain-dependent susceptibility to nerve injury by microarray-based expression profiling M. Swanberg, K. Duvefelt, J. Hillert, T. Olsson, F. Piehl and O. Lidman Karolinska Institute, Stockholm, Sweden Axonal damage is a common feature of many neurological diseases. Although certain regulators of the retrograde axotomy reaction have been identified, information about the regulation of this process on a global genome-wide transcriptional level is limited. It is not known to what extent the genetic background influences the response. Global gene expression pattern was examined after ventral root avulsion (VRA), a well-characterized model of nerve injury-induced neurodegeneration and inflammation, in two inbred rat strains. Gene expression was analysed with Affymetrix arrays in spinal cord tissue sampled from naRve DA and PVG rats at 5 and 14 days after VRA. Regulation was shown in both strains for 359 of 8000 studied transcripts; 104 transcripts significantly differed between strains at one or more of the studied time points. The DA rat displays a higher degree of neuronal loss and more intense glial activation than the PVG strain and this was associated with higher levels of several mRNAs coding for proteins known to be involved in inflammatory reactions. Principal component analysis revealed that the segregation between the strains was almost of the same magnitude as that between naRve and injured rats. These results suggest a common VRA response pattern significantly modified by the genetic background and underscore the importance of a strict control of genetic background in experimental design.
Age-related response of brain cells to pro-inflammatory cytokines in the mouse M. Bentivoglio, A. Sadki and X.-H. Deng University of Verona, Verona, Italy Neurological diseases show a well known age-dependent prevalence; e.g. multiple sclerosis prevails in young and middle age and neurodegenerative diseases increase with aging, indicating that the response to inflammatory and neuronal death-inducing processes varies in the CNS during lifetime. Recent evidence also indicates that aging is characterized by low-grade chronic inflammatory activity, with increased production of pro-inflammatory cytokines. On this basis, we are investigating the brain response to an inflammatory challenge. Intracerebroventricular (i.c.v.) stereotaxic injections of a mixture of recombinant interferon-gamma and tumor necrosis factor-alpha were made in young (2-3.5 months) and old (18-20 months) C57BL/6J mice; saline was injected as control. We examined neuronal activation using Fos as marker, expression of the anti-apoptotic protein Bcl-2, and the glial response with immunocytochemistry. Cytokine injections resulted in Fos induction in neurons at several brain sites, such as the piriform cortex, midline thalamus, hypothalamic structures including the suprachiasmatic nucleus which plays a role of biological clock. At these sites, Fos induction was even more robust after i.c.v. cytokine injection in old mice than in young ones, with some subregional differences. After cytokine treatment, marked activation of astrocytes and microglia, and Bcl-2 induction in neurons and some glial cells, were seen in both age groups, with age-related differences. The data point out that the brain cell response to an inflammatory challenge is age-dependent and involves gene expression in different cell types. Supported by EC Grant QLRT-2001 QLRT- -2258 Advanced glycation and glyco-oxidation conditionate the loss of neuroprotective growth factors and the increased production of neurotoxic cytokines in Alzheimer's disease M. Fioravanti a , A. Lapolla b , S.B. Solerte a a Department of Internal Medicine and Geriatrics, University of Pavia, Pavia, Italy; b Department of Metabolic Sciences and Metabolism, University of Padova, Padova, Italy Advanced glycation end products (AGEs) and glyco-oxidative mechanisms could induce a pro-inflammatory burst of cytokines from immune and microglial cells and a loss of neuroprotection. Within this context, AGEs and Pentosidine were studied in 27 subjects with Alzheimer's disease (AD). We also measured TNF-alpha, IFN-gamma and Vascular endothelial growth factor (VEGF) released in the supernates of natural killer (NK) cells. Both cytokines and VEGF were evaluated after exposure with LPS (1 Ag/ml), beta-amyloid 1-42 (A-beta 1-42: 5 Ag/ml), and LPS/A-beta co-incubated with Glycated human serum albumin (GHSA: 20 Ag/ml). TNF-alpha and IFN-gamma were also determined after GHSA at 5, 10, 20, 50 Ag/ml. AGEs and Pentosidine were significantly increased in AD than in healthy subjects (15F3 Ag/ml and 190F40 pmol/ml vs. 8F2.5 Ag/ml and 115F28 pmo/ml respectively; pb0.001). A significant dose response increase of TNF-alpha and IFN-gamma was demonstrated in AD after GHSA ( pb0.001). Moreover, GHSA amplified the overproduction of both cytokines after LPS and A-beta ( pb0.001). Finally, a significant reduction of VEGF was found in AD subjects compared to healthy subjects ( pb0.001); while the co-incubation of NK cells with GHSA determined a further reduction of VEGF. In summary, advanced glycation and glyco-oxidation are significantly enhanced in AD. These mechanisms could conditionate the overexpression of neurotoxic cytokines, also inducing an impairment of neuroprotection induced by VEGF.
Innate immunity receptor TLR4 plays a key role in Alzheimer's disease S. Walter University of Gfttingen, Göttingen, Germany A pathophysiological hallmark of Alzheimer's disease is chronic microglial activation, which is considered to contribute to progressive neuronal injury by release of neurotoxic products. The Toll-like-receptor 4, TLR4, is localized on the surface of microglia and has recently been shown to play a key role in innate immune responses. Here, we show in studies with murine tlr4 mutant microglia, peritoneal macrophages and human monocytes that TLR4 is crucial for microglial and monocytic activation and that it can contribute to microglial toxicity to neurons. Further, investigations in HEK293 cells demonstrate interaction and define a trimolecular receptor complex consisting of TLR4, MD-2 and CD14, necessary for full cellular response to amyloid peptide fibrils. Suggesting a clinical relevance of TLR4 in AD pathology in vivo, we observed that expression of TLR4 is upregulated in APP transgenic mice, the animal model of AD, in close association with microglia. Together these observations provide first evidence for a role of the key innate immune receptor TLR4 in neuroinflammation in AD pathophysiology as a new potential therapeutic target in AD.
Oxidative stress in rat brain induced by amyloid-beta under continuous light conditions S. Rosales-Corral, G. Ortiz, M. Valdivia-Velázquez Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco, Mexico The brain is especially sensitive to oxidative damage. On the other hand, the chief secretory product of the pineal gland, melatonin, may reduce tissue destruction during inflammatory responses by scavenging free radicals and, indirectly, by lowering the production of cell damaging agents (cytokines and adhesion molecules). With the age, changes in both the amplitude and timing of the melatonin rhythm have been reported in humans. We used male, adult Wistar rats, separated into two groups: (a) continuous light (during 5 days); and (b) continuous darkness, besides an only surgically manipulated control group. Fibrillar amyloid-beta (4 Ag) was intracerebrally injected onto CA1. Hydrogen peroxide-injected rats were used as positive controls. During 5 days of continuous light, melatonin levels in serum decreased significantly compared with the observed concentrations in animals housed in light/dark cycles. This phenomenon was related with an increase in brain lipoperoxides and nitrites. On the other hand, increased serum levels of melatonin, either by exogenous administration or an increment in the endogenous production due to continuous darkness, was related with a significant reduction in oxidative stress induced by amyloid-beta. Even the highly significant production of lipoperoxides and nitrites induced by hydrogen peroxide injection was also reduced by melatonin. Microglial activity, as assessed by immunohistochemistry, showed a marked increase in hippocampus, around the amyloid-beta deposits in CA1 region. Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL, Nasu-Hakola disease) is a recessively inherited disease characterized with the unusual combination of bone cysts and early-onset dementia. Our group has recently established the molecular background of PLOSL by identifying mutations in DAP12 and TREM2-genes. The encoded proteins form an activating signal transduction complex in immune cells, but their function in the target tissues of PLOSL, central nervous system (CNS) and bone, is largely unknown. As a starting point for understanding how loss of function of this activating signaling complex leads to dementia and loss of myelin, we have analyzed the expression of DAP12 and TREM2 in the mouse CNS. Analyses of transcripts in the mouse CNS show that DAP12 and TREM2 are expressed from embryonic stage to adulthood with a highly similar expression pattern. In addition, microglial cells and oligodendrocytes are identified as the major DAP12/TREM2 producing cells in the CNS. Consequently, microglial cells and oligodendrocytes can be considered as the predominant cell types involved in PLOSL pathogenesis. The myeloid origin of both microglial cells and osteoclasts provides a biological link between the CNS and bone pathology in PLOSL patients. Further, the expression of DAP12/TREM2 in oligodendrocytes, the myelin producing cells of the CNS, agrees well with the histopathological and clinical findings of PLOSL patients. These findings provide a basis for the study of the molecular mechanisms behind this inherited dementia and new evidence for the involvement of the immune system in neuronal degeneration. It is known that stress induces increase of chemotaxis, which may contribute to increase migration and recruitment of immune cells to sites of inflammation. Chemokines play a role in neuroinflammation. CXCR4 is a chemokine receptor, which binds to its ligand SDF 1 (stromal cell-derived factor), while CX3CR1 is the only receptor for fractalkine. In this study, we have focused whether these two chemokines systems are involved in stressinduced neurodegeneration. For this purpose, rats were submitted to a chronic stress consistent of daily restraint for 6 h, for 3 weeks. Levels of CXCR4/SDF and CX3CR1/fractalkine were measure in homogenates from several brain structures, and chemokines were quantified by ELISA assay. Protein levels of CXCR4/SDF and CX3CR1/fractalkine were reduced in the hippocampus. However, upregulated CXCR4 levels were found in the cerebellum and olfactory bulb. Interestingly, SDF was significantly increased in the hypothalamus and olfactory bulb whereas fractalkine was only upregulated in the olfactory bulb. Control groups showed a high negative correlation between SDF and hippocampal Bax levels. All together, these data suggest that hippocampal SDF 1 decreased levels might reduce neuroprotection under chronic restraint stress. By contrast, differential chemokine modulation was found in other brain areas. Funded by a UNED Grant, Spain. In Alzheimer's disease (AD), amyloid plaques within the brain are surrounded by activated microglia, astrocytes and dystrophic neurites. Elevated levels of the pro-inflammatory cytokines IL-1beta and IL-6 have been found in AD brains at early stages of the disease, implying that the inflammation is an early event in AD. This inflammation is regulated, at least in part, by the transcription factor NFkappaB. The mechanisms of amyloid-beta (Abeta)-mediated neurotoxicity are unknown but it has been suggested that oxidative stress plays a key role. Although Abeta may act directly on neuronal cells, the increased levels of oxidative stress due to activation of glial cells may be more important. Upon activation, glial cells produce pro-inflammatory cytokines and putatively neurotoxic factors including superoxide anion, hydroxyl radicals and nitric oxide (NO). The reactive oxygen species (ROS) can cause great damage by lipid peroxidation and oxidation of proteins that may lead to cell death. The aim of this study was to investigate the effect of Abeta and the proinflammatory cytokine IL-1beta on glial activation and the production of ROS in rat primary mixed glial cell cultures. We investigated NFkappaB activation and the mRNA levels of IL-1beta, IL-6 and iNOS. NO and H 2 O 2 , secreted into the medium, were analysed in the absence or presence of the anti-oxidant alpha-tocopherol (Vitamin E). Parkinson's disease (PD) is an age-related neurodegenerative disorder of unknown etiology. The involvement of immune mechanisms in the etiopathogenesis of PD is documented. The neuroinflammation is regulated by numerous signal molecules, including nitric oxide. The nitric oxide-synthesizing enzyme nitric oxide synthase (NOS) is present in the CNS in three different isoforms: two constitutive enzymes (neuronal-nNOS, and endothelial-eNOS) and inducible enzyme (iNOS). In the present study, we examined nNOS, eNOS and iNOS gene expression in the striatum of C57BL/6 male mice (12 month old) in a murine model of PD induced by 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP). The animals were sacrificed on 6 h, on 1, 3, 7, 14 days after MPTP intoxication. The levels of mRNA were assayed by RT-PCR method. We observed the increased nNOS gene expression from 1 up to 14 days with the peak within 3-14 days. eNOS mRNA expression showed increase at 24 h and it was continued to 3 days and peaked in this time point. iNOS mRNA expression rapidly increased as early as at 6 h after MPTP intoxication; peaked at 24 h; significantly decreased at 3 days, and was observed until the 14th day.
In conclusion, this work extend our knowledge about the potential role of NOS isoforms in neurodegenerative disorders and may become in the future a target for therapeutic interventions in patients suffering from Parkinson's disease.
Characterization of mononuclear cells isolated from bone marrow for potential transplantation in an animal model of Huntington Disease T. Serrano-Sanchez Centro Internacional de Restauración Neurológica, CIREN, Havana City, Cuba
Huntington Disease (HD) is an inherited neurodegenerative disorder. The intrastriatal administration of neurotoxic has served as the most frequent animal model of HD. Animal models have allowed extensive research into therapeutic strategies for HD. Recently, the cellular source has demonstrated an important role. The bone marrow cells are easily accessible in the adult rat and it is a rich source of stem and progenitor cells. A growing number of recent publications have shown that adult stem cells from the bone marrow are able to exhibit neural phenotypes in vitro and in vivo after transplantation. This study was based on immunocytochemical technique for obtaining these descriptive phenotypic markers available for cell identification. In this report, we have performed the isolation of cells, antibody titration and characterization of cells from rat bone marrow. The phenotype of the cells comprising mononuclear cells system is present in the marrow. This population was characterized as CD3+, CD45+, CD90+, and CD38+. Our results showed the different contribution in the percentage each marker given a major comprehension to the real contribution of each one of this population cells in the effectiveness of transplantation. Gluten sensitivity is an alterated immunological responsiveness to ingested gluten. Extraintestinal organs, such as cerebellum, might be the unique target of this disease as occurs in gluten ataxia (GA) syndrome. We report a 76-year-old patient with ataxia yielding antigliadin antibodies (AGA), who developed a Ramsay Hunt syndrome (RHS). Instrumental exams, biochemical, genetic and histological analyses excluded vascular, inflammatory, metabolic or heredodegenerative ataxia and coeliac disease. Mild limb ataxia with severe gait ataxia, cognitive impairment, bladder dysfunction and high values of AGA without any intestinal involvement, resembled the pattern of GA. The patient showed a temporary clinical improvement to gluten free diet, and AGA became negative. Five months later, her clinical condition rapidly worsened. The patient became bedridden, confused and developed myoclonus and pseudo-absence episodes. Serial EEGs showed a progressive slowing of background activity and multifocal, periodic, epileptiform activity. AGA titer was higher than that at onset. RHS was suggested. Throughout the last 2 months her clinical and electrophysiological conditions improved once again and AGA test became negative. RHS has been previously reported with coeliac disease, but never with GA, considered a mild and monophasic disease. We report an association between AGA and RHS, and between clinical severity of RHS and AGA titer. AGA have also been found in healthy controls and in patients with heredodegenerative ataxia. The significance of their presence and fluctuation needs to be clarified.
The role of anti-ganglioside antibodies in nerve injury M.A. David a , J.M. Spies a , J.D. Pollard a , G. Zhang b , P.J. Armati a and K. Sheikh b a The University of Sydney, Sydney, Australia; b John Hopkins University, Baltimore, Maryland, USA Objective: To determine whether anti-GM1 and anti-GD1a antibodies directly mediate nerve injury. Background: Chronic inflammatory demyelinating polyradiculoneuropathy and Guillain-Barre Syndrome (GBS) are both considered to be organ specific autoimmune disorders. Antibodies have long been suspected of mediating nerve damage in these neuropathies. Antibodies to the major gangliosides GM1 and GD1a are strongly associated with the acute motor axonal neuropathy variant of GBS, implicating these gangliosides as target antigens. Until recently, attempts to develop animal models of anti-ganglioside antibody mediated nerve injury have given inconsistent results. The recent availability of high affinity monoclonal anti-ganglioside antibodies has enabled the development of new animal models to further clarify this issue. Methods: Monoclonal IgG anti-GM1 or anti-GD1a antibodies or control IgG were injected into rat sciatic nerve. Motor nerve conduction studies, stimulating proximal and distal to the site of injection and recording the compound muscle action potential (CMAP) from the foot muscles, were performed before and at 2-day intervals after injection. Results: Injection of anti-GD1a and anti-GM1 IgG caused a significant (40%) decrease in proximal CMAP amplitude with relative preservation of distal amplitude. Preliminary pathological studies show mild axonal degeneration without demyelination. Conclusion: These findings suggest that anti-GM1 and antiGD1a do cause nerve dysfunction. Whether this is via axonal degeneration or conduction failure remains to be determined. The buckthorn (Karwinskia humboldtiana) is a poisonous shrub. Ingestion produces distal, ascending, symmetric flaccid paralysis with slow recovery or death. Buckthorn polyneuropathy is frequently misdiagnosed as GBS or CIDP. Histologically, the peripheral nerve presents demyelination and axonal degeneration. The mechanisms implicated and the participation of the immune system are unknown. We developed a new experimental neuropathy model, which resembles human intoxication stages. Rats were classified in four groups: (1) before the onset of paresis, (2) weakness in limbs, (3) paralysis, (4) paralysis recovered. Sciatic nerves histopathological studies and statistic analyses were performed (hematoxylin-eosin, Toluidine-blue, Klqver-Barrera, Marsland-Glees and Erickson silver stains. CD4+Lymphocyte immunohistochemistry). IgG and IgM-anti-myelin were searched by Western-Blot. Sciatic nerves of treated rats showed demyelination, axonal degeneration, an important increment of mast cells and lymphocytes in comparison with controls. Many lymphocytes were CD4+. Groups 2 and 3 showed great degranulated mast cells at endoneurium and perineurim, shrunken or broken axons. All sera including controls presented IgM-antibodies against four myelin proteins, in contrast only IgG-antibodies of treated animals reacted with such proteins but none IgG from controls. We propose that mast cells, lymphocytes and immunoglobulins participate in the Buckthorn polyneuropathy demyelination. Our model can be useful for pathogenic mechanism studies in this and other polyneuropathies. Grants: CONACYT, PAICYT. The aim of the present study was to investigate cytokine changes after peripheral nerve injury in the rat sciatic nerve and the corresponding sensory ganglions. The left sciatic nerves of 26 male rats were transacted at the level of hip joint under pentobarbital anesthesia. Samples for TaqMan PCR studies were collected proximally and distally from the point of transection as well as from the contralateral intact sciatic nerve 12 h and 3, 7, 14, 35 and 49 days post-operation (PO). The corresponding sensory ganglions were also collected. Non-operated rats served as controls. Studied cytokine RNAs were: IL-1beta, IL-10, IFN-gamma, TNF-alpha and TGF-beta1. IL-1beta, TNF-alpha and IL-10 were markedly expressed during the first week after transection in the distal and proximal areas. The expressions in the sensory ganglions correlated partly with these findings. TGF-beta1 was upregulated in almost all the samples taken from the injured side between days 3 and 49. The most prominent expressions were between 3 and 14 days. Contralateral sciatic nerve showed increased expression of TNF-alpha up to 49 days PO. The contralateral ganglions showed marked expression of IL-10 and TNFalpha in all time points. The expressions patterns of the cytokines IL-1beta, TNF-alpha and IL-10 were similar in the sciatic nerve and in the ganglions of the transected side. The noted strong expression of TGF-beta1 could be related to its property to increase axonal growth or to induce fibrosis.
Effect of nordihydroguairetic acid on thermal hyperalgesia and cold allodynia in STZ-induced diabetic rats and mice M. Anjaneyulu and K. Chopra Pharmacology Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India Although pain is experienced by many patients with diabetic neuropathy, the pathophysiology of painful diabetic neuropathy is not fully understood. Thus study was designed to examine the effect of an antioxidant and lipoxygenase inhibitor, Nordihydroguairetic acid (NDGA), on thermal nociception in streptozotocin (STZ)-induced diabetic rats and mice assessed by tail-immersion and paw withdrawal assays. Diabetes was induced in rats and mice by single injection of STZ (65 and 200 mg/kg/i.p respectively). After 4 weeks of STZ, diabetic rats and mice were subjected to tailimmersion and paw withdrawal assay. Diabetic rats exhibited significant decrease in tail-immersion and paw withdrawal latencies as compared to controls. Similarly in diabetic mice, significant decreases in tail-immersion latencies were observed. In an acute study in mice after 4 weeks of STZ, NDGA (1, 2 and 5 mg/kg/s.c), dose dependently attenuated tail-immersion latencies in diabetic mice. Where as chronic treatment of NDGA (5 and 10 mg/kg/s.c) for 4 weeks starting from 4th week after STZ, a significant attenuation of both tail-immersion and paw withdrawal leniencies were absorbed in diabetics. NDGA did not show any change in control animals' latencies. These results emphasize that role of oxidative stress in the development of significant hyperalgesia and cold allodynia in diabetic animals and points towards the potential of NDGA as a complementary therapy for the prevention/treatment of diabetic neuropathic pain. Regulatory cells and human malignant gliomas A. Waziri a , R.C. Anderson a , J.N. Bruce a and D.E. Anderson b a Columbia-Presbyterian Medical Center, New York, USA; b Harvard Medical School, Boston, MA, USA
We have isolated and analyzed both tumor cells and tumor infiltrating lymphocytes (TILs) present in ex vivo central nervous system (CNS) tumor specimens. We used flow cytometry to examine immunologically important cell surface molecules on glioblastoma multiformae (GBM) tumor cells prior to any culturing in vitro, and screened supernatants from ex vivo GBMs for secreted cytokines. Tumor cells expressed moderate to high levels of both HLA class I and class II molecules, suggesting that tumor cells could directly interact with and influence TILs. In addition, a majority of the GBMs examined (n=10) secreted high amounts of the immunosuppressive cytokine IL-10. Based on these observations, we hypothesize that GBM tumor cells promote the differentiation and/or expansion of IL-10secreting type 1 T regulatory (Tr1) cells, which in turn suppress tumor immunity in situ. Consistent with this hypothesis, additional preliminary observations suggest that a CD25+ population of CD4+ TILs, which appear to represent effector cells activated in situ, infiltrates GBMs and that these cells are notable for their secretion of IL-10. In contrast, CD4+ TILs lacking CD25 expression are notable for their secretion of IFN-gamma and the absence of IL-10. These data suggest a novel mechanism by which the tumor microenvironment influences the function of TILs and hinders their ability to mount an effective tumor response. To determine whether the T-cells infiltrating brain tumors are comprised of oligoclonal T-cell populations, we amplified, cloned and sequenced betachain TCR transcripts from tumor biopsy specimens. In one patient with glioblastoma multiforme, we identified the presence of multiple identical copies of beta-chain TCR transcripts after NPA-PCR amplification, cloning and sequencing, suggesting the presence of oligoclonal populations of T cells. The non-palindromic adaptor (NPA)-PCR method was developed in our laboratory to sequence transcripts with unknown or variable 5Vends, such as those of the immunoglobulins and TCRs. Vbeta13-specific PCR amplification followed by cloning and sequencing revealed the presence of a strong clonal expansion, which accounted for 7 of 20 (35%) of the betachain transcripts sequenced. Similarly, Vbeta22-specific and Vbeta21specific PCR amplification followed by cloning and sequencing revealed clonal expansions accounting for 6 of 14 (35%) and 6 of 21 (28%) of the beta-chain transcripts sequenced, respectively. Oligoclonal expansions of beta-chain TCR transcripts were also identified in two other patients (one with recurring glioblastoma multiforme and one with recurrent malignant astrocytoma) using two-sided Vbeta-specific PCR. The presence of these oligoclonal T-cell populations indicates that the T cells may have undergone antigen-driven proliferation and clonal expansion in situ at the site of the tumor in response to an as yet unknown tumor antigen(s).
Increased IFN gamma secretion from restimulated lymphocytes in patients immunized with autologous glioma cells correlates with prolonged survival P. Siesjf and E. Visse Lund University, Lund, Sweden Introduction: In order to monitor the immune response of patients immunized with IFN gamma secreting tumor cells, peripheral blood leucocytes were assayed after restimulation for 5 days with tumor cells. Methods: Blood samples were collected during the immunizations and PBL were isolated and frozen. After thawing PBL, were restimulated with tumor cells in vitro for 5 days. The restimulated, cells were then assayed for cytokine production by flow cytometry and ELIspot, proliferation by thymidine incorporation and lymphocyte subset distribution by FACS. Results: Samples from five patients were analyzed and compared with survival data, immune infiltration and DTH reactions at the vaccination site. No correlation or discernable pattern could be observed from proliferation and lymphocyte subset analyses. Increased IFN gamma levels measured by flow cytometry and ELIspot were seen in 2/5 patients with a prolonged survival. A decrease in IFN gamma production was observed during tumor recurrence. Detectable levels of IL-10 were observed displaying a weak association with shorter survival. Lymphocyte cytokine production correlated with lymphocyte infiltration but not DTH reactions at the immunization site. Conclusions: IFN gamma secretion from restimulated lymphocytes and lymphocyte infiltration at the immunization site were more prominent in 2/5 patients with a prolonged survival. The results underscore the importance of using relevant parameters in the evaluation of immunotherapy against tumors. Patients with glioblastoma, the most common and lethal intrinsic human brain tumor, are reported here to show decreased expression of the activating immunoreceptor NKG2D in CD8 T and NK cells. A soluble factor released by glioma cells down-regulates NKG2D expression in CD8 T and NK cells in vitro. Unexpectedly, this soluble factor is not a soluble NKG2D ligand, but transforming growth factor-beta (TGF-beta) which inhibits NKG2D gene transcription. Moreover, TGF-beta inhibits the transcription of the NKG2D ligand MICA and promotes the release of matrix metalloproteinases (MMP) by glioma cells which in turn mediate the shedding of MICA from the surface of glioma cells. Interference with TGFbeta synthesis by siRNA technology in glioma cells prevents the downregulation of NKG2D on immune cells and suppresses MMP expression and strongly enhances MICA expression in glioma cells. Furthermore, TGF-beta knock-down leads to a decreased migratory and invasive tumor phenotype. Altogether, the knock-down of TGF-beta results in a glioma cell phenotype which is less motile, more sensitive to immune cell lysis, and non-tumorigenic in nude mice.
Identification of a novel neuroblastoma-associated molecule that protects from NK-mediated lysis R. Castriconi a , A. Dondero a , C. Bottino b and A. Moretta a a Dipartimento di Medicina Sperimentale, University of Genova, Genova, Italy; b Istituto Giannina Gaslini, Genova, Italy
In order to identify novel markers useful for specific recognition of Neuroblastoma cells, we immunized mice with a Neuroblastoma cell line and after cell fusion, an mAb termed 5B14 was selected that stained all Neuroblastoma cell lines tested as well as tumors of different origin, including melanomas and carcinomas. Importantly, in bone marrow aspirates derived from stage 4 patients, infiltrating Neuroblastoma cells were selectively recognized by 5B14 mAb. The molecule recognized by the 5B14 mAb has a molecular mass of approximately 100 kDa (thereafter termed NBp100 molecule). Protein purification followed by in-gel tryptic digestion, liquid chromatography and tandem mass spectrometry allowed the identification of the NBp100 protein. Purified neuroblastoma cells were used as target cells in cytotoxicity assays in which effector cells were represented by polyclonal NK cells. These experiments demonstrated that NBp100 molecules play an important role in protecting tumor cells from NK-mediated lysis. Indeed NK cell-mediated killing was inhibited by the interaction with NBp100. This inhibition was reversed by mAb-mediated masking of NBp100. This observation is reminiscent of the effect of anti-HLA class I mAb that can reverse the NK-mediated lysis of HLA class I+ target cells. Thus it appears conceivable that NK cells may be equipped with specific inhibitory receptors that upon interaction with this new ligand transduce signals that down-regulate cytotoxicity.
Eradication of murine brain tumors by direct inoculation of concentrated high titer-recombinant retrovirus harboring the herpes simplex virus thymidine kinase gene K. Shimizu a , T. Yawata a , T. Tsuchiya a , K.C. Park a , S. Toyonaga a , S. Yamada a , H. Nakabayashi a , E. Nakai a , N. Ikawa a and K. Ikenaka b a Kochi Medical School, Nankoku City, Japan; b National Institute for Physiological Sciences, Okazaki, Japan Implantation of retrovirus-producing cells within a tumor has been demonstrated to eliminate malignant brain tumors effectively in animal models. In our previous study, the implantation of high-titer retrovirusproducing fibroblasts into tumors resulted in highly efficient transduction in vivo. The transduced glioma cells migrated far from the implantation site, potentiating the induction of the remarkable bystander effect. It is also possible, however, that the implantation of murine fibroblast-derived virusproducing cells may induce an immune response in patients. In this study, we prepared retroviruses carrying the herpes simplex virus thymidine kinase (HTK) gene with titers of 1.4-2.5Â10 11 colony-forming units (c.f.u.)/ml, and stereotactically inoculated only 3 Al of the HTK-bearing retroviruses into the brain tumors of mice. Following repetitive ganciclovir (GCV) intraperitoneal injection, effective killing of glioma cells in the mouse brain was observed. The transduction efficiency was nearly as high as that observed for the implantation of high-titer retrovirus-producing fibroblasts. Eighty percent of brain tumor-bearing mice were completely cured by our treatment protocol using concentrated HTK-harboring retroviruses. Our results suggest that repeated inoculations of high-titer retroviruses carrying the HTK gene followed by GCV treatment may be a promising strategy for the clinical treatment of malignant gliomas.
Delivery of interleukin 4 to experimental gliomas by viral vectors and neural stem-progenitor cells P. Tunici and G. Finocchiaro Laboratory of Experimental Neuro-Oncology and Gene Therapy, Istituto Nazionale Neurologico Besta, Milan, Italy
In previous experiments, we showed that retroviral delivery of IL-4 restrains the growth of C6 and 9L malignant gliomas in rats and, subsequently, that the injection of rat-immortalized or murine primary neural stem progenitor cells (NSPC) overexpressing IL-4 could be even more effective for the treatment of C6 or GL261 malignant gliomas. MLVderived retroviral vectors, used in these experiments to deliver the IL-4 cDNA to brain tumors or NSPC, have limitations because they only transduce dividing cells and because LTR-driven expression of the transgene can be silenced. To test if lentiviral vectors could be more effective, we prepared a lentiviral vector for IL-4 gene delivery (plasmids were obtained from the laboratory of Dr. Luigi Naldini). The lentiviral preparation contained 50 ng/Al of p24 and NSPC transduced by 1 Al of lenti-IL-4 produced 90 ng/10 6 cells/24 h of IL-4. We injected 2Â10 4 GL261 glioblastoma cells into the striatum of C57Bl6J mice (seven for each experimental group) and after 5 days 3 Al of lenti IL-4 or 2Â10 5 NSPC-IL-4. Lenti GFP or NSPC-GFP, respectively, were used as controls. Only mice injected with NSPC-IL-4 cells survived significantly longer than controls ( p=0.006, log-rank, Mantel-Cox). This effect, however, decreased if more GBM cells (5Â10 4 ) were injected. These experiments confirmed that NSPC-IL-4 cells have a significant anti-tumor effects on gliomas. However, NSPC transduced by lentivirus did not appear more effective than NSPC transduced by MLV-derived retrovirus and IL-4 delivery by lentivirus did not yield anti-tumor activity. Cancers can be partially attributed to defects in the regulation of apoptotic cell death. Failure to trigger the cellular suicide program not only predisposes to development of malignancies, but also increases resistance of tumors to anticancer drugs and irradiation. From the recent observation that taurolidine, an antibacterial drug, displayed a potent antineoplastic effect on several tumor cell lines, we decided to test taurolidine on human ex vivo glioma cells. We performed cytotoxicity and colony formation assays and found a high susceptibility of the glioma cells. From the high activity of taurolidine in both assays, we elucidated the mechanism(s) of its antineoplastic effect. The results of our studies revealed that taurolidine induces programmed cell death through the generation of intracellular ROI which was prevented by the radical scavenger N-acetylcysteine (NAC), but not by the pan-caspase inhibitor z-VAD-fmk. By confocal laser scanning confocal microscopy, we identified apoptosis-inducing factor (AIF) as the major player in this caspase-independent cell death. Interestingly, taurolidine suppressed the constitutive VEGF production of glioma cells displaying thus an antiangiogenic effect besides its antineoplastic activity. Taken together, taurolidine treatment of glioblastoma cells results in a caspase-independent programmed cell death which was preceded by ROI generation, mitochondrial dysfunction and AIF release from mitochondria. Interestingly, taurolidine-induced cell death is also initiated at the lysosomal level leading to mitochondrial dysfunction and programmed cell death either directly or through autophagy of damaged mitochondria.
Multifunctional fusion cytokines for breaking cancer-induced immune anergy S. Paul Transgene SA, Strasbourg, France
The idea that tumors must bescapeQ from immune recognition contains the implicit assumption that tumors can be destroyed by immune responses either spontaneously or as the result of immunotherapeutic intervention. bNatural selectionQ of heterogeneous tumor cells results in the survival and proliferation of variants that happen to possess genetic and epigenetic traits that facilitate their growth and immune evasion. Several mechanisms have been developed by tumor cells which enable them to escape host immunity due to reduction in antigen presentation by tumor cells or due to a local or general decline in patient immunity. Previous work has shown that cytokine immune effectors can enhance host's immunity, and thus overcome a tumor-induced state of immune anergy. More recently, many studies with both mouse and human tumor models have shown the importance of cytokine combinations in the development of optimal immune responses.
Here we described novel fusion proteins also called bFusokineTMQ, that are useful for enhancing both nonspecific and specific immune responses. The murine fusokine IL-2/IL-18 provide high rate of tumor rejection after intratumoral delivery of encoding adenoviral vectors in various animal models, with evidence of immunostimulation and very limited toxicity. The antitumoral effect of IL-2/IL-18 murine fusokine is correlated with the activation of both CTLs, NK and antigen presenting cells.
Microglia and gliomas may have a symbiotic relationship M. Graeber a and A. Flqgel b a Imperial College London, London, UK; b Max-Planck-Institute of Neurobiology, Martinsried, Germany
The number of microglial glial cells/brain macrophages in malignant astrocytomas is surprisingly high. Based on in vitro findings, it has been speculated that brain macrophages derived from microglial cells may exert tumour cytotoxic functions. However, the biological bsuccessQ of malignant astrocytomas strongly suggests that this potential is not exploited in vivo. We have tested the effect of both native microglia that were isolated from neonatal Lewis rat brain and kept in primary culture for 14 days and of supernatant taken from these cells on C6 glioma cells grown in vitro. We did not use a conventional cell death assay but determined the absolute number of tumour cells which carried the bgreen fluorescent proteinQ (GFP) marker gene using a fluorescence-activated cell sorter (FACS). There was no significant cytotoxicity of microglia. Instead, our experiments showed a clear growth promoting effect on C6 glioma cells. The fact that supernatant alone derived from unstimulated microglia which had not been in contact with tumour cells was sufficient to promote survival of neoplastic cells suggests that there is no need for complicated microglia-tumour interactions to explain survival of glioma cells in the presence of microglia in vivo. The molecular basis for the observed effect is currently being studied. Symbiosis between microglia and a glial tumour seems a distinct possibility as gliomas are known to stimulate microglial proliferation.
Vascular endothelial growth factor (VEGF) and interleukin-10 (IL-10) in brain tumours R. Kumar a,b , L. Madden a , K. Hills b , D. Crooks b , J. Greenman a and D. O'Brien b a University of Hull, Hull, UK; b Hull and East Yorkshire Hospitals, Hull, UK IL-10 is an immunosuppressive cytokine produced by T-lymphocytes, and is reported to be a regulatory molecule for angiogenesis primarily mediated by VEGF. The aim of this work was to investigate the relationship between VEGF and IL-10 in brain tumour patients. A prospective analysis to compare levels in serum, cyst and tumour expression of VEGF and IL-10 in patients with newly diagnosed brain tumours. Immunohistochemistry was performed for VEGF, its receptors and IL-10; 71 patients were recruited. Serum samples were obtained prior to surgery and cyst fluid at surgery. Serum and cyst, VEGF and IL-10 were measured by Quantitative ELISA. Patients were divided into benign, low grade, high grade and metastatic groups. Patients with benign and low grade tumours had a median serum VEGF levels of 211.4 and 274 pg/ml. The median serum VEGF levels in the high grade and metastatic group were 279.9 and 288.3 pg/ml respectively. Serum IL-10 was only detected in 54 patients at 2.0 pg/ml. Immunohistochemistry results to follow. There is a trend towards higher serum VEGF levels with advancing tumour grade, with the highest levels being seen in the metastatic group. Serum IL-10 was detectable in only 76% of patients. There was no relationship with tumour grade. There was no association between serum VEGF and IL-10. There was a significant increase of cyst fluid VEGF ( p=0.002) and IL-10 ( p=0.015) compared to serum. Here a pilot study is presented comparing the levels of a panel of cytokines in intracavitary cyst fluid from patients with brain tumours against their serum and normal serum controls. Design: A prospective analysis of cyst fluid VEGF, TNF alpha, IL-6, IL-8 and IL-10 levels in patients with brain tumours (n=12). Serum was obtained prior to surgery and cyst fluid was obtained at the time of surgery. Outcome measures: Quantitative ELISA was used to measure intracavitary cytokine and serum levels. All samples were analysed in duplicates. All cytokine levels were compared with serum values and normal serum control. Results: The levels for all these cytokines were significantly higher in intracavitary cyst fluid compared with patients and normal serum. The level of VEGF was approximately 480-fold higher than normal serum (mean 131,805 vs. 271 pg/ml); TNFalpha concentration was approximately 2-fold lower (3 vs. 6.7 pg/ml); IL-6 concentration was approximately 6500-fold higher (325 vs. 0.05 pg/ml); IL-8 concentration was approximately 10-fold higher (127 vs. 12.5 pg/ml) and IL-10 was approximately 3-fold higher (6.52 vs. 2 pg/ml). Conclusions: Except for TNFalpha, there was a significant increase in all cytokines studied. The significance of such increases in cytokine levels will require further analysis in a larger series of patients.
Expression of novel B7 molecules by human glioma cells in vitro and in vivo: B7-H1 as a potential mechanism of immune paralysis S. Wintterle a , B. Schreiner a , M. Mitsdoerffer a , D. Schneider a , L. Chen b , R. Meyermann a , M. Weller a and H. Wiendl a a University of Tübingen,Tübingen, Germany; b Mayo Clinic, Rochester, MN, USA Human glioblastoma is a highly lethal tumor that is known for its immune inhibitory capabilities. B7-H1 is a recently identified homolog of B7.1/2 (CD80/86) described to exert costimulatory and immune regulatory functions. We investigated the expression and the functional activity of novel B7 costimulatory molecules in human glioma cells in vitro and in vivo. While lacking B7.1/2 (CD80/86), all 12 glioma cell lines constitutively expressed B7-H1 mRNA and protein. Exposure to IFN-gamma strongly enhanced B7-H1 expression. Immunohistochemical analysis of malignant glioma specimens revealed strong B7-H1 expression in all 10 samples examined whereas no B7-H1 expression was detected on normal brain tissues. To elucidate the functional significance of glioma cell-related B7-H1 expression, we performed coculture experiments of glioma cells with alloreactive CD4+ and CD8+ T cells. Glioma-related B7-H1 was identified as a strong inhibitor of CD4+ as well as CD8+ T cell activation as assessed by increased cytokine production (IFN-gamma, IL-2, IL-10) and expression levels of the T cell activation marker (CD69) in the presence of a neutralizing antibody against B7-H1 (mAb 5H1). B7-H1 expression may thus significantly influence the outcome of T cell tumor cell interactions and represents a novel mechanism by which glioma cells evade immune recognition and destruction. Microglia, in response to brain pathology, undergo an activation process and become capable of phagocytosis, antigen presentation, and lymphocyte activation. Recent studies suggest that microglia accumulation in glial tumors may reflect participation of these cells in supporting and promoting the invasive phenotype. Mutual interactions between microglia and glioma cells are not well established. We therefore studied the effects of soluble factors released by rat microglial cells on proliferation and signaling pathways in rat C6 glioma cells. LPS treated microglia-conditioned medium has a weak activatory effect on C6 glioma proliferation as determined using MTT metabolism assay. In the presence of conditioned medium from resting and LPS-activated microglia cultures, we did not observed changes in the activation of p38 MAPK, Erk and JNK kinases in glioma cells, as analysed by Western blot analysis with specific antibodies. However, a significant activation of Akt signaling pathway was observed. PI-3K/Akt kinase signaling pathway regulates many cellular processes, including survival, proliferation, differentiation, chemotaxis and angiogenesis. Furthermore, we studied the effects of glioma-conditioned medium on resting microglia. We found an activation of morphological changes and stimulation of some MAP kinases in microglia. Our results suggest that glioma cells may activate microglial cells that further exert an activatory effect on glioma cells contributing to glioma pathology. Supported by PBZ-MIN/001/P05/10.
Gene immunotherapy in the brain: looking for anti-tumour activities of microglia A. Abschütz, R. Geibig and A. Régnier-Vigouroux Deutsches Krebsforschungszentrum, Heidelberg, Germany
Common strategies for tumour gene therapy mainly focus at inducing cell death or stimulating the immune system in order to eliminate the tumour mass. In case of brain tumours, new therapies are desperately needed due to poor prognosis of patients and failure of classical therapies. Microglia represent a potential, though poorly exploited arm of defense which antitumour activity could be triggered by tumour-targeted gene immunotherapy. Autonomous parvovirus-based vectors are promising tools for delivery of therapeutical genes into the brain, given the natural oncotropism of these viruses, their oncosuppressive activity in vivo and their capacity for selective delivery of toxic/immunostimulatory genes into tumour cells. We first investigated the permissiveness of primary mouse glial cells to the murine parvovirus. Astrocytes, but not microglia, readily take up the virus and about 10% of the cells express viral or transduced proteins. Nevertheless, the viral life cycle is not completed and cells are not lysed. Second we started to characterize anti-tumour capacities of microglia. We show that microglia can (i) kill glioma cells via soluble compounds after stimulation with lipopolysaccharide and IFN-gamma and (ii) phagocytose living and apoptotic glioma cells. Taken together, our data suggest that microglia do have the potential to fight against brain tumours and that immunostimulatory molecules produced by virally transduced tumour cells and astrocytes could regulate this potential. Glioma cells stimulated with proinflammatory cytokines produce high amount of nitric oxide (NO), a highly reactive free radical with both anticancer and cancer-promoting properties. Aloe-emodin (AE), an herbal compound with tumoricidal activity, strongly down-regulated NO synthesis in rat astrocytoma C6 cells stimulated with IL-1beta, IFN-gamma and TNFalpha. AE affected the expression of both transcription and translation products of NO-producing enzyme inducible NO synthase (iNOS), rather than its enzymatic activity. Treatment with AE significantly reduced cytokine-triggered expression of important iNOS transcription factor IRF-1 in C6 cells, while activation of NF-kappaB remained unaltered. Furthermore, AE partially interfered with the activation of mitogen-activated protein kinase (MAPK) pathway, as activation of ERK1/2, but not c-Jun Nterminal or and p38 MAPK, was suppressed upon AE treatment. In conclusion, AE-mediated down-regulation of IRF-1 and ERK pathways, with subsequent inhibition of iNOS transcription and NO synthesis in glioma cells, might have important implications for potential therapeutic use of this antraquinone. Aloe-emodin (AE) is a naturally occurring compound with wide spectrum of biological properties. This antraquinone induces apoptosis in several tumor cell lines with special affinity to tumors of neuroectodermal origin. High amounts of nitric oxide (NO) released by activated macrophages induce tumor cell death. Therefore, we explored the capacity of AE to modulate macrophage NO-mediated killing of glioma cells in vitro. Interestingly, while AE markedly suppressed NO release from macrophages alone, it significantly potentiated NO production in cocultures of macrophages and rat astrocytoma C6 cells. Accordingly, the viability of C6 cells cocultivated with macrophages was significantly reduced in the presence of AE. The contrasting AE-imposed effects on macrophage NO release were closely related to macrophage density, as its increase readily converted the observed inhibition into potentiation of NO synthesis. Stimulation of NO synthesis in high density macrophages culture by AE was not affected by transcription or translation inhibitors, indicating that enzymatic activity, rather than gene expression, was a target for the drug action. Accordingly, the expression of iNOS in macrophages was not affected by AE. Therefore, the capacity of AE to modulate macrophage NO release and survival of glioma cells might considerably depend on the contact between macrophages or macrophages and tumor cells. Expression of CCR7 in the central nervous system (CNS) and its implications for CNS immunity P. Kivis7kk, D. Mahad and R.M. Ransohoff Cleveland Clinic Foundation, Cleveland, OH, USA It is unclear how immune cells traffic between the lymphoid compartment and the CNS. We defined the expression of trafficking determinants on T cells and dendritic cells (DCs) in the cerebrospinal fluid (CSF) and brain parenchyma as well as on endothelial cells from different compartments within the CNS to gain insight into pathways for immune cell trafficking to the brain. Ninety percent of CSF T cells from patients with multiple sclerosis (MS) or noninflammatory neurological diseases (NIND) were CD4+/CD45RO+/CD27+/CCR7+ central-memory T cells. In contrast, all CD4+ T cells in parenchymal MS lesions lacked CCR7, consistent with an effector-memory phenotype. Postcapillary venules of the choroid plexus and subarachnoid space, but not brain parenchymal microvessels, in autopsy sections from NIND patients expressed adhesion molecules supporting recruitment of T cells. These findings suggest that immune surveillance of the non-inflamed CNS immunity is executed by centralmemory T cells, which enter CSF directly from the systemic circulation, retaining the capacity to differentiate into effector-memory cells upon local antigen restimulation. Inflamed MS lesions contained numerous CCR7+ cells with phenotypic features of mature DCs or activated microglia. CCR7+ DCs were identified in the CSF from MS patients. These findings indicate that the efferent limb of CNS immunity is comprised in part of DCs, which migrate to deep cervical lymph nodes through the CSF. The nature of CCR7+ myeloid cells in the CNS parenchyma is under investigation.
Identification of putative trigger agents in multiple sclerosis using an unbiased approach M. Sospedra a , Y. Zhao a , P. Muraro a , S. Jacobson a , C. Pinilla b and R. Martin a a National Institutes of Health, Bethesda, USA; b Torrey Pines Institute, San Diego, USA Multiple sclerosis (MS) is a T cell-mediated autoimmune disease of the central nervous system (CNS). Viral infections often precede exacerbations, and hence infectious agents have been considered as triggers, although their nature is unknown. Molecular mimicry provides an elegant framework as to how similarities between foreign agents and autoantigens can trigger autoimmune diseases. In this study, we determined the specificity of cerebrospinal fluid (CSF)-infiltrating T cells from a patient with RR-MS in exacerbation using an unbiased approach in order to identify foreign agents that might have activated these T cells. CSF T cells were cloned by limiting dilution using PHA. Subsequently, we analyzed the TCR expression and by CDR3 spectratyping we identified which of these T cell clones (TCC) were clonally expanded in vivo. In vivo expanded TCC were then tested with synthetic combinatorial peptide libraries (ps-SCL), and a recently described search algorithm applied to predict peptides from the entire bacterial-and viral protein databases that might be recognized by each TCC. Interesting peptides were synthesized and their stimulatory potential tested. Several stimulatory natural peptides originating from foreign agents were identified. Interestingly, two out of five TCC recognized multiple peptides from related organisms, mainly human herpes viruses and TT-virus. Our data strongly indicate that ubiquitous viral agents can activate CSF-infiltrating T cells in MS.
Similar low frequency of anti-MOG IgG and IgM in MS patients and healthy subjects V. Lampasona a , D. Franciotta b , R. Furlan a , S. Zanaboni a , R. Fazio a , E. Bonifacio a , G. Comi a and G. Martino a a San Raffaele Scientific Institute, Milan, Italy; b University of Pavia, Pavia, Italy
Anti-MOG antibodies induce extensive demyelination in T cell-mediated experimental autoimmune encephalomyelitis (EAE), and bind to disintegrating myelin in acute multiple sclerosis (MS) and in the marmoset model of EAE. These observations motivate the search for anti-MOG antibodies in MS, in which increased MOG-specific T and B cell responses are reported. Reported data have had a negligible impact on patient care, but Berger et al. showed that serum anti-MOG IgM strongly predicted the early conversion of first demyelinating events to clinically definite MS. We used a liquidphase radiobinding assay to measure serum anti-MOG IgG in 87 multiple sclerosis patients (MS), in 12 encephalomyelitis patients, and in 47 healthy subjects. Anti-MOG IgM were determined in samples obtained at onset from 40 of the 87 MS patients, and in controls. The frequency of positive samples with low titers of anti-MOG IgG (=5.7%) and IgM (=8.3%) was similar in all the groups/subgroups. Binding competition experiments showed that these antibodies had low affinity. Anti-MOG antibodies are not disease-specific. Interactions between complement, immunoglobulins (IgG) and IgGreceptors (FcgammaR) could contribute to inflammation and demyelination in multiple sclerosis (MS). MS lesions were recently classified into four subtypes, one of which is characterized by deposition of complement and IgG. Expression of FcgammaR in active lesions has been described in another study. We investigated the presence of complement, IgG and FcgammaR in different stages of MS lesions. Immunohistochemistry was performed on 55 lesions from 30 patients, derived from frozen autopsy material. We stained for complement, IgG, MHC class II, myelin proteins and FcgammaR (FcgammaRI, FcgammaRII and FcgammaRIII). Immunopositivity was scored semiquantitatively. Lesions with MHC class IIpositive macrophages containing intracellular myelin debris (PLP+ macrophages) were classified as active demyelinating. C1q and C3d, but not C4d, C5b-9 and IgG, were detected on astrocytes in MS lesions of all stages. In contrast, presence of C1q, C3b, C5b-9 and IgG on and within macrophages was restricted to active demyelinating lesions. Complement and IgG colocalized intracellularly. In contrast to earlier publications, complement and IgG deposition were observed in all active demyelinating lesions. Enhanced expression of FcgammaRI, FcgammaRII and FcgammaRIII was observed on both PLP+ and PLPÀ macrophages, always associated with enhanced expression of MHC class II. This suggests that the presence of complement in macrophages is associated with active demyelination, whereas expression of FcgammaRs is associated with inflammation. Double stainings to co-localize complement, IgG and FcgammaR within PLP+ macrophages are currently performed.
New antigenic candidates in multiple sclerosis: identification by serological proteome analysis D. Lefranc a , L. Almeras a , H. Drobecq b , J. de Seze c , S. Dubucquoi a , P. Vermersch c and L. Prin a a Department of Immunology, CHRU of Lille, Lille, France; b Biological Institute of Lille, Lille, France; c Department of Neurology, CHRU of Lille, Lille, France
Myelin antigen targets that are clearly associated with pathogenic events in multiple sclerosis (MS) patients remain to be defined. We recently demonstrated that the analysis of global IgG antibody response against human brain antigens using one-dimensional immunoblotting, allowed us to discriminate MS patients from controls (both healthy subjects and Sjfgren's syndrome). Additionally, this approach also differentiated the three clinical forms of the MS disease. Indeed, 42 brain antigenic bands (26 in healthy brain and 16 in MS brain) showed these discriminant IgG immune responses. The aim of our study was to characterize the 26 discriminant antigenic bands detected on healthy brain. Protein identification was successively performed by one-dimensional (1-D) and two-dimensional (2-D) immunoblottings using sera from 18 MS patients, followed by MS analysis and a database search. One hundred and two antigenic spots were then detected on 2-D immunoblots, with MS sera against healthy brain. Sixty-four spots successfully matched with 2-D coomassie brilliant blue (CBB) stained gels, which were further selected for MS analysis and annotated leading to the identification of 14 of the 26 discriminant antigens. Thus, serological proteome analysis (SERPA) may provide a useful tool for the identification of potentially new MS-associated antigens, whose relevance to physiopathological events remains to be defined. Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system (CNS). Disease mechanisms in MS at the molecular level remain poorly understood and no reliable proteinaceous disease markers are available yet. The goal of the present study is the construction of a protein database of cerebrospinal fluid (CSF) from MS patients. Proteomic approaches are particularly suitable for a molecular dissection of disease phenotypes of the CNS that is largely inaccessible to meaningful mRNA expression based analysis. CSF is a secretion product of several CNS structures and its protein profile can be affected by several diseases of the CNS. Proteomic analysis of CSF could provide more insight into the pathogenesis of these diseases. CSF proteins from relapsingremitting MS patients were sampled and analyzed by means of twodimensional (2-D) gel electrophoresis and 2-D liquid chromatography, both coupled to tandem mass spectrometry. The use of these two complementary techniques resulted in the identification of more than 150 different proteins. The majority of these proteins have been analyzed already in other CSF studies. However, some of these proteins have not been described so far. Further investigation will provide additional information on whether they are implicated in the MS pathogenesis. The constructed CSF database will be used in our future study on differentially expressed proteins. Important applications are the identification of diagnostic, paragnostic and theranostic protein markers.
The role of the HLA-DR2 haplotype in multiple sclerosis K. Kawamura a , L. Fugger b and T. Forsthuber a a Institute of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA; b Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK Multiple sclerosis (MS) is an autoimmune disease strongly associated with the MHC class II genes HLA-DRB1*1501, HLA-DRB5*0101 and HLA-DQB1*0602 which are almost always inherited together as the bHLA-DR2 haplotypeQ. The reason for the association of the HLA-DR2 haplotype with MS is unknown, but it has been postulated that these MHC class II genes could be deficient in their ability to induce central or peripheral tolerance to myelin antigens. Indeed, T cell responses to myelin antigens are readily detectable in MS patients, indicating that self-tolerance or regulatory mechanisms have failed. To understand the role of the HLA-DR2 complex in the induction of tolerance to MBP, we have generated mice that express each of the HLA-DR2 haplotype MHC molecules and are deficient for MBP (MBPÀ/À) or not (MBP+/+). The MBP+/+ or MBPÀ/À mice were immunized with human MBP protein and epitope mapping of MBP was performed by cytokine ELISPOT assay with overlapping sets of MBP peptides spanning the complete MBP sequence. The epitope mapping showed that MBP84-102 was the immunodominant epitope for MBPdeficient HLA-DRB1*1501 and DRB5*0101 Tg mice. Surprisingly, MBP+/+ HLA-DR transgenic mice were tolerant to MBP84-102, suggesting that these MHC class II molecules were not deficient in their ability to induce central or peripheral tolerance to myelin antigens. We will suggest an alternative mechanism to account for the role of the HLA-DR2 haplotype in MS. Microarrays provide a global view of gene expression. In multifactorial diseases with uncertain etiology, they represent a powerful method to disclose unknown causative factors and to place known alterations in the right context. However, microarrays are subjected to many variables that complicate the interpretation of the results. We tried to limit these variables by studying monozygotic twins (four sets; F/M=3/1, mean age 36.25F3.9) discordant for MS. The affected twin was free of immunomodulatory treatments. Following leukapheresis, CD4+ and CD8+ T cells were separated and studied by microarrays (Agilent Human 1A Oligo Microarray Kit). In the affected co-twins, 10 genes were over-while 22 were underexpressed in CD4+ cells; in CD8+ cells, expression was increased in 74 genes and decreased in 62 (fold change N1.3, pb0.05). Differentially expressed genes included transcripts of immunological significance, genes involved in cell structural remodelling and in the regulation of the cellular electrolyte balance. Experiments are in progress, on the same material, with an Affymetrix platform. So far, the results show that differentially expressed genes CD8+ T cells numerically exceed those of CD4+ lymphocytes. Results on the validated genes will be presented.
Functional genomic analysis in multiple sclerosis J. Wuerfel a , A. Weber a , J. Kraetzschmar b , T. Prozorovski a , J. Bellmann-Strobl a , O. Aktas a , C.S. Stuerzebecher b , F. Zipp a and C. Infante-Duarte a a Institute of Neuroimmunology, Charité, Berlin, Germany; b Schering AG, Berlin, Germany
Multiple sclerosis (MS) is a complex multifactorial disease characterized by an enormous variability in its clinical presentation and course, in which clear diagnostic parameters are lacking. In order to identify a gene expression pattern in peripheral immune cells from MS patients with potential diagnostic and prognostic implications, we performed a largescale gene expression analysis using DNA-microarrays (12,000 genes) in relapsing-remitting (RRMS) and primary progressive (PPMS) patients as well as in healthy individuals. The most representative genes differentially regulated in MS were selected according to very stringent selection criteria. Ten genes out of these were subsequently used to confirm the array data by real-time rtPCR in an extended control and patient cohort. By this approach, we defined an MS expression profile which is characterized by an increased expression of genes involved in cellular activity, chemotaxis, adhesion processes and transendothelial migration, and by a diminished expression of factors related to regulation of B-cell response and response to interferons. No factors distinguishing between RRMS and PPMS were detected applying our selection criteria. The identified expression profile indicates important aspects of the MS pathophysiology, highlighting the role of transendothelial migration as well as B-cell and IFN-related processes in MS immunopathology and offers a basis for the development of diagnostic markers in MS.
Human endogenous retrovirus-W envelope abundance and infectivity in multiple sclerosis J. Antony a , M. Izad b , K. Zhang a , K. Warren c , M. Vodjgani b and C. Power a a University of Calgary, Calgary, AB, Canada; b Tehran University of Medical Sciences, Tehran, Iran; c University of Alberta, Edmonton, AB, Canada
Human endogenous retroviruses (HERVs) constitute 8% of the human genome yet their role (s) in health and disease remain uncertain. To determine if disease status is associated with HERV-W envelope (env) abundance, we developed an HERV-W env PCR-based viral load assay for quantitation of provirus and viral RNA copy numbers in brain and blood. Proviral copy number in blood (9.9F8.3 log10 copies/mg DNA) and brain (8F7.3 log10 copies/mg DNA) was significantly increased ( pb0.05) in MS patients (n=33) compared to controls (n=31) (8.6F7.8 and 7.11F6.7 log10 copies/mg DNA respectively). In addition, viral RNA levels in brain of MS patients (n=7) (7.9F7.1 log10 copies/mg RNA) were higher than in controls (n=3) (7.6F6.7 log10 copies/mg RNA). Whether this increased HERV-W env copy number reflected increased infectivity of neural cells was examined by infection of human brain-derived cells by a pseudotyped virus consisting of the HERV-W envelope and an envelope-deleted HIV-1. Pseudotyped virus infected only astrocytes and macrophages but not neurons despite expression of the putative HERV-W receptor, ASCT2 in all cells. These data indicate both HERV-W env provirus and viral RNA levels are increased in MS patients, which may reflect infectivity mediated by HERV-W envelope in astrocytes and macrophages, likely driven by concurrent innate immune activation.
Expression and modulation by cytokines of multiple sclerosis (MS)associated retrovirus (MSRV): epiphenomenon or pathogenic factor? C. Serra a , G. Mameli a , G. Arru b , V. Astone a , S. Sotgiu b and A. Dolei a a Section of Microbiology, Department of Biomedical Sciences, University of Sassari, Sassari, Italy; b Institute of Clinical Neurology, University of Sassari, Sassari, Italy
Multiple sclerosis (MS) has unknown etiology and debated, immunemediated pathogenesis, including genetic and environmental factors. Several viruses were suggested to be involved, particularly HHV-6 and MSRV, a member of the HERV-W family of human endogenous retroviruses, with gliotoxic, fusogenic and superantigenic properties. Extracellular MSRV is detectable in plasma of 100% MS patients from Sardinia, Italian island at very high MS risk. Patients with MSRV-free CSF remain stable, whereas those with MSRV-positive CSF disclose a more severe, treatment-requiring disease. We selected MSRV(+) and MRSV(À) healthy volunteers to evaluate the effects on MSRV release by cultured PBMC exerted by cytokines supposed to have detrimental or beneficial effects on MS immunopathogenesis, evaluating virionic RNAs by semiquantitative RT-PCR. Data show that cultured PBMC from MSRV(+) donors spontaneously release MSRV that accumulates with time and with mitogens, and is cytokine-sensitive. The highest effects are seen with the MS-detrimental TNF-alpha and IFN-gamma cytokines, while IFN-beta is strongly inhibitory. PBMC from MSRV(À) individuals do not release MSRV, neither spontaneously nor after treatments. Although MSRV role in MS is still not defined, an intriguing parallelism between the effects of the above cytokines on MSRV production in vitro and on MS disease in vivo is observed. Multiple sclerosis associated retrovirus (MSRV) seems to be the important candidate for viral etiology of MS. The aim of the study was to analyze MSRV pol sequences in MS and control groups. In the serum RNA MSRV pol sequences have been identified in 31/32 MS patients. MSRV pol sequences were detected in serum cDNA of 9/17 myasthenia gravis patients, 7/16 with Parkinson's disease, 10/21 migraine patients, and 13/27 healthy individuals. MSRV pol sequences were observed also in RNA from lymphocytes of all MS patients, 12/17 myasthenia gravis patients, 9/16 with Parkinson's disease, 14/21 migraine patients and 18 out of 27 healthy donors. In genomic DNA of all studied cases, MSRV pol sequences were found. The pattern of fiber-FISH signals suggests the presence of multiple copies of MSRV pol sequences, tandemly dispersed in the genome. It can be concluded that MSRV pol sequences are endogenous, widespread in lymphocytes DNA and transcribed into RNA of MS patients as well as of other studied patients and healthy individuals. However, more frequent expression of MSRV sequences detected in lymphocytes RNA, as well as their presence in higher frequency in serum of MS patients may suggest the involvement of MSRV in the etiopathogenesis on MS. Clonally expanded CD8 populations have been identified in brain specimens, peripheral blood and the CSF of patients with MS. Although their antigen-specificity is unknown to date, genuine CNS autoantigen(s) or viral antigens are the most obvious candidates. Notably, disease onset and exacerbations have been correlated to concomitant viral infections, especially EBV. MHC-class-I tetramers were synthesized for dominant epitopes of EBV and CMV herpes viruses. Antigen-specific T cells were analyzed by multicolour flow-cytometry in the peripheral blood of MS patients and controls; 28 MS patients were compared with 24 healthy donors (HD) using an HLA-B7 EBV peptide (RPPIFIRRL) tetramer. Whereas 93% of the MS patients showed reactivity to this dominant EBV epitope, only 15 out of 24 HD (62.5%) were positive. Additionally, frequency of EBVreactive CD8-cells in the MS group was higher than in controls (mean 0.52% against 0.12%). EBV-specific CD8-T cells predominantly belonged to the effector memory phenotype as assessed by costaining with CD45RA/CCR7. Overall, MS patients showed a significant downregulation of CD28 in EBVreactive CD8-cells suggesting a chronic in vivo stimulation. In two out of seven patients with acute relapses, the majority of EBV-reactive T-cells consisted of CD45RA+/CCR7À cytotoxic effector cells. In contrast to EBV, CMV-specific CD8-T cells did not show significant differences between MS and controls. Taken together, EBV-specific CD8-T cells in MS patients differ from HD in their frequency and phenotype further corroborating their putative involvement in the immunopathogenesis.
CD8 T cells from acute multiple sclerosis patients display selective increase of adhesiveness in brain venules: a critical role for P-selectin glycoprotein ligand-1 L. Piccio a , L. Battistini c , B. Rossi a , S. Bach a , L. Ottoboni a , E. Scarpini b , G. Borsellino c and G. Constantin a a University of Verona, Verona, Italy; b University of Milan, Milan, Italy; c IRCCS Santa Lucia Foundation, Rome, Italy Lymphocyte migration into the brain represents a critical step in the pathogenesis of multiple sclerosis (MS). CD4 and CD8 T cells were obtained from healthy donors and relapsing-remitting (RR) MS patients at the relapse onset. Adhesion molecules expression was studied by flow cytometry. Intravital microscopy studies and in vitro rolling assays were performed. In patients with RRMS CD8, but not CD4, T cells display increased rolling and arrest in inflamed murine brain venules and increased rolling on P-selectin in vitro. Anti-l-selectin antibodies block the recruitment of CD8 and CD4 T cells from MS patients in vivo. Anti-P-selectin glycoprotein ligand-1 (PSGL-1) antibodies dramatically inhibit the recruitment of CD8 cells from MS patients in brain vessels. Moreover, vascular cell adhesion molecule-1 (VCAM-1), but not PSGL-1, is critical for the adhesion of CD4 cells from MS patients. FACS analysis and functional data indicate that a large fraction of CD8 cells from MS patients display a memory-effector phenotype. Our results show that CD8, but not CD4, T cells from MS patients in the acute phase display increased ability to be recruited in inflamed brain venules. PSGL-1 is a key molecule mediating CD8 cell migration into the brain. The dichotomy in the adhesion mechanisms of CD8 versus CD4 lymphocytes provides important information for the selective blocking of the recruitment of lymphocyte subpopulations in acute MS.
Dopamine fails to regulate T cell activation in multiple sclerosis. Effects of IFN beta M. Giorelli, P. Livrea and M. Trojano Department of Neurological and Psychiatric Sciences, University of Bari, Bari, Italy Objective: To assess whether the dopamine network of lymphocytes is involved in the pathophysiology of Multiple Sclerosis (MS). Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 14 stable relapsing-remitting MS patients, 6 relapsing MS patients, 13 IFNbeta-1a (8) or IFNbeta-1b (5) treated MS patients, 6 patients affected from Other Inflammatory Neurological Diseases, and 11 healthy controls. We assessed the expression of dopamine receptors (D3R, D5R), and investigated the dopamine (0.001-1 Ag/ml) capability to regulate T cell proliferation, expression of IFN gamma, MMP-9, and Th1/Th2 cytokines. Results: The D5R was reduced in PBMCs from both stable and IFNbeta treated MS patients ( pb0.0001 to 0.05). The D3R was expressed at normal levels in PBMCs from stable MS patients, but it was reduced in PBMCs from MS patients undergoing to IFNbeta treatment ( pb0.0001). Dopamine inhibited T cell proliferation, MMP-9 expression, and enhanced T1/Th2 cytokines mRNA in PBMCs from HD but not from MS patients. Differently, the regulatory effects of dopamine were renewed in PBMCs from IFNbeta treated MS patients. Conclusions: Differential expression of the dopamine receptors subtypes D5R and D3R underlies the loss of the inhibitory effects of dopamine on T cell proliferation, expression of MMP-9 mRNA and secretion of IFN gamma in MS. Availability of agonists/antagonists for dopamine receptors and effects of IFNbeta might open novel paths to therapeutic intervention. In humans, T cells that utilize a Vdelta2Vgamma9 T cell receptor respond to phosphate antigens found in pathogens such as Mycobacterium tuberculosis by rapidly producing large amounts of proinflammatory cytokines, chemokines and cytotoxic factors. For this reason, they have been implicated as contributors to inflammatory diseases, and depletion of gamma-delta T cells in mice delays or protects against experimental autoimmune encephalomyelitis, a model for multiple sclerosis (MS). Using transcriptional profiling of phosphate-antigenactivated Vdelta2 cells, we observed potent upregulation of proinflammatory cytokines and cytotoxic factors, but also noted upregulation of the vitamin D receptor (VDR) and confirmed by western blotting that was dependent on the PKC and ERK signaling pathways. The VDR has been implicated in downregulation of Th1-type cells by promoting the production of regulatory activity of dendritic cells. Less is known about the direct effects of VitD on T cells. We now show that VitD significantly reduced IPP-induced IFNgamma, CD25 expression and IL-2-signaling. VitD also increased activation-induced cell death, as determined by indicators of apoptosis. Given the recent data that support a protective effect of VitD intake on the risk of developing MS (Munger, K.L. et al., Neurology, 2004; 62: 60) , our results suggest that effects of VitD on reducing the proinflammatory activity of gamma-delta T cells may contribute to this effect.
Spreading of autoreactivity from antigens present in the thyroid gland to the spinal cord or vice versa: an alternative pathogenic mechanism in multiple sclerosis? J.M. Greer and M.P. Pender University of Queensland, Brisbane, Australia
We have identified a subgroup of patients with multiple sclerosis (MS) who have coexistent autoimmune thyroid disease (AITD). In most patients, the AITD developed prior to the onset of MS. All of these patients have shown predominant involvement of the spinal cord from the outset of their MS, with much less involvement of the cerebrum and optic nerves than is normally seen. These patients also have a high prevalence of autoimmune disease (especially AITD) in their family members. Class II HLA alleles carried by the patients are linked to the autoimmune disease that first arises in these patients, i.e. patients who develop MS first tend to carry HLA-DRB1*1501, whereas patients who first develop AITD carry DR3, DR4 or DR7. Increased T cell and antibody reactivity can be detected in these patients against several antigens that are present predominantly, but not exclusively, in both the spinal cord and the thyroid gland. Increased reactivity to these antigens is not found in patients with only MS or only AITD. These findings suggest the possibility of interorgan spread of autoimmunity from the thyroid gland to the nervous system or vice versa in individuals who have a genetic susceptibility to autoimmune disease.
Expression of K+ channels by CD4+ T-lymphocytes from multiple sclerosis patients P. Sarchielli , L. Greco, A. Candeliere, Ar. Floridi, Al. Floridi and G. Capocchi University of Perugia, Perugia, Italy Aim: This study was aimed to assess the expression of K+ channel Kv1.3 mRNA levels by CD4+ cells in patients with relapsing-remitting (R-R) multiple sclerosis. Patients and methods: 15 R-R MS patients were assessed in a stable clinical phase and 15 R-R MS patients during a relapse. The control group consisted of 15 age-matched healthy individuals. Expression of KTv1.3 was assessed by RT-PCR, lymphocyte proliferation assay, measurement of IL-2 and IFN-gamma by ELISA and Ca+ measurement by Fura-2 method were also carried out. Results: RT-PCR analysis showed a higher expression of the KTv1.3 band in CD4+ Tcells of MS patients, particularly during relapses, than in healthy controls. The higher expression of KTv1.3 band was present in MBP+ CD4+ Tcells. K+ blockers KTXK, ShK, Shk-Dap and MgTx all inhibited mitogen-and antigen-specific proliferation of total CD4+ and MBP+CD4+ T cells in MS patients. The same K+ blockers also reduced Ca+ intracellular levels and production of IL-2 and IFN-gamma. Discussion: We confirmed the expression of Kv1.3 channel by CD4+ T-cells and MBP+ encephalitogenic T cells of MS patients particularly during the active phase of the disease. These channels can be a relevant target for immunosuppression and blockers of these channels can be putative drug candidates for treating MS. Multiple Sclerosis is an autoimmune disorder of the Central Nervous System, in which the immune attack is directed against myelin antigens. A role for both gamma/delta T lymphocytes and NK cells in disease pathogenesis has been suggested. Upon exposure to phosphoantigens, human Vdelta2 T lymphocytes enter a lineage differentiation pattern that determines the generation of memory cells with a range of effector functions. Here, we show that within the effector memory Vdelta2 population, based on expression of CD16 two distinct and complementary subsets with regard to phenotype, mode of activation and type of responses can be identified: Vdelta2TEMh cells, and Vdelta2TEMRA cells. The latter are refractory to phosphoantigen but respond to activation via FcgammaRIII and are highly active against tumoral target cells. CD16+ gamma/delta cells likely represent the final differentiation step of effector cells. Interestingly, on a cohort of 27 MS patients, we have found an expansion of the CD16+ fraction within the Vdelta2 T cell population: implications on disease pathogenesis are discussed. Dendritic cells (DCs) are essential in orchestration of immunity and tolerance. We investigated, if the immunoregulatory function of freshly isolated plasmacytoid dendritic cells (pDCs) is impaired in multiple sclerosis (MS). We studied pDCs from untreated MS patients and healthy controls. In an allogenic co-culture assay with irradiated peripheral blood mononuclear cells (PBMCs) always obtained from the same healthy donor, increasing ratios of pDCs (1:100, 1:20 and 1:10) were added to autologous PBMCs (=effector cells) that were before depleted of pDCs. The response of effector cells was measured by proliferation. Proliferation was calculated as stimulation index (SI) representing the x-fold increase compared to PBMCs depleted of pDCs. We found a dose dependent increase in proliferation by adding increasing numbers of pDCs in co-cultures from healthy controls. Increases were 1.2-fold (SDF0.3) for pDCs 1:100, 1.7 (F0.6) for pDCs 1:20 and 2.2 (F1.3) for pDCs 1:10. In MS patients, no modulatory effects could be observed. There was a statistically significant difference between MS and controls for the pDC ratios 1:20 and 1:10 ( pb0,007, Mann-Whitney U-test). In co-cultures supplemented with IL3, we also found a dose dependent difference between MS patients and controls reaching significance for the 1:20 ratio. Our results demonstrate that pDCs derived from MS patients have an altered regulatory function compared to controls that may have an influence on immune imbalance in MS.
Reactivation of immune response causes increased cerebral blood volume K.A. Broom a , D. Anthony b , A. Blamire a , S. Waters b , V.H. Perry b , P. Styles a and N. Sibson a a University of Oxford, Oxford, UK; b University of Southampton, Southampton, UK It has long been postulated that environmental factors cause exacerbation of multiple sclerosis (MS). We have used Magnetic Resonance Imaging (MRI) to investigate whether systemic inflammation, induced by peritoneal injection of bacterial cell wall products, can reactivate a quiescent CNS lesion bearing many of the hallmarks of an MS lesion; 5Â10 5 heat-killed BCG organisms/Al were injected stereotaxically into the striatum of male Lewis rats. Additionally, 0.2 Al of BCG was deposited on the cortical surface to initiate a spontaneous delayed type hypersensitivity (DTH) response. Once the lesion had resolved (day 42), 100 Ag/ml (i.p.) of E. coli lipopolysaccharide (LPS) endotoxin was injected. MRI was performed throughout the acute DTH response and following LPS challenge at 6, 24, and 72 h. The initial intracerebral injection caused an acute DTH inflammatory response, including an~8-fold increase in regional cerebral blood volume (rCBV), blood-brain barrier (BBB) breakdown, and oedema accumulation, all of which resolved by 42 days. At 6 h after LPS challenge, a new increase in rCBV was observed throughout the injected striatum ( pb0.005 when compared with pre-challenge values). Additionally, increased BBB permeability was observed 24-72 h post-LPS challenge. We have shown that a systemic inflammatory response can reactivate a Th1-dominated DTH lesion in the rodent brain, by increasing rCBV and altering BBB permeability. These findings may have implications for MS patients, and suggest that monitoring regional perfusion may be valuable in detecting early CNS inflammation. During the course of Multiple Sclerosis (MS), the gene expression levels of some immune system genes are modified. The aim of this study was to assess the differences in gene expression levels for a set of eight immune system genes in peripheral blood mononuclear cells (PBMCs) in patients with MS. We assessed the gene expression levels of HLA-DQB1, HLA-DRA, IFN-gamma, IL4, TNF-alpha, IDO, soluble CTLA4 and full length CTLA4 in PBMCs from 20 MS patients and 20 healthy controls by realtime PCR. We found that the gene expression levels of IFN-gamma were significantly lower in patients than in controls ( p=0.018) and the ratio IFNgamma/IL4 was significantly lower in MS patients ( p=0.028). We also found a correlation between IFN-gamma and TNF-alpha expression levels in both groups ( p=0.041, r=0.46) and a reduction of the ratio IFN-gamma/ TNF-alpha in patients ( p=0.037). Finally, IFN-gamma levels were lower in untreated than in treated MS patients ( p=0.030). No differences were found between groups for the other studied genes. Our results show an imbalance of Th1 cytokines in the PBMCs of patients with MS. Whether such decrease indicates that IFN-gamma producing cells are decreased or sequestered in peripheral tissues (such as the brain) requires further studies.
Cerebrospinal fluid and serum levels and intrathecal production of active matrix metalloproteinase-9 are elevated in multiple sclerosis patients with disease activity M. Castellazzi, T. Bellini, M.C. Manfrinato, E. Baldi, E. Paolino, E. Granieri, F. Dall'occhio and E. Fainardi University of Ferrara, Ferrara, Italy
We measured cerebrospinal fluid (CSF) and serum levels of matrix metalloproteinase-9 (MMP-9) and their tissue inhibitor TIMP-1 by Activity Assay System ELISA, respectively, in 37 relapsing-remitting (RR), 15 secondary progressive (SP) and 9 primary progressive (PP) multiple sclerosis (MS) patients, stratified according to clinical and Magnetic Resonance Imaging (MRI) evidence of disease activity, and in 96 patients with other inflammatory neurological disorders (OIND) and noninflammatory neurological disorders (NIND). Intrathecal synthesis of active MMP-9 were more frequent in MS than in NIND ( pb0.02) and in clinically and MRI active than in clinically and MRI stable MS patients ( pb0.05 and pb0.02, respectively). Serum active MMP-9/TIMP-1 ratio were higher in MS and OIND than in NIND ( pb0.02) and in clinically and MRI active than in clinically and MRI inactive MS patients ( pb0.001). CSF active MMP-9/TIMP-1 ratio was more elevated in MS than in NIND ( pb0.02) and in MRI active than in MRI stable MS patients ( pb0.01). Our results suggests that an intrathecal synthesis of active form of MMP-9 could represent a sensitive marker of disease activity in MS in association with CSF and serum active MMP-9/TIMP-1 ratio.
Brain plaques from patients with multiple sclerosis (MS) of short duration contain oligoclonal populations of infiltrating T cells X. Zhang, W.L. Lin, C. Platsoucas and E. Oleszak Temple University, Philadelphia, USA
We have amplified by NPA-PCR and/or Valpha-or Vbeta-specific PCR alpha-and beta-chain TCR transcripts of T cells infiltrating acutely demyelinating brain lesions of four patients with chronic progressive MS who died within 2 to 8 years after clinical diagnosis. The amplified transcripts were cloned and sequenced. Sequence analysis revealed multiple identical copies of TCR transcripts, strongly suggesting the presence of clonally expanded populations of T cells. Limited numbers (1-2) of clonally expanded T-cell clones have been found in brain plaques from patients who died 2 years after diagnosis or from patients who died of acute or severe necrotizing MS 6 to 7 years after diagnosis. In contrast several alpha (Valpha9, Valpha22, Valpha23, and Valpha24)and beta-chain (Vbeta3, Vbeta13, Vbeta14 and Vbeta5) TCR transcripts were clonally expanded in brain plaques from a patient who died of chronic MS 8 years after diagnosis. These results demonstrate the presence of oligoclonal populations of T cells in brain plaques from these patients with chronic progressive MS, comprised of a few numbers of expanded T-cell clones. Multiple sclerosis is considered to predominantly affect the white matter of the human CNS. However, demyelination of grey matter structures is prominent in some MS cases. In the present study, we systematically analyzed cortical demyelinated plaques in a large sample of multiple sclerosis brains from different stages of the disease. Hemispheric and double hemispheric paraffin-embedded tissue sections were examined for cortical demyelination and offered the unique opportunity to evaluate disease involvement of large cortical areas. Demyelinated plaques-in both the cerebral and cerebellar cortex-were abundant in patients with primary or secondary progressive MS, but were virtually absent in patients with acute or relapsing-remitting MS. The extent of demyelination in the white matter was almost the same in the four groups. Additionally, in primary and secondary MS a characteristic pallor of the myelin of the normal appearing white matter was observed, which was associated with significant inflammation and also microglia and macrophage activation. These pathological changes were absent in acute and RRMS brains. Incidence and distribution of cortical lesions and diffuse white matter injury in multiple sclerosis may be pathological correlates of disease progression and suggest that fundamentally different pathological mechanisms may play a role in different stages of the disease.
In vivo dynamic neuropathology of brain new lesions in multiple sclerosis A. Barilaro a , F. Zellini a , F. Caleri a , A. Passeri a , C. Gasperini b , C. Pozzilli c and L. Massacesi a a University of Florence, Florence, Italy; b San Camillo Forlanini Hospital, Rome, Italy; c University of Rome b La SapienzaQ, Rome, Italy
The present study describes in relapsing-remitting multiple sclerosis (MS) patients, the natural history of new brain lesions visualized with Gd enhanced (Gd+) MRI scans by two different Gd dosages. Persistent lesions were evaluated monthly for 6 months and once more after 6 months; other persistent lesions were analyzed weekly for 6 weeks. In each lesion, two different enhancing volumes were observed according to the different Gd dosage administered. These volumes were smaller than the volume evaluated at the same time with the T2 w scan, but larger than the corresponding T2 outcome evaluated after N6 months. Therefore, during the acute phase, tissue damaging factor(s) are present mainly in the centre of lesions. Interpolating the volume kinetic of each lesion, with a multiparametric fitting suitable for representing diffusive expansion and contraction biological phenomena, random walk type curves were obtained. Noteworthy kinetic of the T2 volumes was the result of two different kinetics, in part overlapping. Correlation of the T2 outcome of each lesion with the corresponding enhancing lesion, was high provided that the zenith Gd+ volume was considered. This correlation was usually high also in each patient, and patient specific correlation coefficients were observed. These data indicate that brain MS lesions follow common biological rules. In addition, these data indicate that the T2 outcome correlates with the corresponding Gd+ lesion, better than previously reported. are an effort to simplify the diagnostic process of MS and to incorporate magnetic resonance imaging (MRI) into the diagnosis. Diagnosis continues to require two attacks separated in space and time but can utilize MRI to establish new MS activity. Cerebrospinal fluid (CSF) analysis and evoked potentials studies may still be employed to provide paraclinical evidence of the diseases. Materials and methods: We measured tau protein and beta-amyloid42 concentrations in CSF in patients with MS and control group of patients and the results were statistical analysed. Tau protein and beta-amyloid42 were measured by a double antibody sandwich ELISA (Innogenetics, Ghent, Belgium). Results: The outcomes of diagnostic process decided patients into two groups. The CSF tau and beta-amyloid42 concentrations of patients with MS were not different from controls. We did not find a correlation with age of patients and with duration of the disease. Conclusions: The concentrations of tau protein and beta-amyloid42 in two groups of patients with and without MS were not statistical different.
The role of immune mechanisms in multiple sclerosis (MS) patients with epilepsy D. Kountouris and K. Koutsobelis Neurological and Diagnostic Center, Athens, Greece Background: The aim of this study was to check any role of the immune mechanisms in MS patients with epilepsy. Method: Electroencephalograms (EEG) and sleep EEGs were performed in eight MS patients (two males and six females) with epilepsy of mean disease duration 8.4F3.6 years at different stages of the disease and in a period of 1 year's time. The partially pathological EEG findings and the immunological parameters were collected and assessed at the end of the study. The Kurtzky EPSS were also taken into consideration. Results: We found a significant increase of CD8 and lymphocytes in relation to the sleep EEG and EDSS findings pb0.05. We also noticed an analogy between the clinical symptoms and the EEG findings. Conclusion: The results showed that in MS patients with epilepsy, deterioration of the general clinical image and the EEG is due to occasional immunological disorders. Therefore, treatment should be accordingly directed.
Multiple sclerosis associated with other autoimmune diseases T. Petkovska-Boskova, V. Bojkovski and V. Daskalovska Clinical Centre, Skopje, R. Macedonia Background: Multiple sclerosis (MS) is commonly associated with other autoimmune diseases, such as: uveitis, thyreoiditis, Crohn's disease, etc. Case history: We present seven cases of definite MS. Three of them, in whom the disease has been ranged 5 to 7 years, developed uveitis, one patient with relapsing-remitting (R-R) MS, presented two attacks of erythema nodosum (EN), one of them developed Crohn's disease, 3 years after diagnosing MS, another one after 10-year duration of psoriasis developed MS. The last one presented coexistence of diabetes mellitus type 1, and R-R form of MS. The mean disability degree was 2.0 by the EDSS. Conclusion: Intermediate uveitis may belong to a constellation of HLA-DR15 related disorders which include MS. Although MS and chronic inflammatory bowel disease (IBD) may share common predisposing factors, unsufficient information is available to speculate about possible mechanisms. If an individual has a genetic susceptibility to infections, the downregulation of an inflammation does not occur in a proper way. This initiates the autoimmune process EN which is a self-increasing cycle. It was found that haplotype DRB1*0401 and DQB1*0302 is linked to type1 diabetes mellitus and that is increased among patients with the primary progressive subtype of MS. Whether patient's autoimmunity or genetics, associated MS with another disease, remains indeterminate, with a possibility that some environmental factor could bcomplicateQ this condition.
Antiphospholipid syndrome, livedo reticularis and multiple sclerosis? C. Dikova Medical University, Sofia, Bulgaria
In the last couple of years, patients with multiple sclerosis (MS) and antiphospholipid syndrome (APS) have been reported because of the presence of migraine, miscarriages, livedo reticularis (LR) and increased level of anticardiolipin antibodies (ACA). We present 10 women with livedo reticularis, diagnosed with multiple sclerosis. The purpose of this study is to find out clinical or laboratory data for APS. Five women (50%) have elevated IgM anticardiolipin antibodies (ACA), two have elevated IgG ACA, six have miscarriages, two are migraine sufferers, two have tension type of headache, three have had deep vein thrombosis, three have had occasionally high blood pressure. Astrocyte loss in Rasmussen's encephalitis J. Bauer a , C. Elgar b , T. Pietsch b , J. Schramm b , H. Urbach b , H. Lassmann a and C.G. Bien b a Brain Research Institute, Vienna, Austria; b University of Bonn, Bonn, Germany Rasmussen's encephalitis is an inflammatory, unihemispheric epileptic brain disorder. The current histopathological criteria include the presence of T-cell dominated inflammation, microglial activation and microglial nodules as well as neuronal loss and astrocyte activation. Anti-GluR3 antibodies have been implied to play a pathogenic role in RE. In mixed cell cultures, these antibodies could activate the glutamate receptor and were shown to destroy neurons and astrocytes. This prompted us to investigate astrocyte cell loss in situ in RE. Respective epilepsy surgery or diagnostic brain biopsies from 16 patients were studied for active degeneration (apoptosis) and loss of GFAP+ and S100+ astrocytes by fluorescence confocal and light microscopy. Astrocyte apoptosis was found both in cortical areas, inseparable from neuronal loss, as well as in white matter areas, resulting in astrocyte deficient lesions. Like other parenchymal cells (neurons and microglial cells), astrocytes in these tissues revealed MHC class I expression. Furthermore, Granzyme-B positive lymphocytes were found in close apposition to astrocytes on the border of astrocyte-deficient lesions. Granzyme-B positive granules in these lymphocytes were polarised and facing the astrocytic membrane. We therefore suggest a specific attack by cytotoxic T lymphocytes as a possible mechanism responsible for astrocyte degeneration. The loss of astrocytes, important in homeostatic regulation of glutamate and other neuronal transmitters, in addition to other cytotoxic mechanisms, might play a role in neuronal dysfunction, seizure induction and enhancement of neuronal cell death. Rasmussen's encephalitis (RE) is a severe epileptic disorder that may respond transiently to immunotherapies suggesting a possible autoimmune aetiology. Antibodies to glutamate receptor type 3 (GluR3) have been reported, but in a recent comprehensive study we failed to confirm the presence of GluR3 antibodies in sera from RE and intractable epilepsy (IE) patients (Watson et al., in press ). Here we looked for antibodies to neuronal alpha 7 acetylcholine receptor (alpha7 nAChR). Sera from patients with RE (n=30), IE (49) and healthy individuals (23) were tested by a range of techniques for antibodies to the alpha7 nAChR. Two sera from patients with RE blocked the agonist-induced currents of alpha7 nAChRs expressed in Xenopus oocytes. Additionally, these sera, and their derived IgGs, inhibited the agonist-induced calcium ion influx into an alpha7 nAChR-transfected human endothelial cell line. To confirm that the antibodies were directed against the alpha7 nAChR, we used immunoprecipitation of 125I-alphabungarotoxin-labelled detergent extracts of these cells. Anti-alpha7 nAChR antibodies were present during active disease, but were negative later in the disease process when the patients were in bburn-outQ. Our results demonstrate for the first time alpha7 nAChR antibodies in RE. These antibodies inhibit nAChR channel function and have the potential to be pathogenic. Ongoing studies are looking for these antibodies in patients with different forms of severe epilepsy, and testing their pathogenicity in vivo.
HLA studies indicate immunogenetic susceptibility to adult Rasmussen's I.K. Hart a and J. Darroch b a University of Liverpool, Liverpool, UK; b Royal Liverpool University Hospital, Liverpool, UK Rasmussen's syndrome (RS) causes severe, drug resistant epilepsy and neurologic and cognitive deficits. It can be present in childhood or adulthood. Autoimmunity is implicated in its pathogenesis, although the crucial mechanisms are not clear. Objective: To study HLA associations in adult RS. Methods: We performed DNA based HLA typing on six RS adults. All had responded to immunomodulatory therapy. Results: Compared with control data-the allele prevalencies in the general UK population-our patients had increased frequencies of HLA A2 (100% versus 24.1%,), HLA B44 (66.7% versus 10.3%), HLA DR4 (83.3% versus 12.8%), and HLA DQ8 (66.7% versus 5%). While only 2.2% of the general population are HLA A2, B44, DR4 positive, 66.7% of our patients had this haplotype. Conclusions: These findings provide further evidence, albeit indirect, that autoimmunity contributes to the triggering or maintenance of adult RS and that the immune dysregulation is genetically dependent. Further studies will establish whether HLA typing can help identify those adults presenting with focal epilepsy who are at risk of progressing to severe chronic focal autoimmune encephalitis and who may benefit from immune therapy.
Morphological and functional analysis of the effect of GAD-antibody positive sera on rat hippocampal neurons in culture M. Vianello a , C. Mucignat a , S. Vassanelli a , K. Fountzoulas a and B. Giometto b a Department of Human Anatomy and Physiology, University of Padova, Padova, Italy; b Department of Neurology, Hospital of Treviso, Treviso, Italy
In addition to Stiff-Person Syndrome (SPS) and Ataxia, GAD-antibodies (Ab) were detected in Drug-resistant Epilepsy (EP). We tested reactivity of sera on cultured rat hippocampal neurons and recorded post-synaptic inhibitory potentials (IPPS) in these cells to assess the pathogenetic role of these autoantibodies. We tested sera from GAD-Ab positive patients with EP on cultured neurons using an immunohistochemical (IHC) method. As control we tested sera from GAD-Ab positive patients with SPS, Ataxia and diabetes and GAD-Ab negative epileptic individuals. Sera from GAD-Ab positive epileptic and diabetic patients and normal controls were selected and IPPS recorded on cultured hippocampal neurons before and after application of diluted sera, in a patch clamp study. On IHC, a specific reactivity on the soma of neurons was detected when we applied sera from GAD-Ab epileptic patients. Different staining was obtained with sera from SPS and ataxic patients. No specific labelling was found in patients with GAD-Ab negative epilepsy and diabetic controls. A significant reduction in amplitude and an increase in frequency of IPPS were observed after application of serum from the positive epileptic case, while no effect was noted using controls. The observed morphological and functional effect on hippocampal neurons may suggest a pathogenetic role of GAD-Ab in the development of the disease.
Cytotoxic T cells in the pathogenesis of paraneoplastic encephalitis, nonparaneoplastic limbic encephalitis and Rasmussen's encephalitis C.G. Bien a , H. Lassmann b , C.E. Elger a and J. Bauer b a University of Bonn, Bonn, Germany; b Medical University of Vienna, Vienna, Austria
Recently, the immunohistochemical investigation of the immunopathology of Rasmussen's encephalitis (RE) has provided evidence for a cytotoxic T cell reaction against neurons. In the present study, we sought to determine if the composition of inflammatory cells in patients with paraneoplastic encephalitis (PE) and nonparaneoplastic limbic encephalitis (NPLE), two other chronic encephalitic conditions that have been suggested to be T cell mediated disorders, are similar to that in RE. We studied paraffin-embedded brain specimens from 8 PE, 5 NPLE and 21 RE cases. Specimens were stained immunohistochemically with antibodies against CD3, CD8, Granzyme-B, CD20, CD138, immunoglobulins, C9neo, CD68, Fas, and MHC class I. Quantitative evaluation of cell densities were performed and statistically analyzed using nonparametric and parametric tests as appropriate. The ratios of CD8+/CD3+ and GrB+/CD3+ ratios and the frequency of Fas+ cells did not differ between the three diseases. MHC class I+ neurons, astrocytes and oligodendrocytes were observed at similar proportions. The PE specimens however contained significantly higher densities of T cells, B cells, plasma cells and macrophages compared to NPLE and RE. We conclude that T cell cytotoxicity forms a bbackboneQ of these conditions. The higher inflammatory activity and broader spectrum of inflammatory cells in PE may be an indicator of inflammatory mechanisms beyond T cell cytotoxicity. This work was supported by a grant from the Austrian science foundation FWF (P16063-B02). Since the first description in 1999, there have been reports on 12 patients with anti-Ma (anti-PNMA1 and PNMA2) and 27 patients with anti-Ta reactivities (anti-PNMA2 only). Here we update the clinical information on these two reactivities with 9 additional anti-Ma and 10 additional anti-Ta positive patients whom we have identified between 12/99 and 04/04. Sera were tested using Western-Blot analysis with semi-purified recombinant PNMA1 and PNMA2 protein, and the clinical data collected retrospectively. The nine anti-Ma positive patients (6/9 female, mean age 62) presented with brainstem/cerebellar (6/9), limbic (5/9), extrapyramidal symptoms (2/9) and polyneuropathy (2/9). In 6/9 tumors were identified (each twice: renal rhabdosarcoma, breast, lung). In the patient with lung tumor, an overlapping anti-Hu antibody was detected. The 10 anti-Tapositive patients (9/10 male, mean age 50) presented with limbic syndrome (7/10, one with obsessive-compulsive symptoms), brainstem/cerebellar symptoms (4/9), polyneuropathy (1/9) and with signs of motor neuron involvement (1/9). In 8/10 tumors were identified including germ cell tumors (5/10, one extragonadal, one at orchiectomy, one at autopsy), and one each of melanoma, lymphoma and lung-carcinoma. One patient has a past history of seminoma without known relapse. Another recognized a suspicious testicular swelling which disappeared spontaneously before biopsy. Anti-Ma and anti-Ta reactivities prove to be of high clinical relevance in patients presenting with subacute brainstem or limbic symptoms. If present, a thorough tumor search is warranted.
Proteasome antibodies in paraneoplastic cerebellar degeneration A. Storstein, A. Knudsen and C.A. Vedeler Haukeland University Hospital, Bergen, Norway Paraneoplastic neurological syndromes (PNS) are often associated with specific onconeural antibodies, but the pathogenic importance of the humoral immune response is unclear. Activated specific T lymphocytes in paraneoplastic cerebellar degeneration (PCD) and in paraneoplastic encephalomyelitis/sensory neuropathy (PEM/SN) suggests that cellular immune responses participate in the pathogenesis. In this study, we detected proteasome antibodies in sera from 7 of 10 patients with PCD by Western blot, but in only 1 of 8 sera from patients with PEM/SN. Proteasome antibodies were not found in any control serum (10 blood donor sera, a serum pool of 100 blood donors and 20 patients with cancer and no PNS). By immunohistochemistry, we found that serum antibodies from 8 of the 10 PCD patients stained cytospins of various cancer cell lines, and the staining co-localised with the proteasome-associated promyelocytic protein. The PCD sera also stained living cancer cells, but did not affect the cell viability. Proteasome antibodies were not detected in the cerebrospinal fluid from one PCD and three PEM/SN patients. The proteasome is important in nonlysosomal protein degradation and in antigen processing and presentation by major histocompatibility complex class I molecules. The proteasome antibodies in PCD may be of pathogenic importance by regulating the cellular metabolism and the immune response. Conformational epitopes can prevent the recognition of target antigens of autoantibodies in paraneoplastic syndromes. As such, the target antigen of anti-TR antibodies-characterizing patients with paraneoplastic cerebellar degeneration (PCD) and Hodgkin's disease (HD)-is still elusive owing to the failure of its identification using conventional proteomics approaches. We applied new procedures, which are based on direct tissue isoelectric focusing and gradient electrophoresis in non-denaturing polyacrylamide gel, to test the hypothesis that anti-TR antibodies recognize a conformational epitope. We studied sera of two patients with PCD and HD, using rat and human cerebellum as the source of antigens. Both the sera recognized a sharp band of approximately 39 kDa both in rat and human tissues. With mass spectrometry (MALDI-TOF), we identified a cytoplasmic protein of a corresponding mass that was the same in rats and humans. This protein is a candidate ligand for anti-TR antibodies.
CRMP3 antibodies associated with limbic encephalitis and thymoma A. Knudsen a , G. Bredholt a , A. Storstein a , L. Oltedal b , S. Davanger b and C.A. Vedeler a a Haukeland University Hospital, Bergen, Norway; b University of Bergen, Bergen, Norway Introduction: Paraneoplastic neurological syndromes arise as remote effects of several different tumors. Thymoma can be associated with various antibodies such as anti-AChR, anti-titin, anti-RyR, anti-VGKC and anti-CRMP5. Methods: A 52-year-old patient developed limbic encephalitis with focal epileptic seizures. Screening for occult cancer revealed a thymoma. Paraneoplastic antibodies related to limbic encephalitis such as anti-Hu, anti-amphiphysin, anti-Ma2, anti-CRMP5 and anti-Ri and those related to thymoma were negative. The patient serum was tested on tissue sections of rat brain and on primary cultured neurons of the hippocampus and used to screen a cDNA expression library of rat cerebellum. A rabbit polyclonal antibody against CRMP1-4 was used to stain the hippocampus. Results: The patient serum stained cellular regions of the hippocampus, synaptic boutons and nuclei of cells in the dentate gyrus and similar staining was observed on the pyramidal cells i culture. The CRMP1-4 antibody also stained the hippocampus. Western blot of rat brain extract incubated with the patient serum revealed two bands of approximately 40 and 60 kDa. Screening of the cDNA library identified two different clones, one coding for CRMP3, and the other for nuclear protein XAP-5. Discussion: CRMP3 antibodies were associated with thymoma and limbic encephalitis. CRMP3 is present in the hippocampus and may be relevant for the pathogenesis of limbic encephalitis. Seizures involve complex neurophysiological and neurochemical events that are involved in the detrimental consequences and reoccurrence of seizures, and the establishment of chronic epilepsy. These events include neuroinflammation, which manifests itself quickly following seizures. Although inflammation in the brain following seizures has been well characterized, its role, which could be adaptive or detrimental, is still under debate. In order to clarify this problem, we treated mice with corticosterone (a potent suppressor of inflammation) and characterized its effect on seizures and their associated neurological and neuroimmune consequences (in the well-established model of pilocarpine administration in mice) using in situ hybridisation, immunocytochemisty and various markers of cell death and damage. We found that even if corticosterone was able to suppress neuroinflammation associated with seizures, these seizures and their associated neuronal cell death still occurred. However, suppressing the immune system spared a specific population of neurons in the hippocampus, and the expression of NPY in this region (believed to be an indices of mossy fiber sprouting and neuroprotective) was greatly affected. Thus we conclude that neuroinflammation could play a major role in the reorganization that takes place in key regions following seizures and this could have implications in possible immune interventions to prevent the detrimental events that follow seizures and lead to epilepsy.
Antiepileptogenic and anticonvulsant activity of interleukin-1B in amygdala-kindled rats M. Sayyah a and S. Beheshti b a Institute Pasteur of Iran, Tehran, Iran; b Tehran University, Tehran, Iran Ischaemic, excitotoxic and traumatic brain injuries have been associated with the occurrence of epileptic seizures. Microglia, the principal immune cells in the brain, produce a variety of proinflammatory and cytotoxic factors especially interleukin-1 (IL-1) early after an acute insult. We studied the effect of IL-1h (1 pg-10 ng/rat, i.c.v.) on seizure acquisition and on fully kindled seizures in amygdala kindling model of epilepsy. IL-1h retarded acquisition of kindled behavioral seizures and growth of afterdischarges (AD). IL-1h also exhibited significant anticonvulsant effect on established kindled seizures and AD duration. Pretreatment of the kindled animals with nitric oxide synthase inhibitor, N G -nitro-l-arginine methyl ester, or cyclooxygenase inhibitor, piroxicam, completely reversed the anticonvulsant effect of IL-1h. Although most of the previous studies indicate a proconvulsant or convulsant property of IL-1, our results support a protective and antiepileptogenic role of IL-1h. Seizures are common sequel to brain insults in cases such as stroke, trauma and infection where there is a certain neuroinflammation. Intracerebroventricular (i.c.v.) administration of lipopolysaccharide (LPS) induces an inflammatory state in brain that is used as a model of neuroinflammation. We studied the effect of LPS (0.25 and 2.5 Ag/rat, i.c.v.) on development of electrical kindling of the amygdala and on fully kindled seizures. LPS, at the doses used, had no effect on fully kindled seizures and afterdischarge (AD) duration at 0.5, 2 or 4 h after administration. However, daily injection of LPS (2.5 Ag/rat) retarded acquisition of kindled behavioral seizures. This antiepileptogenic effect could be due to the release of inflammatory mediators from microglia and the related morphological and functional changes in synaptic neurotransmission. Increased serum levels of interleukin-8 in patients with epilepsy L. Bastone, A. Bagalà, T. Ferraro, M. Casaletto, N. Romeo and L. Crescibene Institute of Neurological Sciences, CNR, Mangone (CS), Italy
There are some evidences that inflammatory processes may be involved in clinical and neuropathological manifestations of epilepsy. Proinflammatory cytokines (such as IL-1, IL-6, TNF-alpha) are known to have indirect or direct effects on neurons and neurotoxic neurotransmitters released during excitation or inflammation. Studies of patients with epilepsy and animals with experimental induced seizures indicate that cytokines may influence the electrophisiological properties of neurons. In adult rats, status epilepticus induces cytokine production by glia especially when seizures are associated with neuronal injury. This suggests that cytokines may play a role in seizures-induced neuronal damage. Interleukin-8 is a chemokine produced by monocytes, endothelial cells and other cells that acts as a chemotactic and activator for neutrophilis. Its effects are manly proinflammatory. We investigated the levels of interleukin-8 as proinflammatory cytokines in serum of normal and epileptic patients by using an enzymelinked immunosorbent assay. We found elevated concentrations of interleukin-8 in serum of epileptic patients. Our data support the hypothesis that cytokines groups is activated. Whether these elevated cytokines levels have some role in the pathogenesis of epilepsy must be investigated further. Experimental studies have demonstrated that epileptic seizures are followed by increased production of cytokines. In human patients, increased levels of IL-6 and IL-1ra have been reported in CSF and plasma after seizures. However, whether this increase is caused by seizures per se or some other underlying disease causing seizures is controversial. Electroconvulsive seizures (ECS) are commonly used experimental approach in antiepileptic drug development. ECS is also employed in treatment of drug refractory depression. In the present study, we determined the plasma levels of cytokines IL-1h, IL-1ra and IL-6 before and at several time points after administration of ECS. The levels of IL-1h were significantly increased at 1-3 h time points ( pb0.05) and returned to basal levels by 24 h. The levels of IL-1ra were unchanged. The levels of IL-6 were increased significantly at 3-6 h time points ( pb0.05) and returned to basal levels by 24 h. Our results clearly demonstrate that ECS are followed by transient increase in systemic levels of pro-inflammatory cytokines. This finding provides further evidence about seizure induced production of cytokines in CNS. In addition, activation of cytokine cascade after ECS may have importance in the therapeutic effect of ECS in major depression.
Antinuclear, cytoskeletal, antineuronal antibodies and C-reactive protein levels in the sera of children with tic disorders and obsessive compulsive disorders I. Gorker, G. Akman-Demir, N. Gqrel Polat, R. Eker Ö meroglu, S. Icoz, P. Serdaroglu and U. Tuzun Istanbul Medical Faculty, Istanbul, Turkey 650 Immunohistochemical analysis of serum antibodies to central nervous system antigens in antiphospolipid syndrome E. Aykutlu a , E. Tqzqn b , B. Baykan a , N. Polat-Gqrel a , S. Icoz a , P. Christadoss b , C. Gqrses a , M. Inanc a , G. Akman-Demir a and A. Gfkyigit a a Istanbul Medical Faculty, Istanbul, Turkey; b University of Texas Medical Branch, Galveston, TX, USA In order to detect antibodies against central nervous system (CNS) antigens in 12 patients with seizures and primary or secondary antiphospholipid (aPL) antibody syndrome, serum samples were tested by immunohistochemistry. Four out of 12 cases (33.3%) with aPL syndrome and seizures showed binding to neurons. The binding was predominantly nuclear in two cases with strong staining and cytoplasmic in two cases with moderate staining. The strength of immunohistochemical staining was correlated with both the age of the patients and the duration of their seizures. Patients with strong nuclear staining also had SLE. Three patients had partial epilepsy, whereas one patient had generalized epilepsy. In one of the patients with strong staining, seizures were a sequel of aPL syndrome related-cerebral infarct, whereas in the remaining three cases seizures were not related to any focal cerebral lesion. In aPL syndrome patients, serum antibodies against CNS might have emerged due to certain antiepileptic drugs, disruption of blood-brain barrier by stroke or in certain cases, they might be playing a causal role. Strong nuclear staining might simply be related to the anti-nuclear antibodies frequently detected in aPL syndrome or SLE patients, whereas cytoplasmic staining might be indicating the presence of CNS-specific antibodies developing either as an epiphenomenon during the course of disease or as a contributing factor to epilepsy.
Autoantibodies to central nervous system in cases with Lafora disease-late onset variant E. Aykutlu a , E. Tqzqn b , G. Akman-Demir a , N. Bebek a , S. Icfz a , P. Christadoss b and B. Baykan a a Istanbul Medical Faculty, Istanbul, Turkey; b University of Texas Medical Branch, Galveston, TX, USA Sera from 4 siblings (aged 24-31 years) with Lafora disease (LD) late onset variant, diagnosed by axillary skin biopsy, who also had high titers of anti-gliadin IgG antibodies were tested by immunohistochemistry for antibodies capable of binding to mouse central nervous system (CNS) antigens. Serum samples of three out of four patients with LD showed binding to the neurons including cerebellar Purkinje cells and cortical neurons. No staining was observed beyond 1/800 dilution or with control serum samples. The binding was predominantly nuclear in all three cases. Notably, these patients also had high titers of anti-gliadin IgG antibodies, although coeliac disease could not be demonstrated by intestinal biopsy. All four patients were treated with valproate, clonazepam and lamotrigine. The strength of immunohistochemical staining was correlated with neither the severity nor the length of the PME syndrome. Since one patient from the same family with a similar clinical condition did not reveal detectable amounts of antibodies against CNS, it is more likely that these autoantibodies emerged as a by-product of the ongoing disease rather than being primary or causal factors. Coexistence of LD and coeliac disease have previously been reported and our results are providing further support that these two diseases might be associated. Introduction: CRMP5 is an onconeural antibody that is associated with small cell lung cancer (SCLC) and thymoma. The prevalence of CRMP5 antibodies in patients with such tumours, as well as the correlation with the prognosis is largely unknown. Patients and Methods: Sera from 200 patients with SCLC, 83 patients with thymoma and myasthenia gravis and from 200 healthy blood donors were examined for CRMP5 antibodies by an in vitro transcription-translation (ITT) based immunoprecipitation assay. Positive sera were also examined by Western blot using CNS extract and recombinant CRMP5, and by a commercial dot blot kit. Results: CRMP5 antibodies were found in 14/200 (7.5%) of the SCLC, 9/83 (10.8%) of the thymoma and in none of the healthy control sera by the ITT immunoprecipitation assay. High CRMP5 index (N500) was found in seven of the thymoma and four of the SCLC patients. Most of the high-index sera were also positive by Western blot and dot blot. Conclusion: CRMP5 antibodies were detected in approximately 8% of the SCLC and 11% of the thymoma patients by the ITT immunoprecipitation assay. This assay was more sensitive than Western blot and dot blot. Background: Presence of anti-neuronal autoantibodies (Abs) has been well described in association with late onset cerebellar ataxia (LOCA) as a paraneoplastic manifestation. They are identified in the presence of certain cancers but may also rarely be seen in the absence of neoplasia. However, the frequency of these Abs and relevance in wider LOCA population remains unclear. We have explored their significance in a prevalent, population-based sample of patients with idiopathic LOCA (ILOCA) in South Wales. Methods and materials: Sera of 84 patients (mean disease duration: 7.8 years; range: 1 to 35) were analysed using indirect immunohistochemistry and/or Western blot (IHC/WB) for anti-Hu, Ma, Ri, Tr and Yo Abs, and using immunoprecipitation (IP) for P/Qtype voltage-gated calcium channel (VGCC) and voltage-gated potassium channel (VGKC). Results of each assay were compared with known negative, normal and positive controls. Results: On IHC/WB, 11/84 sera showed staining patterns initially suggestive of anti-Yo (n=4), Hu (n=3), Ma (n=3) and Tr (n=1), but were excluded on WB. In further 13/84 sera, these revealed batypicalQ staining patterns. On IP, none was positive for anti-VGCC and anti-VGKC Abs. Discussion: No ILOCA patients were positive for the anti-neuronal Abs screened (anti-Hu, Ma, Ri, Tr, Yo, VGCC and VGKC). However, 13/84 (15%) showed atypical staining patterns of unknown significance indicating the possibility of an undetermined immunological pathology in a significant proportion of patients. Neuroprotection in multiple sclerosis R. Diem, M. Hobom and M. B7hr Department of Neurology, University of Gfttingen, Gfttingen, Germany Long-term disability in multiple sclerosis (MS) patients is primarily caused by axonal and neuronal lesions, but up to date no neuro-or axono-protective therapy is available. We previously demonstrated that neuronal apoptosis occurs early during myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). High-dosage methylprednisolone treatment has been established as the standard therapy of acute inflammation of the optic nerve (ON). To investigate the influence of methylprednisolone therapy on the survival of retinal ganglion cells (RGCs), whose axons form the ON, we used (MOG)-induced EAE. ON involvement was assessed by recording visual evoked potentials (VEPs), and RGC function was monitored by measuring electroretinograms (ERGs). Methylprednisolone treatment significantly increased RGC apoptosis during MOG-EAE and did not lead to a recovery of ERGs or VEPs. The underlying molecular mechanism of RGC death involved suppression of mitogen-activated protein kinase phosphorylation, which was calcium-dependent. Hence we provide evidence for negative effects of steroid treatment on neuronal survival. Thus, besides immunosuppressive strategies, neuroprotective approaches should be designed. To that end, we could show that systemic treatment with erythropoietin of rats with MOG-EAE including severe optic neuritis reduced the clinical severity of the disease. Moreover, survival and function of RGCs, as documented by histology and ERG measurements were significantly improved.
Neurodegeneration in multiple sclerosis: high vulnerability of neurons to Th1 but not Th2 lymphocytes F. Giuliani a , A. Bar-Or b and V.W. Yong a a University of Calgary, Calgary, Canada; b McGill University, Montreal, Canada MS is considered a T cell-mediated disease of the CNS and is characterized by inflammation, demyelination, and neuronal degeneration. The current objective is to determine whether Th1 and Th2 polarized T cells have different capacity for neurodegeneration in vitro and in EAE. We used a coculture system of human leukocytes and human fetal neurons. T cells were activated in an antigen-specific manner and polarized into lines of Th1 or Th2 bias. Following a period of co-culture, neurons were stained with anti-MAP-2 antibody and counted to assess cytotoxicity. Antigen-specific mixed Th1/Th2 populations induced 50% of neuronal death, whereas the Th1 polarized population induced 90% of death within 24 h. Th2 polarized cells were unable to kill neurons. EAE was further induced in C57/BL6 mice that were clinically scored on a daily basis and then sacrificed for histological analysis. Co-localization of inflammation and axonal loss showed a significant correlation in the spinal cord of EAE animals. Overall, these data demonstrate that Th1 cells are toxic for human neurons while Th2 lines are not, and that axonal loss occurs in vivo in EAE in areas of T cell inflammation. We suggest that when T cells are appropriately activated, they can traffic into the CNS and induce neuronal death. The shift of T cells towards a Th2 phenotype decreases their potential toxicity.
Direct impact of T cells on neurons as potential damage mechanism in neuroinflammatory disorders O. Aktas a , E. Pohl b , A. Smorodchenko a , C. Infante-Duarte a , R. Nitsch b and F. Zipp a a Institute of Neuroimmunology, Humboldt University Medical School Charité, Berlin, Germany; b Institute of Anatomy, Humboldt University Medical School Charité, Berlin, Germany
The precise mechanisms of neuronal damage in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE) are poorly understood. Here we applied two photon laser-scanning microscopy to track the direct interactions of proteolipid protein (PLP)specific T cells within the complex cellular network of living brain tissue. CD4+CD8-CD25+CD69+CD44+ Th-1-like T cell lines releasing high amounts of IFN-gamma and TNF-alpha (as used for induction of EAE) were labeled with red fluorescent dye CMTMR and applied on living brain slices, which had been incubated with the calcium-sensitive dye Fluo-4. PLP-specific T cells directly contacted neurons in which they induced calcium oscillations finally resulting in a lethal increase in neuronal calcium levels. Prevention of the calcium overload was achieved by blockade of perforin-mediated cytotoxicity of the T cells by concanamycin-A. Wash-in experiments, in which the glutamatergic neuronal connectivity in the hippocampus was inhibited using blockers of the different glutamate receptors, further reversed the T cell-induced calcium oscillations. The effects were independent of antigen presentation since OVA-specific T cells were also able to induce an increase in neuronal calcium. In contrast, unstimulated T cells did not elicit this effect. Thus, our data provide direct insight into the detrimental impact of T cells on neurons and shows that stimulation of T cells independent of their target antigen enables them to induce collateral damage in neurons via direct cell-cell contact within the brain parenchyma. Complement activation is involved in the initiation of antibody-mediated inflammatory demyelination in experimental autoimmune encephalomyelitis (EAE). At a sublytic dose, the C5b-9 membrane attack complex (MAC) protects oligodendrocytes from apoptosis. Using C5-deficient mice (C5-d), we previously showed a dual role for C5: enhancement of inflammatory demyelination in acute EAE, and promotion of remyelination during recovery. In this study, we investigated the role of C5 in apoptosis in myelin-induced EAE. In acute EAE, C5-d and C5-sufficient (C5-s) mice had similar numbers of total apoptotic cells, whereas C5-s had significantly fewer than C5-d during recovery. In addition, while both groups of mice displayed TUNEL+ oligodendrocytes, there were significantly fewer in C5-s than in C5-d during both acute EAE and recovery. Gene array and immunostaining of apoptosis-related genes showed that FasL expression was higher in C5-s. In C5-s mice, Fas+ cells were also higher than in C5-d mice in acute EAE; however, these cells were significantly reduced during recovery. Together, these findings are consistent with the role of C5, possibly by forming the MAC, in limiting oligodendrocyte apoptosis in EAE, thus promoting remyelination during recovery.
The JNK activation is involved in TRAIL-induced death of adult human oligodendrocytes M. Matysiak, A. Jurewicz, S. Andrzejak and K. Selmaj Medical University of Lodz, Lodz, Poland TRAIL has been reported to induce apoptosis in tumour cell lines and in normal cells. TRAIL was demonstrated on autoreactive T cells and may be involved in immune mediated apoptosis of brain target cells. Susceptibility or resistance to TRAIL-induced death is related to differences in TRAIL receptors expression or/and differences in involvement of intracellular apoptotic molecules. To assess death of adult human oligodendrocytes (ahOl) after TRAIL stimulation we used Annexin and PI staining. The JNK activation after TRAIL stimulation was assessed by using antibodies against phosphorylated form of kinase in Western blot. The c-jun phosphorylation in cell lysates was assessed by autoradiography. Caspase pathway activation was analysed by Western blotting and using general caspase inhibitor. The role of JNK activation in oligodendrocytes death was determined by inhibition of JNK using dominant-negative mutant of MKK-4/SEK. TRAIL induces ahOl death, but only in condition on protein synthesis inhibition or pre-treatment with IFNgamma. Microglia cells are resistant to TRAIL mediated cytotoxicity. We did not detect caspase pathway activation in ahOL after TRAIL stimulation and caspase inhibitor did not prevent ahOl from death. We have observed persistent activation of JNK in oligodendrocytes, but not in microglia cells, after TRAIL stimulation. Inhibition of JNK activity protects oligodendrocytes from TRAIL-induced death. These results may implicate JNK pathway involvement in TRAIL-induced selective oligodendrocytes demise in inflammatory/demyelinating conditions. An inappropriate cross-talk between activated T lymphocytes infiltrating the central nervous system (CNS) and neural cells can sustain the onset and progression of demyelination and axonal degeneration in neuroinflammatory diseases. To mimic this deleterious crosstalk, we designed an experimental paradigm consisting of transient cocultures of T lymphocytes chronically activated by retrovirus infection (not virus productive) with human multipotent neural precursors or primary oligodendrocytes from rat brain. We showed that activated T lymphocytes induced apoptotic death of multipotent neural progenitors and immature oligodendrocytes after a progressive collapse of their process extensions. These effects were reminiscent of those induced by brain semaphorin on neural cells. Blockade by specific antibodies of soluble Sema-4D/CD100 released by activated T cells, or treatment with recombinant sSema4D/CD100, demonstrated that this immune semaphorin has the ability to collapse oligodendrocyte process extensions and to trigger neural cell apoptosis, likely through receptors of the plexin family. The specific presence of sSema4D/CD100 in the CSF and of Sema4D/CD100 expressing-T lymphocytes in the spinal cord of patients suffering with neuroinflammatory demyelination pointed out to the potential pathological effect of sSema4D/CD100 in the CNS. Thus, our results show that Sema4D/CD100 is a new important element in the deleterious T cellneural cell crosstalk during neuroinflammation and suggest its role in demyelination or absence of remyelination in neuroinflammatory diseases including multiple sclerosis and HTLV-1-associated myelopathy.
Proteinase-activated receptor-2 expression is neuroprotective in lentivirus-induced neuroinflammation F. Noorbakhsh a , N. Vergnolle a , M. Vodjgani b , M. Hollenberg a and C. Power a a University of Calgary, Calgary, Canada; b Tehran University of Medical Sciences, Tehran, Iran Proteinase-activated receptors (PARs) are a family of G-protein coupled receptors that are activated by the proteolytic cleavage of their aminoterminus. Previous studies from our group have shown a pathogenic role for PAR-1 in lentivirus-induced neuroinflammation. To explore further the role of these receptors we analysed the role of PAR-2 in the context of lentivirus (human immunodeficiency virus-HIV, and feline immunodeficiency virus-FIV)-induced neuroinflammation. PAR-2 mRNA and protein levels were increased in both HIV-and FIV-infected brains, with principally neuronal localization ( Pb0.05), as analysed by real-time RT-PCR and immunocytochemistry. Treatment of human and mouse neuronal cell lines with recombinant IL-1beta or TNF-alpha increased PAR-2 mRNA and protein levels ( Pb0.05). Overexpression of PAR-2 in neuronal cells decreased neuronal cell death induced by exposure to HIV Tat-induced neurotoxins ( Pb0.05). Intracerebral implantation of PAR-2 activating peptide (SLIGRL) decreased the neurobehavioral effects of HIV Tat. Indeed, PAR-2 null animals exhibited more prominent astrocytosis and neuronal loss, together with greater neurobehavioral effects than wild-type littermates, following the implantation of HIV Tat-induced neurotoxins. These studies indicate that PAR-2 exerts a neuroprotective effect against lentivirus-induced brain disease that is augmented by pro-inflammatory cytokines. MoMuLV-ts1-mediated neuronal death in mice is likely due both to loss of glial support and to release of cytokines and neurotoxins from ts1-infected glial cells. We investigated whether ER stress signaling is involved in ts1mediated neuronal loss in the brain of infected mice. ts1-infected brainstems were found to show significant increases in phosphorylation of the double-stranded RNA-dependent protein kinase-like ER kinase and eukaryotic initiation factor 2-a. In addition, increased expression of growth arrest DNA damage 153 (GADD153), glucose-regulated protein 78, and caspase-12 were accompanied by increases in processing of caspase-12 and its downstream target, caspase-3. All of these events are markers of ER stress. We observed that GADD153 and cleaved caspase-3 were present in degenerative neurons in the lesions of infected mice, but not in uninfected controls. Phosphorylated calmodulin-dependent protein kinase II-a was significantly increased, and was co-expressed with GADD153 in a large proportion of neurons undergoing early and advanced degenerative changes. Finally, neuronal degeneration in spongiform lesions was associated with increase in calcium (Ca2+) accumulation in mitochondria. Together these results suggest that ts1 infection-mediated neuronal degeneration in mice may result from activation of ER stress signaling pathways, presumably initiated by perturbation of Ca2+ homeostasis. Our findings highlight the importance of the ER stress signaling pathway in ts1 infection-induced neuronal degeneration and death.
The radical scavenger edaravone prevents oxidative neurotoxicity induced by peroxynitrite and activated microglia M. Banno, T. Mizuno, G. Zhang, J. Kawanokuchi, R. Kuno, S. Jin, H. Kato, H. Takeuchi and A. Suzumura University of Nagoya, Nagoya, Japan
The free radical scavenger edaravone has been used as an anti-oxidative agent in acute ischemic brain disorders. We examined the effect of edaravone on the production of nitric oxide (NO), reactive oxygen species (ROS) and proinflammatory cytokines by activated microglia, and we also examined its neuroprotective role in cortical neuronal cultures oxidatively stressed by the peroxynitrite donor N-morpholinosydoniamine (SIN-1) or activated microglia. Edaravone significantly suppressed the production of NO and ROS by activated microglia, though it did not suppress production of inflammatory cytokines. In addition, edaravone significantly suppressed neuronal cell death and dendrotoxicity induced by either SIN-1 or activated microglia in a dose dependent manner. These results suggest that edaravone may be a useful neuroprotective agent counteracting oxidative neurotoxicity arising from activated microglia, as occurs in either inflammatory or neurodegenerative disorders of the central nervous system.
Effects of nitric oxide on adult neural stem cells R. Covacu, M. Aguilar-Santelises, T. Olsson and L. Brundin Karolinska Institutet, Stockholm, Sweden Aim: To determine the long and short time effects of nitric oxide on adult neural stem cells. Background: Nitric oxide, NO, is produced in high concentrations in the CNS of multiple sclerosis patients, where it contributes to the tissue damage seen in MS. Methods: Adult neural stem cells (rat) were exposed to Deta-NONO:ate 0.01-2.5 mM for 2, 8 and 24 h. NO release was calibrated using a NO sensitive electrode. The NO-effects on cell viability and differentiation were assessed after 2, 8 and 24 h. Necrosis and apoptosis was studied using propidium Iodide/Annexin V (flow cytometry) and JC-1 staining which also was assessed by immunocytochemistry. The effects of NO on differentiation were assessed by immunolabeling the cells for neuronal (Tuj), oligodendrocyte-(O4) and astrocyte-(GFAP) lineage markers. Results: Nitric oxide administered to stem cell cultures had dose dependent effects on survival and differentiation of the cells. There was dose dependent damage to the cells as determined by flow cytometry for annexin V/propidiumiodide and JC-1. The levels of NO released from the donor above 1 mM is comparable to the levels found in patients. Above 1 mM, the capacity of the stem cell to form neurons and oligodendrocytes is lost and only astrocytes are formed. NO-depleted DETA-NONO:ate was used as control. Conclusion: The CSF nitric oxide levels of MS patients affect stem cell survival and differentiation.
Neurotoxicity induced by LTA-activated glial cells is mediated by nitric oxide production and caspase activation A. Kinsner a , S. Coecke a , T. Hartung a,b and A. Price a a ECVAM, European Commission Join Research Centre, Ispra (VA,) Italy; b Dept. of Biochemical Pharmacology, University of Konstanz, Konstanz, Germany In central nervous system (CNS) pro-inflammatory potential of lipoteichoic acid (LTA) derived from the Gram-positive bacterial cell wall is still controversial. We investigated whether LTAFMDP (muramyl dipeptide) could activate rat cortical glial cells (astrocytes and/or microglia) by measuring IL-1beta, TNF-alpha and nitric oxide (NO) production that subsequently could induce neuronal cell death and if so by what mechanisms. Indeed LTA in synergy with MDP (active principle of peptidoglycan) induced strong inflammatory response of both types of glial cells as time-and dose-dependent production of IL-1beta, TNF-alpha and NO was observed. To study whether activated glia could induce neuronal cell death, we exposed cerebellar granule cells (CGCs) cultured in the presence of glia (~15% non-neuronal cells) to LTA (30 Ag/ml) and MDP (100 ng/ml). After 72 h, significant neuronal cell death was observed (70-90%) as assessed by propidium iodide and Hoechst staining. Indeed, the death of CGCs was caused by activated glia as in the absence of glia (treatment with 7.5 AM cytosine-d-arabinoside to inhibit non-neuronal cell proliferation) LTA+MDP did not cause significant cell death (~20%). The neuronal cell death induced by LTA and MDP-activated glia was significantly blocked by iNOS inhibitor (100 AM 1400 W) and broad-spectrum caspase inhibitor (50 AM z-VAD-fmk) suggesting that neuronal cell death was mediated by NO production and caspase-activation. This mechanism may contribute to neurodegeneration observed in CNS infections caused by Gram-positive bacteria. Aim: Cytotoxic T cells are involved in West Nile virus (WNV) encephalitis, and are the predominant lymphocyte population infiltrating the brain. We aimed at determining the role of the Tc cell cytolytic pathways, granule exocytosis (mediated by perforin and granzymes) and Fas-triggered killing, in either virus clearance or immunopathology. Methods: C57BL/6J wildtype and mutant mice defective in cytolytic effector molecules were infected i.v. with WNV (Sarafend strain). Virus was detected by plaque titration and immunohistochemistry. Cytotoxicity of splenocytes was measured by in vitro 51Cr-release. Results: Infection of CD8+ T cell-deficient mice (beta2mÀ/À) with high doses of WNV (10 8 PFU) resulted in reduced mortality with prolonged mean survival time (MST), whereas infection with low doses (10 3 PFU) led to increased mortality with prolonged MST. Transfer of WNV-immune CD8+ T cells reduced WNV-mediated mortality. Mice with defects in the granule exocytosis pathway of cytotoxicity are at higher risk of encephalitis and death, but perforin by itself does not play a crucial role in the recovery from WNV infections. Deficiency of either Fas or FasL did not markedly affect the susceptibility of mice to WNV. However, mice with deficiency in Fas and perf (perfÀ/Àgld) displayed a higher incidence of encephalitis and death after infection. Conclusions: Granule exocytosis as well as the Fas pathway of cytotoxicity are involved in recovery and immunopathology in mouse WNV infection. TMEV induces, in SJL mice, early acute disease followed by late chronic demyelinating disease. During early acute disease, the virus is partially cleared from the CNS by CD3 T cells. These T cells express Fas, FasL and negligible levels of Bcl-2. At 3 days post infection (dpi), but not at 8 dpi, CD3 T cells infiltrating the CNS of SJL mice express significantly higher proportions of CTLA-4 versus those in B6 mice. In vivo labeling with BrdU revealed that 15% of CNS-infiltrating T cells is cycling during early acute disease. Large proportion of these cells showed a phenotype of activated effector cells (CD44hi CD64lo/-, CD25+). These T cells were undergoing AICD resulting in downsizing of the inflammatory response. TUNEL assay revealed that 30.1% of CD3 T cells were undergoing apoptosis at 8 dpi. In contrast, during late chronic demyelinating disease only few mononuclear cells were apoptotic. CNS infiltrating T cells expressed high levels of Fas, FasL and Bcl-2. 26% of these CD3 T cells were memory cells while 98.6% were arrested in G0/G1. Lack of in situ proliferation of T cells expressing high levels of Bcl-2 in the CNS and the paucity of apoptosis are likely responsible for the accumulation of these T cells in the CNS during late chronic demyelinating disease in SJL mice.
Identification of astrocyte-derived genes that induce apoptosis of autoreactive T cells H. Hara and T. Tabira National Institute for Longevity Sciences, Obu City, Japan
In EAE, apoptosis of T cells is mainly seen at the site of CNS inflammation. There are some reports that astrocytes may render T cells susceptible for induction of apoptotic cell death. In this study, we have identified and cloned the genes derived from IFN-gamma-treated astrocyte cell line that induce apoptosis of autoreactive T cells. We made the subtracted cDNA libraries from IFN-gamma-treated astrocyte cell line and got 100 positive clones. All clones were sequenced and 50 genes with interest were transfected to CHO cells individually. Activated PLP-reactive CD4+ T cell line were co-cultured with each transfected CHO cells, then, T cell were harvested and stained with Annexin V-FITC and PI and analyzed by flow cytometry. We identified two candidate genes that induced apoptosis of PLP-reactive T cell lines. First gene is unknown gene, named as astrocytederived immune suppressor factor (AdIF) and its length is 726 bp, 228 amino acids. Recombinant AdIF protein was made by Drosophilae expression system. After acute EAE was induced in C57BL6/J mice by immunization with MOG peptide plus CFA, AdIF protein was injected intravenously. The treated group showed significant ameliorations of clinical EAE scores and pathological changes in the CNS. This AdIF may be effective in the treatment of multiple sclerosis.
Regulation of natural killer cell stimulatory and inhibitory molecules in mouse nervous system E. Backström a , A.F.J. Drenth a , H. Sjflin b , H.G. Ljunggren c and K. Kristensson a a Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden; b Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden; c Center for Infectious Medicine, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden Expression of ligands for stimulatory and inhibitory natural killer (NK) cell receptors may determine whether neurons are susceptible or resistant to NK cell-mediated cytolysis. By using real time PCR, the expression of ligands for the stimulatory receptor NKG2D as well as ligands for NK cell inhibitory receptors in dorsal root ganglia (DRG) were analyzed in vivo, after a peripheral nerve lesion, and in vitro, in DRG neuronal cultures exposed to interferon (IFN)-gamma or LPS, or infected with murine cytomegalovirus (MCMV) or a neurotropic influenza A virus strain, WSN/33. Peripheral nerve lesions are associated with a decrease in levels of RAE1 and murine UL16-binding protein-like transcript 1 (MULT1), but an increase in levels of classical MHC class I transcripts in DRG of adult mice. The study also shows that IFN-gamma and LPS caused a marked induction of classical and nonclassical major histocompatibility complex (MHC) class I encoding transcripts in DRG cultures. Cultures infected with either MCMV or WSN/33 showed a significant increase in the expression of transcripts encoding RAE1 and classical and nonclassical MHC class I. Exogenous agents as well as peripheral nerve lesions can alter the expression of NK cell stimulatory and inhibitory ligands in nervous tissue.
Mechanism of resistance of microglia to quinolinic acid-mediated cell death C.M. Figueiredo, M. Faria Pais and S. Chatterjee Instituto Gulbenkian Ciência, Oeiras, Portugal Brain macrophages, microglia, respond to traumatic injury or to pathogens in Central Nervous System by migrating to the site of injury, where they may proliferate and release inflammatory cytokines, and potential neurotoxins. In addition, microglia when activated releases a metabolite of the tryptophan-kynurenine pathway, quinolinic acid (QA), which can be implicated in neurodegenerative diseases. QA can induce neurotoxicity through N-methyl-d-aspartate receptor (NMDAR). NMDAR is a heteromeric complex composed of two classes of subunits, the essential NR1 and NR2A to D that potentiate NR1 activity and confer functional variability to the NMDAR. Interestingly, unlike neurons primary microglia is resistant to QA challenge; therefore analysis of the mechanism of resistance of microglia to quinolinic acid-mediated cell death was performed. Results from fluorescence staining with propidium iodide, calcium imaging, Western Blot and RT-PCR analysis showed that QA stimulates NMDAR primary cerebellar granule neurons but not microglia; inducing an increased Ca2+ influx and activation of the extracellular signal regulated kinase, and consequently cell death. The two cell types possess same protein levels of NR1 subunit, but microglia did not express mRNA of the other NMDAR subunits. A specific NR1-NR2B complex blocker, ifenprodil, rescues cell death, suggesting that QA may bind to NR2B, a subunit absent in microglia. Characterization and functional implication of the NMDAR in microglia and in neurons in perspective of the differential response to QA will be discussed in this presentation.
Beta amyloid 1-40 peptide selectively downregulates MMP-9 production in differentiated SH-SY5Y neuronal cell line M. Ruggieri a , F. Roselli a , C. Pica a , A. Lia a , C. Avolio b , M. Trojano a and P. Livrea a a University of Bari, Bari, Italy; b University of Foggia, Foggia, Italy Objective: Neuronal Matrix Metalloproteinases (MMPs) may be involved in memory consolidation processes and synaptic plasticity. MMP-9 expression has been detected in dentate gyrus neuron cell bodies and dendrites. Amyloid accumulation in strategic brain areas induce memory dysfunctions, but the effect of amyloid peptides on neuronal MMPs are unknown. We tested whether amyloid peptides could alter MMPs (or their inhibitors) production by neurons in vitro. Methods. SH-SY5Y human neuroblastoma cell cultures were treated with 1-40 Abeta at 0.5 and 5 AM or by vehicle. Supernatants were collected after 24 and 48 h incubation and MMP-9, MMP-2 (activity assay) and TIMP-1, TIMP-2 (ELISA) were detected. Cell death was evaluated by vital dye exclusion test. Results. MMP-9 levels were markedly reduced in both 0.5 and 5 AM Abeta treated cell supernatants versus negative controls at 24 h ( pb0.002) and 48 h ( pb0.002); no differences were detected between 0.5 and 5 AM Abeta treated cells. MMP-2, TIMP-1 and TIMP-2 were unchanged between samples. Vitality did not differ between treated and untreated cells. Conclusions: 1-40 Abeta selectively inhibits MMP-9 production in neurons. This effect could not be due to cell toxicity because it is restricted to MMP-9, with unchanged MMP-2 and TIMPs and with unchanged cell death in treated versus untreated cells. The down regulation of MMP-9 induced by 1-40 Abeta may play a role in age related memory impairment and in Alzheimer disease. Immunization with myelin oligodendrocyte glycoprotein (MOG 35-55) slows down the recovery of dopaminergic neurons injured with MPTP I. Kurkowska-Jastrzebska a , E. Balkowiec-Iskra b , I. Joniec b , A. Czlonkowski b and A. Czlonkowska a,b a Institute of Psychiatry and Neurology, Warsaw, Poland; b Medical University of Warsaw, Warsaw, Poland 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration causes an injury of nigrostriatal dopaminergic neurons. The inflammatory reaction follows the dopaminergic neurons impairment, and is believed to contribute to neuronal death and survival. As it is postulated that additional brain inflammation may modulate neurodegenerative process, we would like to examine an influence of immune system enhancement on the recovery after toxic insult caused by MPTP. Male, 2 months old C57BL10 mice received 40 mg/kg MPTP i.p. and after 7 days they were immunized with MOG 35-55 peptide in CFA. Mice receiving MPTP showed a 63% decrease of dopamine content in striatum on the 7th day following MPTP administration as compared to control mice. From the 14th day dopamine content increased and was less than in the control by 52%, 42% and 18% on 14th, 28th, and 50th day, respectively. In group of mice receiving additional immunization the recovery was however slower as compared to nonimmunized mice, and dopamine content decrease was less than in the control by 75%, 59% and 33% on the same days, respectively. Thus immunization with MOG after MPTP intoxication resulted in deeper dopamine depletion and the worse recovery of dopaminergic neurons after the insult. Tumor necrosis factor (TNF) induces apoptotic-like cell death of oligodendrocytes, cell target in multiple sclerosis (MS). Human adult oligodendrocytes (hOLs) and microglial cells (MG) were prepared from human brain specimen obtained during neurosurgical procedures. The cells were stimulated with TNF and cell death was detected by annexinV-FITC and PI staining and flow cytometry. We defined that intracellular transduction pathway involved in TNF-induced death of hOLs is noncaspase dependent as evidenced by lack of generation of active subunits of caspase-8, -1 and -3 detected on Western blot as well as the lack of cleavage of fluorogenic substrates of caspase-1 and -3 measured by flow cytometry. In addition, the general caspase inhibitor ZVAD.FMK did not prevent TNFinduced hOLs death. Also calpain inhibitor ZLLY.FMK, serine proteases inhibitor, TPCK, and cathepsin inhibitors, ZFL and ca-074-Me, were inefficient in prevention of TNF-induced hOLs cell death. Electrophoresis of TNF-exposed hOLs DNA revealed a large scale DNA fragmentation characteristic for apoptosis inducing factor (AIF)-induced cell death. In TNF-stimulated hOLs AIF was translocated to nuclei. Accordingly, hOLs death was prevented by inhibition of AIF using antisense strategy (asAIF). Inhibition of AIF with asAIF led to significant decrease of AIF expression in hOLs but did not affect the TNF-induced change of mitochondrial membrane potential. These results indicate that TNF-induced hOLs death depends critically on AIF activation and might have significant importance in designing new molecules to protect hOLs in multiple sclerosis.
Leukemia inhibitory factor is produced by myelin reactive T-cells and protects against cytokine induced oligodendrocyte cell death J. Vanderlocht a , N. Hellings a , F. Vandenabeele a , M. Moreels a , J. Antel b , J. Raus a and P. Stinissen a a Limburgs Universitair Centrum, Diepenbeek, Belgium; b McGill University, Montreal, Canada Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are members of the neuropoietic family of neurotrophins and ameliorate experimental autoimmune encephalomyelitis (EAE). We examined whether human T-cells specific for myelin basic protein (MBP) and myelin oligodendrocyte protein (MOG) are able to secrete LIF. 10/19 myelin reactive T-cell lines/clones (TCL) from eight multiple sclerosis (MS) patients and 5/11 TCL from five healthy controls (HC) produced LIF upon stimulation. The mean net LIF secretion of MS TCL did not significantly differ from HC TCL. LIF expression was confirmed by RT-PCR. In addition, TCL specific for tetanus toxoid, monocytes, CD4+ and CD8+ Tcells, but not B-cells secreted LIF. LIF secreting T-lymphocytes and macrophages could also be identified immunohistochemically in active and chronic active MS lesions. We further demonstrated dose-dependent protective effects of both CNTF and LIF on TNF-alpha induced apoptosis in HOG cells and rat oligodendrocyte cultures. At concentrations of 10-20 ng/ml both neurokines completely protected oligodendrocytes against cytokine induced apoptosis. Post-treatment with neurokines was as effective as pre-treatment. In conclusion, our in vitro studies indicate that LIF and CNTF are possible candidates for therapeutic interventions in MS. Factors that enhance the production of neurotrophins by T cells may provide new tools for MS therapy. To elucidate pathogenic circuits between immune and neuroendocrine systems, we are examining the emergence of behavioral deficits that temporally coincide with impaired dopamine catabolism in lupus-prone MRL-lpr mice. Our hypothesis is that systemic autoimmunity affects central dopaminergic systems in diseased mice. The functional damage of the nigrostriatal pathway was assessed from rotational behavior after a single injection of apomorphine. Amphetamine was employed to further probe the relationship between mesolimbic damage and depressive-like behavior. Neurodegeneration in the midbrain was estimated by Fluoro Jade B (FJB) staining and tyrosine-hydroxylase (TH) immunocytochemistry. In comparison to young and age-matched controls, diseased MRL-lpr mice showed increased circling following an apomorphine injection and failed to increase palatable food consumption after receiving amphetamine. Increased FJB-positive neurons and a profound reduction in TH-positive somas confirmed mesencephalon cell loss in behaviorally impaired mice. Our study coincided with the death of a NP-SLE patient. Neuropathologic changes observed in the patient's basal ganglia were similar to MRL-lpr neuropathology, strengthening the validity of our NP-SLE model. In summary, results suggest that systemic autoimmunity damages dopaminergic systems and induces behavioral deficits. Electronic microscopy is presently being employed to elucidate the mode of neuronal death and results will be reported at the meeting. Supported by funds from NIH and CIHR to B.S. and D.A.B.
Immunoglobulins in cytotoxic CSF from autoimmune mice M.M. Sidor and B. Sakic
Neuropsychiatric lupus (NP-SLE) is a complex manifestation of unknown etiology that can occur during the course of systemic lupus erythematosus. We use the lupus-prone MRL-lpr murine model to study the etiology of NP-SLE. Our previous experiments have revealed that leukocyte infiltration into the brain, cell loss in periventricular areas, and cytotoxic cerebrospinal fluid (CSF) coincide temporally with behavioural deficits in diseased MRLlpr mice. The presence of B-cells in the choroid plexus and toxicity of IgGrich CSF fractions suggest intrathecal synthesis of autoantibodies to neuronal tissue. The present study examines whether cytotoxic CSF from diseased MRL-lpr mice exhibits a unique protein profile. Western blotting has revealed distinct IgG bands in the CSF of 16-to 20-week-old MRL-lpr mice, with a 25-kDa band occurring more frequently than in age-matched controls (79% vs. b1%). This band was absent in all asymptomatic groups. Intensity of the IgG bands show positive correlation with splenomegaly, suggesting that progress of systemic autoimmunity and inflammation are associated with changes in the CSF content. At the meeting we will report whether in vitro CSF toxicity is associated with presence of the 25-kDa band, whether this band is produced intrathecally, and whether it is a product or cause of brain damage. This work was supported by funds from the NIH to B. Sakic, recipient of the Father Sean O'sullivan Research Centre career development award.
Glatiramer acetate induces pro-apoptotic mechanisms involving Bcl-2, Bax and Cyt-c in multiple sclerosis treated patients M. Ruggieri a , S. Scacco a , C. Pica a , A. Lia a , S. Papa a , P. Livrea a , M. Trojano a and C. Avolio b a University of Bari, Bari, Italy; b University of Foggia, Foggia, Italy Apoptotic deletion of autoreactive T cells is defective in patients with Multiple Sclerosis (MS). Glatiramer acetate (GA) may be effective to induce apoptosis of detrimental T cells. We analyzed pro-(Bax, Cyt-c, APAF-1) and anti-(Bcl-2) apoptotic proteins in peripheral blood mononuclear cells (PBMNCs) cytosol and mitochondria membranes during GA treatment in relapsing-remitting (RR) MS patients. Blood samples from eight RR MS patients were collected before and every 3 months during 9 months of GA treatment and from six healthy controls (HC). PBMNCs Bcl-2, Bax, Cyt-c and APAF-1 were quantified by Western blot followed by densitometric scanning. The percentage of apoptotic cells was assessed by a dye exclusion test. T-cells were in vitro tested for oxigen consumption by a respirometric analysis. A decrease ( p=0.005) in Bcl-2 and an increase ( p=0.02) in Bax during GA treatment were observed. Cytosolic Cyt-c trended to increase ( p=0.05) during treatment, suggesting a release of Cyt-c from mitochondria membranes to cytosol, whereas APAF-1 remained unchanged. An increase in the percentage of apoptotic PBMNCs was observed during treatment. A reduction by 58% in oxygen consumption by T-cells was evident after GA treatment in vitro. Our findings suggest that GA might exert a regulatory effect on peripheral T lymphocytes through pro-apoptosis mechanisms involving the downregulation of Bcl-2 and an upregulation of Bax and cytosolic Cyt-c.
Modulation of soluble Fas levels in patients with Herpes simplex encephalitis (HSE) F. Sabri, A. Hjalmarsson, F. Granath, K. Lfvgren and B. Skfldenberg Karolinska University Hospital, Stockholm, Sweden
Herpes simplex encephalitis (HSE) is the most common cause of sporadic, severe viral encephalitis. There is evidence of a vigorous intrathecal immune response during the acute phase of HSE and at long-term follow up. We hypothesize that the severity and the progression of the cerebral injury resulting from HSE can be evaluated by quantitative measurement of different compartments of immune activation molecules, such as soluble Fas (sFas), a molecule involved in apoptosis. Cerebral spinal fluid (CSF) and serum sFas levels in 242 consecutive samples from 42 HSE patients in a Vidarabine -Acyclovir trial were analyzed by capture ELISA. Interestingly, the sFas levels in CSF were highest in HSE patients who died, and lowest in those with no or mild sequels and in-between in patients with moderate or severe outcome. No detectable sFas found in CSF samples from 35 patients with non-HSE encephalitis or in 18 persons with headache. The sFas levels in the last serum sample obtained were higher in HSE patients with comatose state compared to those being semicomatose or lethargic at presentation. A similar pattern was observed in serum samples with highest sFas during disease progression in the three HSE groups. These observations enforce the role of immune activation state, observed in serum, during the acute phase of HSE and in CSF, in particular, at disease progression, which might reflect the apoptotic state in the brain. Oxidative stress and NF-kB activation are suggested to be linked to acute and chronic inflammations. Recent studies showed that activation of the receptor for advanced glycated end products (RAGE) by its specific ligand (CML) results in activation of NF-kB and production of proinflammatory cytokines. To determine the possible role of this pathway in the pathogenesis of vasculitic neuropathy we investigated the immunolocalisation of CML, RAGE, NFkB and IL6 in sural nerve biopsies of 12 patients with vasculitic neuropathy and eight controls (normal, HMSN). CML, RAGE, NFkB and IL6 could be localized in infiltrating mononuclear cells in epineurial and endoneurial vessels and in the perineurium in vasculitic neuropathy. Controls showed sporadic weak staining for the four antigens in vessel walls and for CML/IL6 in the perineurium. Colocalisation studies demonstrated a CML-, RAGE-, and IL6 expession in CD4+, CD8+ and CD68+ cells in vasculitis. Activated NFkB was expressed by CD4+, CD8+ and CD68+ mononuclear infiltrating cells. Our data suggest that CML induced, RAGE mediated NF-kB activation resulting in elevated IL6 production is a proinflammatory mechanism in the pathogenesis of vasculitic neuropathy. The therapeutic use of antioxidants like vitamin E, alpha-lipoic-acid or benfotiamine causing a reduction of intracellular oxidative stress and extracellular CML formation as shown in various models for late diabetic complications should be evaluated in vasculitic neuropathies.
NGF and NGF-receptor in myastenia gravis thymus L. Aloe a , T.S. Marinova b , D. Petrov c and L. Manni a a Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy; b Department of Biology, Medical University, Sofia, Bulgaria; c Department of General and Clinical Pathology, Medical University, Sofia, Bulgaria
We have previously reported that autoimmune inflammatory disorders are associated with altered levels of nerve growth factor (NGF), that immune cells produce NGF and express NGF-receptors (NGF-R), and prospected the hypothesis that these two NGF markers should be considered as signals for cell-to-cell recognition in immune systems. We have now analyzed the distribution of NGF and NGF-R in the thymus of subjects with Myasthenia Gravis (MG). Thymuses from healthy and MG subjects, removed in accordance with Helsinki Convention, and Ethical Medical Board consent, were used for structural, ultrastructural, biochemical and molecular analysis. The results showed the presence of NGF and of NGF-R in thymic epithelial cells and in mast cells of control subjects, and an altered expression of these NGF markers, both at protein and gene expression levels, in the MG thymuses. These observations and studies showing that administration of NGF in laboratory animals retards thymic cell death suggest that thymic cells regulate NGF through autocrine mechanisms. Thus NGF represents an important biological mediator not only for nerve cells but also for immune cells and most probably for transmitting signals back and forth between nervous and immune systems. Supported by: Progetto di Ricerca Multicentrico Triennale, CARISBO, Italy.
Spinal neuroimmune activation induces behavioral sensitization in the animal models of chronic pain V. Raghavendra and J. DeLeo Anesthesiology/Pharmacology, Dartmouth-Hitchcock Medical Center, Lebanon, USA Recent evidence suggests that activation of intrinsic inflammatory immune responses in the CNS contribute to the development and maintenance of behavioral sensitization. In this study, we examined the pattern of glial activation and proinflammatory cytokine expression at the lumbar spinal cord in a rat model of neuropathic and chronic inflammatory pain. Further, the role of spinal neuroimmune activation in the development of allodynia and hyperlagesia was investigated. Gene expression, studied by real-time RT-PCR, for glial markers (GFAP and S100B for astrocytes, and Mac-1, TLR4 and CD14 for microglia), and prinflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) showed a differential upregulation following nerve injury or inflammation. Protein quantification (by immunohistochemistry, ELISA and Western blot analysis) in these rats also revealed glial activation and enhanced proinflammatory cytokine expression at the lumbar spinal cord. Central administration of dexamethasone 21-phosphate attenuated both neuroimmune activation and the development of behavioral allodynia and hyperalgesia in the animal models of neuropathic and inflammatory pain. This study provides data to implicate a role of glia and proinflammatory cytokines in the development of behavioral hypersensitivity during peripheral nerve injury or inflammation. Immunological mechanisms of autologous hematopoietic stem cell transplantation as a therapy for multiple sclerosis P.A. Muraro a , K. Chung a , D. Douek a , H.F. McFarland a , R.K. Burt b and R. Martin a a National Institutes of Health, Bethesda, MD, USA; b Northwestern University, Chicago, IL, USA High-dose immunosuppressive therapy followed by autologous hematopoietic stem cell transplantation (HSCT) has shown strikingly beneficial effects on the disease course in a subset of patients with severe autoimmune diseases. The mechanism of action of HSCT, however, has remained elusive so far. We studied seven patients with multiple sclerosis (MS) after HSCT who had no toxicities or infectious adverse events and no signs of residual inflammatory disease activity during follow-up. After 2 years, naRve CD4+ T cells more than doubled at the expense of central memory T cells. T cell receptor excision circle analysis confirmed that the output of thymus-derived naRve CD4+ T cells after immune reconstitution substantially increased, reaching levels comparable to healthy age-matched controls. Peripheral mechanisms contributing to recovery of immune tolerance included propensity of T cells to apoptosis and proliferative senescence of expanded CD8+ T cells. Our findings demonstrate that abrogation of disease activity after HSCT is associated with a profound reconfiguration of the immune system and is not simply the effect of a protracted immunosuppression.
Intense immunosuppression followed by autologous stem cell transplantation as treatment for severe multiple sclerosis G.L. Mancardi, A. Uccelli and the Italian Gitmo-Neuro intergroup on ASCT for multiple sclerosis University of Genoa, Genoa, Italy
Based on studies on experimental models of autoimmunity, intense immunosuppression followed by autologous stem cell transplantation (ASCT) has been recently proposed as treatment for rapidly progressing multiple sclerosis (MS) unresponsive to conventional therapies. It has been reported that such therapy can effect on MRI parameters of disease activity and halt disease progression in such severe MS forms, despite of relevant mortality risk. Here we report the MRI and clinical follow up of 19 rapidly worsening MS patients with a high number of gadolinium-enhancing lesions at T0, treated with cyclophosphamide 4 g/m 2 as mobilizing regimen and BEAM (BCNU, Cytosine-Arabinoside Etoposide and Melphalan) as conditioning treatment. The mean follow up is now 29 months, with a range of 2-54 months. The treatment was relatively well tolerated. More importantly, it strikingly abolished any MRI activity although it did not influence the progression of brain atrophy over time. In most patients we observed also a clinical improvement or stabilization of disease. The present results demonstrate that intense immunosuppression followed by ASCT has an effect on inflammation detected by MRI not comparable to any other currently available therapy, may successfully stabilize disease course and thus should be considered as therapeutic option for those MS subjects with poor prognostic factors not responding to conventional therapies.
Anti-ergotypic T cell responses in healthy controls and multiple sclerosis patients N. Hellings, C. Govarts, J. Raus and P. Stinissen Biomedisch Onderzoeksinstituut, Liburgs Universitair Centrum, Diepenbeek, Belgium
Peripheral regulatory mechanisms may prevent the expansion of autoreactive T-cells that play a role in autoimmune diseases such as multiple sclerosis (MS). Anti-idiotypic and anti-ergotypic T-cells take part in these regulatory networks and can be boosted by T-cell vaccination (TCV). We showed that TCV induces both long-term clonotypic and temporal antiergotypic responses (to activated T-cells). This study aims to further characterize anti-ergotypic T-cells in MS patients and healthy controls (HC). PBMC were stimulated with PHA-activated T-cell blasts as source of activated cells. Proliferative anti-ergotypic responses were found in all HC and MS patients tested. Anti-ergotypic short-term lines with a mixed CD4+/ CD8+TCRab+ phenotype produced high amounts of IFN-gamma, but no IL-4/IL-10 and were predominantly HLA-DR restricted. Depletion experiments showed that CD4+ T-cells were mainly responsible for the observed in vitro anti-ergotypic responses. Cytokine receptors including interleukin-2 receptor (IL-2R) have been identified as candidate targets of anti-ergotypic T-T interactions in EAE. We tested reactivity to a peptide derived from the IL2R alpha chain containing a dominant epitope for HLA-DRB1*1501. We were able to isolate T cell lines (TCL) specific for this IL2R peptide from four HC. The TCL had a Th1/Th0 phenotype and proliferated strongly to activated but not resting tetanus toxoid specific TCL. We are currently testing whether IL2R-specific TCL with similar properties can be isolated from MS patients before and after TCV. This will help unravel their actual role in T-cell immunoregulation.
Boosting regulatory T cells in multiple sclerosis: immunogenicity and safety of a trivalent BV5S2, BV6S5 and BV13S1 CDR2 vaccine in incomplete Freund's adjuvant A. Vandenbark a , R. Bartholomes b and D. Bourdette c a Veterans Affairs Medical Center, Portland, OR, USA; b Immune Response Corporation, Carlsbad, CA, USA; c Oregon Health and Science University Oregon Health and Science University, Portland, OR, USA T cell receptor (TCR) peptide vaccination represents a novel approach to boosting regulatory T cells in multiple sclerosis (MS). Development of TCR peptide vaccination as a treatment for MS has been hindered by the relatively low immunogenicity of previously available TCR peptide vaccines. For example, intradermal vaccination with a single CDR2 BV5S2 TCR peptide given without adjuvant induced regulatory TCR reactive T cells in 20-60% of MS subjects. In an effort to develop a more effective vaccine, we compared intramuscular injections of a trivalent TCR vaccine containing BV5S2, BV6S5 and BV13S1 CDR2 peptides emulsified in incomplete Freund's adjuvant (IFA) with intradermal injections of the same peptides without IFA. Six monthly injections of the trivalent/IFA vaccine induced vigorous T cell responses in 100% of subjects (9/9) and was significantly more immunogenic than the peptide/saline vaccine, which induced responses in only 20% of subjects (2/10) ( p=0.002). Aside from injection site reactions, there were no significant adverse events attributable to the treatment. While there were no significant clinical changes over the course of this short trial, subjects who had T cell responses to the trivalent TCR peptide vaccine showed a trend towards reduced MRI activity. The trivalent TCR peptide in IFA vaccine represents a significant improvement over previous TCR peptide vaccines and warrants investigation of its ability to treat MS.
Atorvastatin regulates T cell anergy via the extracellular signal-related kinase pathway: a novel immunomodulatory function of 3-hydroxy-3methylglutaryl-CoA reductase inhibitors? S. Waiczies, C. Infante-Duarte, T. Prozorovski, S. Pikol and F. Zipp Institute of Neuroimmunology, Neuroscience Research Center, Charité, Humboldt University, Berlin, Germany
The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR) inhibitors, statins, are orally administered cholesterol-lowering drugs, which have been shown to exhibit immunomodulatory properties. We have previously reported that in vivo atorvastatin treatment could prevent and reverse disease progression in the animal model of multiple sclerosis. However, the mechanism of immunomodulation by statins has yet to be fully clarified. In the human system we report an influence of atorvastatin on cell cycle progression in peripheral T cells and antigen-specific T cell lines. We could explain this by a failure of atorvastatin-treated cells to downregulate the negative cell cycle regulator p27Kip1 following a productive stimulus. p27Kip1 is specifically required for the induction and maintenance of T cell anergy. In fact, we report here that following incubation with atorvastatin, T cells lose their capacity to respond to their specific antigen. Anergy induction was shown to be dependent on HMG-CoAR, since we could reverse this with mevalonate, an intermediary product of this pathway. Preliminary results also show that atorvastatin induces an unexpected activation of the extracellular signal-related kinase pathway, which we show, via blocking experiments, to be necessary for the induction of anergy. Our results indicate that atorvastatin regulates T cell expansion via an induction of anergy and portray a novel immunomodulatory function of HMG-CoAR inhibitors in the treatment of autoimmune diseases such as multiple sclerosis.
Glatiramer acetate (Copaxone) therapy induces an oligoclonal CD8+ T cell response with cytotoxic ability R. Mehta, S. Yan, B. Biegler, S. Ortega, M. Racke and N. Karandikar UT Southwestern Medical Center, Dallas, TX, USA Multiple sclerosis (MS) is an inflammatory demyelinating disorder of the central nervous system with features suggestive of T-cell-mediated autoimmunity. Treatment with glatiramer acetate (GA, Copaxone) decreases the rate of exacerbation in relapsing MS. We have shown that GA therapy upregulates GA-specific CD8+ T-cell responses to levels found in healthy subjects. In this study, we evaluated the functional profile and clonal complexity of GA-induced T-cells. Sequencing of T-cell receptor-beta chains of flow-sorted GA-specific CD4+ and CD8+ T-cells revealed strikingly oligoclonal GA-reactive CD8+ responses in MS patients. Over the course of therapy, the same dominant clones of CD8+ T-cells persisted over long periods of time in certain patients. In contrast, GA-reactive CD4+ T-cells were polyclonal and revealed different clonal composition at different time points during therapy. The heightened CD8+ T-cell response corresponded with the longitudinal upregulation of ex vivo cytotoxic activity of GA-specific CD8+ T-cells. GA therapy also induced a change in functional molecular profiles of both CD4+ and CD8+ T-cells. These results demonstrate that a few dominant clones of CD8+ T-cells contribute to the GA-induced response and may mediate its immunomodulatory effect through direct cytotoxic killing of relevant immune populations. This also raises important questions about the immunologic effects of chronic GA therapy and the development of T-cell senescence. Supported in part by grants from the NIH, NMSS and Wadsworth Foundation.
Effect of Rituximab on the peripheral blood and cerebrospinal fluid B cells in patients with primary progressive multiple sclerosis N. Monson, K. Hawker, E. Frohman and M. Racke UT Southwestern Medical Center, Dallas, TX, USA Context: Rituximab is an anti-CD20 monoclonal antibody that depletes CD20+ B cells. Whether the efficacy of Rituximab in peripheral neurological diseases can be translated to neurological diseases of the central nervous system (CNS) with possible autoimmune B cell involvement remains unknown. Objective: To determine what effect Rituximab has on the cerebrospinal fluid (CSF) B cell population in patients with Multiple Sclerosis (MS), a chronic inflammatory, demyelinating disease of the CNS that likely involves an autoimmune mechanism. Design: Four patients with Primary Progressive MS (PPMS), a subtype of MS that presents as a slow deterioration of neurological function without relapses, were treated with Rituximab. Peripheral blood (PB) and/or CSF were collected from each patient pre-and post-Rituximab. The PB and CSF cells were analyzed by flow cytometry to identify B cell subsets. Results: CSF B cells were not as effectively depleted as their peripheral counterparts, most likely because the majority of CSF B cells are highly activated CD19dim advanced memory or plasma B cells. Rituximab suppressed the activation state of the CSF B cells. CD19dim B cells in the periphery and CSF expanded beyond their normal frequencies post-Rituximab. Conclusion: The effect(s) of Rituximab on the CSF B cell compartment are limited in comparison to the periphery, but will need to be confirmed in a larger group of MS patients. Current therapies of multiple sclerosis are only moderately effective. We have recently shown that a humanized monoclonal antibody against the interleukin-2 receptor alpha chain, Daclizumab, substantially reduces brain inflammation in MS patients with incomplete response to standard interferon beta therapy. The aim of this study was to determine in vivo immune changes that are responsible for this profound therapeutic effect. We prospectively analyzed the functional status of lymphocytes in patients before and during therapy with Daclizumab by evaluating surface expression of activation markers, chemokine receptors and cytokine chains, and by employing CFSE-based proliferation assay. Additional in vitro experiments required cellular separations by magnetic beads, intracellular cytokine staining and cytotoxicity assays. We demonstrated that Daclizumab does not act via the anticipated direct functional-and activation blockade of CD4+ T cells, a central population in MS pathogenesis. Instead, Daclizumab therapy led to prominent expansion of immunoregulatory CD56bright natural killer (NK) cells and to a gradual decline in CD8+ and CD4+ T cells. These changes were highly correlated to and predictive of MRI outcome. We will describe the mechanism by which CD56bright NK cells may mediate peripheral tolerance in vivo. Our data reveal an intriguing pathway how to manipulate cytokine circuitry. These findings have important implications for the treatment of autoimmune diseases, transplant rejection and toward modification of tumor immunity.
Immunomodulatory effects of simvastatin in relapsing-remitting multiple sclerosis S. Markovic-Plese a,b , X. b , S. Giri c , D. Sujkowski a and I. Singh c a University of North Carolina, Chapel Hill, USA; b Yale University, New Haven, USA; c Medical University of South Carolina, Charleston, USA Objective: To study immunomodulatory therapeutic potential of simvastatin, CNS penetrating HMG-CoA reductase inhibitor, in relapsing-remitting multiple sclerosis (RR MS). Results: Statins exhibit dose-dependant antiproliferative effect in activated peripheral blood mononuclear cells (PBMCs). Simvastatin decreased IFN-gamma-inducible expression of MHC class II DR on CD14+ monocytes. In addition to the effect on antigen presentation, simvastatin decreased surface expression of activation markers (CD25, CD69), memory and naRve subset markers (CD45RO, CD45RA), and chemokine receptor (CCR5) on the effector CD4+ and CD8+ lymphocytes. Described effects were reversed by mevalonate, the product of HMG-CoA reductase, confirming that the effect on lymphocyte activation is specific for HMG-CoA reductase inhibition. Treatment with simvastatin induced a shift from Th1 to Th2 cytokine production. We measured a significantly decreased IFN-gamma, TNF-alpha and IL-2 secretion, and increased IL-4 production following in vitro PBMC stimulation. Further analysis revealed consistently decreased expression of T-bet, transcription factor that regulates Th-1 cell differentiation. Gene arrays were subsequently used in order to capture treatment-induced changes in the expression of multiple cytokine and chemokine genes. We identified a significant increase in IL-1A, IL-3, and SDF1, and decrease in IL-6, IL-7, IL-8, and TNF-beta gene expression in simvastatin-treated cultures. Findings were confirmed by RT-PCR. Conclusions: Due to multiple immunomodulatory mechanisms of action, statins represent a promising treatment for MS, a chronic inflammatory CNS disease. Multiple sclerosis is a chronic inflammatory disorder of the central nervous system (CNS). Auto-aggressive T-cells drive inflammation leading to focal myelin destruction, loss of oligodendrocytes and acute axonal transection. Campath-1H is a monoclonal antibody that induces prolonged T-cell lymphopenia. After Campath treatment, there is a 90% reduction in relapse rate and an initial improvement in disability (as assessed by the EDSS score), which may represent release from conduction block due to residual inflammation. However, there is continued improvement in disability between 12-24 and 24-36 months. Although there are many possible explanations for this, we hypothesised that immune cells, regenerated after Campath-1H, secrete growth factors which promote neuronal survival and repair. We have shown that patients' PBMCs secrete neurotrophic factors ex-vivo in response to various stimuli and that PBMC-conditioned medium supports the survival of neurons derived from rat embryonic cortices. At 6 months post-treatment we found an increase in ciliary neurotrophic factor (CNTF); a factor recently shown to enhance myelin formation. Additionally, we found a dramatic reduction in insulin-like growth factor-1 (IGF-1) secretion. IGF-1 mediates protection from Fas-induced cell death, a process essential for immune system homeostasis by clearing activated T cells. This finding suggests that Campath's immuno-modulation may, in part, be due to the apoptotic elimination of activated T cells. Increasing evidence indicates that the Epstein-Barr virus (EBV) has a role in the pathogenesis of many autoimmune diseases, including multiple sclerosis (MS). We have proposed a new hypothesis that chronic autoimmune diseases occur in individuals genetically susceptible to the effects of B-cell infection by EBV, with a resultant increase in the frequency of EBV-infected autoreactive B cells, which not only produce autoantibodies but also inhibit activation-induced apoptosis of autoreactive T cells in the target organ [Trends Immunol. 24 (2003) 584 ]. This study aims to determine whether MS patients have an increased frequency of EBV-infected immortalized autoreactive B cells leading to the development of MS. We are using realtime PCR to measure the EBV DNA load in peripheral blood mononuclear cells (PBMCs), and ELISA and myelin opsonization-for-phagocytosis assays to measure antimyelin antibodies. In addition to detecting EBV DNA in PBMCs of MS patients, we have found that, compared to healthy subjects and patients with other neurological diseases, MS patients have higher levels of circulating antibodies to two overlapping peptides of myelin proteolipid protein (PLP), namely PLP184-199 and PLP190-209, and increased levels of opsonizing antimyelin antibodies. One MS patient with high circulating levels of opsonizing antimyelin antibodies and severe progressive spinal cord involvement refractory to intravenous methylprednisolone and glatiramer acetate improved following B cell depletion by rituximab.
Primary progressive multiple sclerosis converted to relapsing remitting multiple sclerosis after intensive immunosuppressive therapy E.J. Bartholomé a , M. Vokaer a , D. Bron b , M. Toungouz a and M. Goldman a a Hô pital Erasme, Brussels, Belgium; b Hô pital Bordet, Brussels, Belgium Primary progressive multiple sclerosis (PPMS) is a devastating form of MS resulting in progressive handicap without remission. It is less frequent than relapsing-remitting MS (RRMS) and some studies suggest it may represent a totally separated disease. We provide evidence of conversion from PPMS to RRMS in a patient after intensive immunosuppression. Our patient born in 1966 suffered from MS since 1986. Clinical diagnosis of PPMS was confirmed by nuclear magnetic resonance, evoked potential and lumbar puncture findings. Between 1998 and 2000, aggressive evolution despite corticoids, azathioprine and cyclophosphamide therapy resulted in severe handicap development. In December 2000, the patient became wheelchair dependant. Peripheral blood stem cell were mobilized, harvested and highly purified in February 2001. In March 2001, the patient received 200 mg/kg Cyclophosphamide followed by autograft rescue with successful haematological recovery. Handicap progressively recessed to stabilize by the end of 2001. Then, the patient was able to walk alone more than 100 m. However, the patient experienced two relapse in 2003 (optic neuritis, left arm neuropathic pain). Both relapses were followed by partial remission over 3 months and confirmed by nuclear magnetic resonance and ophthalmologic findings. This case proves that PPMS may convert to RRMS after intensive immunosuppression, suggesting that PPMS and RRMS are not separate entities but distinct expression of a common immunological disorder.
Identification of novel lead compounds in modulation of pathogenic immune responses in multiple sclerosis M. Massa a , R. Campanelli a , A. Uccelli b , V. Meli c , P. Lanza c , R. Billetta c , A. Martini d and S. Albani e a IRCCS S. Matteo Hospital, Pavia, Italy; b University of Genoa, Genoa, Italy; c Androclus Therapeutics, San Diego, USA; d IRCCS G. Gaslini Institute, Genoa, Italy; e University of California, San Diego, USA Immune responses to bacterial heat shock proteins (HSP) may play a role in autoimmunity. By applying a computer algorithm for the identification of panHLADR binder motifs, we have defined two sets of peptides from bacterial HSP hsp60 and Escherichia coli dnaJ, which may function as relevant T cell epitopes in multiple sclerosis (MS). A PanDR-peptide was tested as control peptide. PBMC from 33 MS individuals were stimulated with individual proband and control peptides. We found that peptides B1, B5, B6, B7 from E. coli dnaJ were able to induce an increased expression of CD69 ( pb0.04) or T cell proliferation ( pb0.02) compared to the control peptide. In addition, while peptide B1 induced an increased intracellular production of IFNgamma ( p=0.04) and TNFalpha ( p=0.02) by stimulated PBMC, peptide B6 elicited bulk cultures to produce intracellular IL10 ( p=0.04) or IL10 and IFNgamma ( p=0.03). A significantly higher percentage of IFNgamma and IL10 producing cells was also found following incubation with hsp60 derived peptides P10 or P11 than with control peptide. Thus, HSP-derived peptides are capable of inducing both pro-inflammatory and regulatory responses in MS subjects. These results suggest that responses to HSP peptides may play a modulating role during CNS autoimmunity and may be exploited at tailoring immunomodulatory compounds for the treatment of MS. Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system caused by an autoimmune attack directed against CNS myelin antigens. Vdelta2 cells form a minor component of the circulating peripheral lymphocyte pool and also home to sites of inflammation, including the brain. It has been reported that in vitro activation of human Vdelta2T cells by nonpeptidic ligands rapidly induces production of IFNgamma. High amounts of IFN-gamma that is produced rapidly by T cells after bacterial infection can have adverse effects for the development of intracellular pathogens. On the other hand, gamma\delta T cells may contribute to the immunopathology associated with chronic inflammatory or autoimmune disorders through a high and sustained release of inflammatory cytokines and chemokines. Here, we have evaluated gamma/delta T cell responses to pyrenil-pyrophosphate antigen with and without ST1959, a compound of the triazoles class of molecules, in healthy and MS individuals. Our results show that ST1959 significantly inhibits proliferation and cytokine production in gamma/deltaT cells. Furthermore, ST1959 efficiently interferes with antigen specific T cell activation. We conclude that ST1959 represents a novel immunomodulatory agent in the treatment of T cell mediated autoimmune diseases, such as multiple sclerosis. T cell-mediated action against self-antigens is implicated in a variety of autoimmune diseases. This T cell activation is dependent on two signals: T cell receptor (TcR) engagement and co-stimulation. Co-stimulation is therefore a potential target for treating autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. We have previously shown that a series of small compounds, discovered at Avidex, bind exclusively to CD80 with nanomolar affinity (SPR technology) and block its interaction with CD28 (time resolved fluorescence resonance energy transfer assay). In the current study, we demonstrate the effects of these compounds in a range of cellular assays using both transformed cells and primary human T cells. The compounds inhibit cytokine release from Jurkat cells (interleukin-2 (IL-2)) and primary CD4+ T cells (IL-2, interferon-gamma and tumour necrosis factor-alpha) with IC50 in the sub-micromolar range. T cell activation is therefore reduced by the presence of molecules bound to CD80 and these compounds represent promising leads for the development of novel autoimmune therapies.
Influence of estriol therapy on inflammatory responses on brain endothelial cell tight junction molecules in multiple sclerosis I.V. Korzh, I.F. Fedotova and V.D. Nemtsova Kharkov Medical University, Kharkov, Ukraine
The protective effect of pregnancy on putative Th1-mediated autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis, is associated with a Th1 to Th2 immune shift during pregnancy. The hormone estriol increases during pregnancy and has been shown to ameliorate experimental autoimmune encephalomyelitis and collagen-induced arthritis. To elucidate mechanisms of endothelial cell (EC) dysfunction in CNS inflammatory responses and beneficial effects of oral estriol therapy in multiple sclerosis (MS), we analyzed effects of individual and combinations of soluble inflammatory mediators on the intracellular localization of the EC tight junction-associated molecules zonula occludens-1 and -2 (ZO-1 and ZO-2) in human brain ECs. The cytoplasm in the majority of cells in control EC cultures was clear; ZO-1 and ZO-2 were localized peripherally near sites of cell contact and associated with submembranous cytoplasmic filaments. H2O2 induced reversible time-and concentration-dependent translocation of ZO-1 and ZO-2 to a random distribution within EC cytoplasm and retraction of EC borders. For low concentrations, these effects were accompanied by less prominent submembranous filaments but not by evidence of cytotoxicity, increased cell death or altered amounts of ZO-1. Tumor necrosis factor-beta induced similar alterations but estriol did not. Co-treatment with either cytokine increased H 2 O 2 effects, whereas estriol therapy reversed H 2 O 2 -induced effects. In control white matter samples, EC cytoplasm was clear and ZO-1 was located on cell borders. In inflammatory/ demyelinating lesions, EC ZO-1 was diffuse, indicating that the alterations induced in vitro mimic those in active MS lesions. These findings suggest that in MS patients, estriol treatment may counteract inflammatory mediator effects on CNS EC tight junction molecules, thereby preserving EC barrier function. Type 1 interferons such as IFN-beta appear critical to trigger immunoregulatory mechanisms lying at the interface between innate and adaptive immunity. The therapeutic effect of IFN-beta could be due to restoration of immunoregulatory mechanisms that are defective in multiple sclerosis (MS) patients such as regulatory NKT cell/CD1d pathway. Aim: to analyze the NKT cell defect in patients affected by MS and to test whether treatment with IFN-b1a improves immunoregulatory NKT cell function. Design: NKT cells percentages in peripheral blood lymphocytes (PBL) and secretion of cytokines (IFN-gamma, IL-4, IL-10, IL-12 and TGF-beta) by NKT cells stimulated with their model antigen agalactosylceramide were measured in RR MS patients before and after treatment with IFN-b1a. Individuals affected by other neurological diseases and healthy subjects were used as controls. Results: Our preliminary data showed that percentages of NKT cells are reduced in MS patients prior to IFN-b1a treatment compared to controls. In some patients percentage of NKT cells in PBL increased with IFNb-1a treatment. Moreover, IFN-b1a increased secretion of cytokines by NKT cells of MS patients without shifting NKT cell cytokine profile from an inflammatory Th1 toward a protective IL-4secreting type.
Neutralizing antibodies have a higher affinity to interferon-beta than non-neutralizing antibodies P. Tripp, C. Gneiss and F. Deisenhammer Innsbruck Medical University, Dept. of Neurology, Innsbruck, Austria Background: Patients with multiple sclerosis receiving recombinant interferon-beta (IFN-beta) may develop binding antibodies (BAB) against IFN-beta. Two types of BAB can be distinguished, neutralizing (NAB) and non-neutralizing antibodies (NNAB). NAB are associated with greater BAB titers than NNAB, suggesting a quantitative influence on NAB development. The correlation between NAB and BAB is weak and there is evidence that IFN-beta neutralization also depends on qualitative components. In this study the affinity of NAB vs. NNAB was investigated. Methods: Thirty-eight serum samples with BAB of 27 MS patients receiving IFN-beta-1a or 1b were included in this study. Of these, 21 samples were NAB-positive and 17 were NAB-negative. The relative affinity values (RAV) of IFN-beta antibodies were determined using an affinity assay as described earlier [J. Neuroimmunol. 66 (1996) 85] . The determination of RAV is based upon disrupting interactions between antibody and antigen using increasing concentrations of sodium isothiocyanate. Results: RAVs were significantly higher in NAB-positive (266.6F5.884) than in NAB-negative (223.8F15.96) serum samples. The amount of antibodies as measured by optical density did not differ between both groups (0.9803F0.035 vs. 0.9771F0.033). No significant correlation was observed between RAV and BAB or NAB titers. Conclusions: After exclusion of confounding factors these results indicate that NAB have a higher affinity to IFN-beta than NNAB.
Evaluation of functional disability in multiple sclerosis patients during interferon beta treatment M. Bošnjak Pašic, M. Lisak, Z. Trkanjec and V. Demarin Sestre Milosrdnice University Hospital, Zagreb, Croatia Introduction: Fifteen patients (10 females and 5 males) with remittingrelapsing multiple sclerosis (RRMS) were treated with interferon beta at University Department of Neurology. Ten patients were treated with interferon beta 1a (6 MIU three times weekly) and five with interferon beta 1b (9,6 MIU every other day). Methods: We used the Expanded Disability Status Scale (EDSS) to evaluate functional disability in RRMS patients. An average EDSS score was recorded before interferon beta therapy and 6 months after initiation of interferon beta therapy. Results: In interferon beta 1a group average EDSS score before therapy was 3.00 and after 6 months of therapy it decreased to 2.92. In interferon beta 1b group an average EDSS score was 3.07, before treatment, and after 6 months of therapy it was 3.00. There was no statistically significant difference between interferon beta 1a group and interferon beta 1b group, before and after 6 months of therapy. The average EDSS score was slightly smaller after 6 months of treatment in both groups, but the difference was not statistically significant ( p=0.17 in interferon beta 1a group and p=0.36 in interferon beta 1b group). Conclusions: Results favor interferon beta therapy in these small groups of RRMS patients. Further follow-ups are required to obtain data about functional status during interferon beta treatment after longer period of time. IFN-beta has shown to be an effective treatment for multiple sclerosis (MS), however, some patients fail to respond fully. The biological activity of IFNbeta is exerted through the IFN receptor, composed of two subunits, IFNAR1 and IFNAR2. Deficiency of one or both of these subunits may lead to a loss of interferon activity. To evaluate differences in IFNAR1 and IFNAR2 expression in MS patients treated with different IFN-beta molecules we quantified IFNAR1 and IFNAR2 mRNA by real-time RT-PCR in peripheral blood mononuclear cells. Clinical disease activity was based on annual relapse rate and EDSS. A total of 210 MS patients, 45 treated with IFN-beta1b (Betaferon), 46 with IFN-beta1a (Avonex), 68 with IFN-beta1a (Rebif) and 30 without treatment were included. The results were analysed by comparisons with a healthy control group. A nonparametric test showed significant decrease in patients treated with IFNbeta compared to patients without treatment and healthy controls in both IFNAR1 and IFNAR2 expression. There was no difference in the IFNAR1 or IFNAR2 expression between patients with any of the treatment used. We found a decrease in both IFNAR1 and IFNAR2 expression in MS patients compared to healthy controls, and also a decrease in this expression was observed in treated MS patients compared to non-treated MS patients. Naturally occurring regulatory CD4+CD25+ T cells at a crossroads of neuro-immune interactions J. Kipnis, M. Cardon, H. Avidan, S. Mordechay, L. Gil and M. Schwartz Weizmann Institute of Science, Revohot, Israel CNS injury activates autoimmune T cells, which accumulate at the site of injury. These T cells were believed to mediate processes of secondary degeneration of the neural tissue. Our group has recently showed that these autoimmune T cells have a beneficial protective effect on neurons and are protecting non-damaged vulnerable to death neurons from degeneration. Autoimmune T cells are kept in the periphery under tight control of naturally occurring regulatory T cells and a mechanism underlying an ability to evoke autoimmune response to CNS injury without developing an autoimmune disease is largely unknown. In this study we show that certain neurotransmitters, increased after CNS injury, can preferentially act on naturally occurring CD4+CD25+ regulatory T cells through a specific receptors, expressed by these cells, and alleviate their inhibitory activity. The same neurotransmitters also affect the adhesion and migration of these cells in a mild manner, namely not eliminating, but rather weakening those functions. The effect of suppression is mediated through reduced expression of CTLA-4 and reduced production of TGF-beta and IL-10, however, the adhesion and migration are reduced by decrease in expression of the specific receptors for adhesion molecules and chemokines. This effect on multiple functions of regulatory T cells involves ERK1/2 dependent pathways. High sensitivity of regulatory T cells to neurotransmitters might represent the bmissing linkQ between the immune and the nervous systems interactions. A bfine tuningQ on a level of regulatory T cells by neurotransmitters could be a promising therapy for autoimmune and neurodegenerative disorders. Human CD4+CD25+ Treg cells expressing the highest levels of CD25 (2-4% CD4+ T cells) are the most efficient suppressors of CD4+CD25À T cell activation. Since these Treg cells are believed to control auto-reactivity, experiments were performed to determine whether CD4+CD25high Treg cells isolated from the peripheral blood of patients with multiple sclerosis exhibited alterations in frequency or function as compared to those from healthy controls. While there was no difference in the frequency of CD4+CD25high cells between patients and controls, Treg cells isolated from patients with MS were significantly less suppressive. Furthermore, Treg cells from MS patients were less able to suppress responder T cells from allogeneic healthy controls, while Treg cells isolated from healthy controls could strongly inhibit responder T cells isolated from MS patients. Functional analysis of non-activated CD4+CD25high cells expressing only high levels of CD62L demonstrated an even greater functional disparity between patients and controls. Furthermore, two subsets within the CD4+CD25high Treg population differing by HLA-DR expression, exhibit striking temporal differences in suppression and regulation of Th2 cytokines indicating that subsets of CD4+CD25high Tregs may function through different mechanisms. As skewing towards Th2 responses has important implications for many autoimmune diseases, this investigation is being expanded to Treg subsets isolated from patients with MS. The central role of T cells in inflammatory reactions of the central nervous system (CNS) is well documented. Much less is known about the few T cells found within the intact organ: In particular, the contribution of CD4+ and CD8+ T cells to the infiltrating lymphocyte pool, their preferential target sites within the CNS, and their responses to environmental cues provided by neurodegeneration are largely undefined. To address these points, we used the Lewis rat as model organism, and studied T cells in the intact and degenerative spinal cord. In the intact spinal cord, T cells were preferentially located to the CNS gray matter, and CD8+ T cells were more numerous than CD4+ lymphocytes. In cases of degeneration, T cells increased in numbers and preferentially infiltrated affected CNS areas. These effects were more pronounced for the CD4+ than for the CD8+ T cell subset. Collectively, these data provide evidence for a clear cellular and compartmental bias in immune cell infiltration and in the immune response to degeneration within the CNS. To learn whether T cells recruited to sites of neurodegeneration represent single or multiple T cell clones, we are currently performing T cell receptor analysis, combining spectratyping, single cell PCR of laser microdissected T cells and immunohistochemistry.
Active and differential regulation of autoreactive CD4+ and CD8+ T cell responses by the CNS: A threshold for autoimmunity within the CNS C.S. Anglen, C. Ploix and M. Carson The Scripps Research Institute, La Jolla, CA, USA To define how the CNS regulates autoreactive T cell responses, we used transgenic mice expressing hemagglutinin (HA) exclusively in CNS astrocytes (GFAP-HA mice) or exclusively in pancreatic beta cells (INS-HA mice). Ins-HA mice that also express CD4+ (6.5) or CD8+ (clone4) HA-specific TCRs develop spontaneous autoimmune diabetes; whereas GFAP-HA mice co-expressing either HA-specific TCR do not develop CNS autoimmunity. Similarly, transfer of HA-specific CD4+ T cells into lymphopenic INS-HA mice led to autoimmune diabetes, but failed to cause CNS autoimmunity in GFAP-HA mice. In transgenic mice expressing HA in both the CNS and pancreas (GFAP-HAxINS-HA mice), encounter with CNS-expressed antigen led to clonal diversion of HA-specific CD4+ T cells, decreased diabetes incidence, and infrequent CNS inflammation. In contrast, co-transfer of HA-specific CD4+ and CD8+ T cells did trigger diabetes and modest signs of clinical ataxic disease in GFAP-HAxINS-HA mice. Autoreactive CD4+ and CD8+ T cells also differentially responded to adjuvant challenge. Co-administration of pertussis toxin (PT) with HA-specific CD4+ T cells triggered rapid lethal CNS inflammation in GFAP-HA mice, while co-administration of PT with HA-specific CD8+ T cells failed to produce CNS disease. These results suggest that regulation of autoreactive T cell responses is not only tissue-specific, but that the CNS actively exerts differential regulation of CD4+ versus CD8+ T cells.
A paradoxical role of APCs in the induction of intravenous tolerance in experimental autoimmune encephalomyelitis G.-X. Zhang, B. Gran, S. Yu, Y. Li, E. Ventura and A. Rostami Thomas Jefferson University, Philadelphia, PA, USA APCs, based on their phenotypes, have been considered to be important in determining Th1/Th2 differentiation. Here we define whether immunoregulatory function of T cells in the induction of antoantigen-specific tolerance is mediated via APCs. Intravenous (i.v.) tolerance against experimental autoimmune encephalomyelitis (EAE) was induced by administering myelin oligodendrocyte glycoprotein (MOG) 35-55, and APCs/CD4+ T cells from MOG-i.v. or PBS-i.v . mice were purified to analyze their functions in vitro and in vivo. Co-culture of purified APCs and CD4+ T cells showed that T cells, but not APCs, play a predominant role in i.v. tolerance induction. In vivo, APCs of MOG-i.v. mice induced a non-specific Th1-type response in naRve mice, and were less effective in suppressing clinical EAE than APCs of PBS-i.v. mice. Further, increased levels of IL-12p40, IL-23p19 and IL-23 receptor mRNA expression were found in APCs of MOG-i.v. mice. We conclude that induction of i.v. tolerance is a T cell phenomenon and that APCs are not tolerized, but indeed immuno-stimulatory, upon systemic exposure to autoantigen. Blocking this APC activation, therefore, would enhance the effectiveness of tolerance induction and block the potential reversal of tolerance in preventing and treating EAE. Using retrovirally transduced, GFP labeled T cells we followed the fate of encephalitogenic effector cells in the clinical phase of EAE. We had learned masses of the pathogenic effector T cells invade deeply into the CNS. Using life-imaging techniques, we now have followed green autoreactive MBP specific T cells within the brain parenchyma. Many of the cells (60-70%) cruise with unexpected speed (bmotile typeQ) and in seemingly undirected fashion. Other cells (30-40%), however, attach and begin to toss around some central object (bstationary typeQ). We speculate that the motile cells are in search of their brain autoantigen, and that the stationary cells just have found their autoantigen, that their arrest reflects presentation and recognition of the antigen. Several observations support our interpretation. First, T cells, which do not recognize brain antigens, such as ovalbumin specific T cells, move almost exclusively (98%) in a motile fashion. Second, the motility pattern correlates with the local reactivation level as well as with the pathogenic potential of the transferred effector T cells. Thus, T cells with a high pathogenic potential, as the MBP specific, display a relatively high proportion of stationary cells, while T cells of low pathogenic potential (e.g. specific for other brain autoantigen such as S100beta), show a significantly lower proportion of stationary cells (15%). This tightly correlates with in situ activation as assessed by T cell receptor down-modulation, up-modulation of activation markers, pro-inflammatory cytokines and chemokines. Impaired function of CD4+CD25high regulatory T cells in patients with multiple sclerosis J. Haas a , T. Vetter a , L. Milkova a , A. Viehfver a , A. Hug a , C. Falk b , E. Suri-Payer c , B. Fritz a , P. Krammer c and B. Wildemann a a Department of Neurology, University of Heidelberg, Heidelberg, Germany; b GSF-Institute of Molecular Immunology, Munich, Germany; c DKFZ, Heidelberg, Germany
Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS). Since activation of circulating self-reactive T cells with specificity for myelin components is considered to be an important disease initiating event, effective immunoregulation of peripheral T cells may be critical to prevent autoimmunity within the CNS. Immunoregulatory T cells of the CD4+CD25high phenotype (Treg) suppress T cell function and protect rodents from organ-specific autoimmunity. The precise role of Treg in human autoimmunity is unclear. We assessed prevalence and suppressor function of Treg isolated from peripheral blood of patients with clinically active relapsing-remitting MS and healthy controls using flow cytometry, gene expression analysis, and in vitro proliferation assays. We demonstrate that the suppressive potency of patient-derived Treg is impaired, as their inhibitory effect on allogeneic and polyclonal T cell proliferative responses as well as on antigen specific T cell proliferation induced by myelin oligodendrocyte glycoprotein is significantly reduced compared to Treg from controls. Number and cell surface phenotype of Treg were not altered and no differences as to the composition and activation state of CD4+CD25À responder T cells were observed. These results suggest that an impaired inhibitory effect of Treg on autoreactive T cell clones might be involved in the pathogenesis of MS.
Identification of a novel natural regulatory CD8 T cell subset and analysis of its mechanism of regulation E. Xystrakis, A. Dejean, I. Bernard and A. Saoudi INSERM U563, Toulouse, France
Normal immune system contains bprofessionalQ natural regulatory T cells that control physiological as well as pathological immunity. Although the suppressive function was historically attributed to CD8 T cells, most current reports about natural regulatory T cells focus on CD4 T cells. In the present study, we identify a new subset of natural CD8 regulatory T cells in normal healthy animals. This subset expresses low level of CD45RC at its surface (CD45RClow), produces mainly IL-4, IL-10 and IL-13 cytokines upon in vitro stimulation and is not cytotoxic against allogeneic targets. In coculture experiments and after in vitro stimulation with allogeneic accessory cells, this subset suppresses the proliferation and differentiation of autologous CD4 T cells into type-1 cytokines producing T cells. This regulatory subset mediates its suppression by cellcell contact and not through secretion of suppressive cytokines. The regulatory activity of CD8 CD45RClow cells is also demonstrated in vivo in a rat model of CD4-dependent Graft-versus Host Disease. Collectively, these data demonstrate for the first time that freshly isolated rat CD8 CD45RClow T cells contain T cells with regulatory properties, a result that enlarges the general figure of T cell mediated regulation. The role of this cell subset in the regulation of central nervous system autoimmunity is under investigation. Campath-1H, a monoclonal antibody used in the treatment of multiple sclerosis (MS), induces persistent T lymphopenia and remission of autoimmune disease activity. During lymphocyte repopulation, a third of patients develop autoimmune thyroid disease. The interaction of lymphocytes with MHC class II: self antigen complexes and cytokines such as interleukin-7 (IL-7) are critical for the maintenance of T cell number and diversity. We assessed the impact of Campath on these homeostatic mechanisms and their possible involvement in the subsequent development of Graves' disease. We demonstrate greater proliferation of unstimulated lymphocytes from untreated patients, suggesting increased homeostatic proliferation (HP). However, IL-7 serum levels were equivalent in patients and controls. Lymphopenia induced a significant increase in serum IL-7 levels, which remained at 12 months. An increased percentage of unstimulated lymphocytes, from patients 1 year following treatment, proliferate when left for 10 days in culture. This result is mimicked using peripheral blood mononuclear cells from controls, variably depleted ex vivo of T lymphocytes, suggesting the involvement of lymphopenia-driven HP. Proliferation was prevented if the interaction between TCR and MHC: self peptide is blocked. In conclusion, MS patients respond to lymphopaenia appropriately. Variation in responses between patients, and the differential effect of IL-7 on various T cell subsets may predispose to the development of new autoimmune phenomenon. Also, we have evidence that HP may be exaggerated in patients with MS perhaps suggesting a mechanism by which autoimmunity may arises. During autoimmune inflammation of the central nervous system (CNS) as occurs in multiple sclerosis (MS), self reactive immune response is compartmentalized, and oligoclonal expansion of B cells has been observed. Probably equivalent expansion occurs in T cell subpopulations because ongoing antigen stimulation. In order to assess whether clonal composition of ex vivo unstimulated cerebrospinal fluid (CSF) T-cells might be related to pathogenic potential, molecular analysis of T-cell receptor (TCR) Vh transcripts of 46 functionally rearranged genes and global comparison of the complementarity-determining region 3 length distribution (CDR3-LD), was carried out ex vivo, contemporarily in unstimulated CSF cells and PBMCs. The method, consisting in a twostep Multiplex RT-PCR, allowed expansion of the whole TCR Vh gene repertoire from as few as 10(5) cells, indicating that it was sensitive enough to evaluate CSF T cells. As skewed usage of the TCRVh chain CDR3 segment is a marker of antigen-driven expansion, the expansions allowed identification and separation by monoclonal Abs and magnetic microbeads, of a CSF T-cell subpopulation that should be enriched of Ag specific cells and allow generation at high frequency of CNS Ag-specific T cell clones. These bpathogenic relevantQ T-cells from CSF are being examined to characterize their phenotypic and functional properties. Preliminary findings suggest that the strategy adopted is a powerful method for sensitive and accurate detection and characterization of the few relevant Tcells in CNS inflammation.
Multiple sclerosis brain-derived cDNA expression libraries allow the identification of novel autoantigens for CSF-infiltrating CD4+ T lymphocytes D.M. Park a , M. Sospedra a , X. Wang a , J. Quandt a , C. Raine b , H.F. McFarland a and R. Martin a a National Institutes of Health, Bethesda, USA; b Albert Einstein College of Medicine, New York, NY, USA CD4+ T cells are believed to play a central role in the pathogenesis of multiple sclerosis (MS). Although extensive research is aimed at characterizing myelin-reactive CD4+ T cells, it remains unknown whether myelin components are the principal or only targets of the autoimmune response in MS. We therefore tried to identify MS-related autoantigens in an unbiased genetic-based approach to determine the targets of cerebrospinal fluid (CSF) infiltrating CD4+ T cell clones (TCC). MS plaque tissue-derived cDNAs were cloned into an expression system designed for human leukocyte antigen (HLA) class II-restricted antigen presentation. A peripheral blood derived myelin-basic protein (MBP)-specific CD4+ TCC served as control. Three CSF-derived TCCs were isolated from an MS patient during exacerbation and characterized in detail with respect to HLArestriction and phenotype. Each of the CSF-derived TCCs was in vivo clonally expanded as shown by complementarity determining region 3 spectratyping. The MBP-specific clone recognized MBP from the plaquederived library, confirming the technical feasibility of the approach. The CSF-derived clonally expanded CD4+ TCCs recognized an array of interesting targets. These findings were further supported by data generated with positional scanning synthetic peptide combinatorial libraries. Our data indicate that likely disease-relevant CSF-infiltrating T cells recognize a much broader range of antigens within MS brain tissue than previously anticipated.
Putative mechanisms of statins in multiple sclerosis-preferential inhibition of activated T lymphocytes O. Neuhaus a , O. Stuve a , J.J. Archelos b and H.-P. Hartung a a Heinrich Heine University, Düsseldorf, Germany; b Karl Franzens University, Graz, Austria Objective: To investigate the effects of statins on human T and B lymphocytes. Background: It was recently demonstrated that statins, 3hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, have immunomodulatory properties and ameliorate disease severity in experimental allergic encephalomyelitis. Evidence is emerging that statins reduce MRI disease activation markers in MS patients. Methods: Mitogenactivated, antigen-specific, and naRve peripheral blood leukocytes obtained from patients with relapsing-remitting MS or from healthy donors were treated with various statins. The proliferative responsiveness and the presence of surface markers, including activation markers and adhesion molecules were evaluated. Results: Statins reduced proliferative activity of mitogen-activated and antigen-stimulated T cells in a dose-dependent manner, simvastatin being most potent, followed by lovastatin and mevastatin. Proliferation of B cells was not affected. There was a marked alteration in the surface marker expression of activated T cells, with a decrease in the ratio of CD45RO+ cells. Following statin-treatment, the phenotype of activated T cells approached that of non-activated T cells. Conclusions: Our results support the prevalent hypothesis regarding mechanisms of action of statins: intermediates of the cholesterol biosynthesis pathway downstream of HMG-CoA promote isoprenylation, i.e. post-translational lipid attachment of intracellular proteins that regulate the activation of lymphocytes. The activation of autoreactive encephalitogenic T cells may be affected by statin treatment in MS patients.
The frequency of CD4(+)CD25(+) T regulatory cells is increased in the CSF but not in the blood of MS patients U. Feger, C. Luther, B. Schreiner, A. Melms, E. Tolosa and H. Wiendl Department of Neurology, University of Tübingen, Tübingen, Germany CD4(+)CD25(+) regulatory T cells contribute to the maintenance of peripheral tolerance by active suppression. They control the reactivity of potentially harmful, self-reactive T cells and prevent autoimmune disease. Multiple sclerosis (MS) is an inflammatory autoimmune disease thought to be mediated by T cells recognizing myelin protein peptides. To address the question if regulatory T cells are involved in the immunopathogenesis of MS, we measured the frequency of CD4(+)CD25(+) regulatory T cells by 4-colour flow cytometry in the blood and the CSF of MS patients in comparison to healthy donors and to patients with non-autoimmune neurological disease. In agreement with other studies, we did not observe any difference in the amount of blood circulating CD25À high CD4+ cells in patients with either chronic or remitting-relapsing MS in comparison to normal donors. By contrast, a higher frequency of regulatory T cells was found in the CSF from the same MS patients, but not in the CSF from patients with other neurological disorders. Selective elevation in the CSF compartment of MS patients therefore suggests a role of CD4(+)CD25(+) regulatory T cells in the development of autoimmune CNS demyelination.
Pathologic relevance of degenerate foreign and self antigen recognition by a myelin-specific HLA-DR2-restricted T cell receptor J.A. Quandt a , J. Huh a , D. Park a , C. Pinilla b , K. Ito c and R. Martin a a National Institutes of Health, Bethesda, USA; b Torrey Pines Institute for Molecular Studies, San Diego, USA; c University of Medicine and Dentistry of New Jersey, Piscataway, USA Degenerate antigen recognition by T cells may contribute significantly to autoimmunity through activation of auto-reactive T cells via molecular mimicry and the subsequent development of disease. Two unbiased approaches were used to demonstrate the degenerate antigen recognition of a clone recognizing the immunodominant myelin basic protein (MBP) epitope restricted by a multiple sclerosis (MS)-associated HLA DR2 allele. Screening of positional scanning combinatorial peptide libraries (PSCL) and an MS brain cDNA expression library with a MBP83-99 specific HLA-DR2a-restricted T cell clone have identified numerous infectious/foreign proteins as well as ubiquitous autoantigens as ligands. PSCL studies predicted several bacterial and viral epitopes as well as myelin-associated proteins as molecular mimics of MBP83-99. Proliferation and cytokine studies confirmed PLP, MOG and MBP epitopes previously described as HLA DR2 immunodominant epitopes as well as others to be ligands for this T cell receptor (TCR). These reactivities were conserved in the humanized TCR/HLA DR2 transgenic mouse sharing this TCR, and studies are currently underway to test the pathologic relevance of these mimics in vivo. Together, these studies emphasize the highly degenerate antigen recognition of autoreactive T cells implicated in MS and describe an important means to identify bnovelQ antigens relevant to MS pathogenesis.
IL-12 suppresses CD4+CD25+ regulatory T cells: a possible mechanism of promoting EAE I. King and B.M. Segal University of Rochester, Rochester, USA
The IL-12p40 family of monokines plays a critical role in the differentiation of T lymphocytes into Th1 effector cells. We have previously demonstrated that IL-12p40 monokines are also critical for the development of relapsing-remitting experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis that is mediated by myelin-specific Th1 cells. In the current study we investigated the effects of IL-12 on CD4+CD25+ regulatory T cells (Treg), a thymusderived subpopulation of CD4+ T cells with immunosuppressive properties. Adoptive transfer of Treg prevents EAE in myelin-sensitized wildtype mice or mice bearing a T cell receptor transgene specific for myelin basic protein. Here we show that Treg express both subunits of the IL-12 receptor (beta1 and beta2 chains). Furthermore, recombinant IL-12 directly reverses Treg-mediated suppression of effector T cell proliferation in vitro. Endogenous Treg appear to infiltrate the CNS during peak EAE and to persist at high levels during remissions as indicated by the upregulation of Foxp3, a Treg-specific transcription factor, in spinal cords of sensitized mice. We conclude that one of the mechanisms by which IL-12 promotes EAE might be through inactivation of an endogenous population of Treg. Furthermore, Treg spontaneously infiltrate the CNS during EAE, suggesting that they might participate in triggering remissions.
Regulation of Th1/Th2 cytokine balance and inhibition of nuclear factor kappa B (NF kappa B) activation by cannabinoid in encephalitogenic T cells A. Garcia-Merino, P. Gonzalez, A. Arriaga and A. Sanchez Puerta de Hierro University Hospital, Madrid, Spain
Cannabinoids have been reported to downregulate the production of T helper 1 (Th1) associated cytokines, including IL-2, IL12, interferongamma (IFN-gamma) and tumor necrosis factor-alpha, and to increase the production of Th2 associated cytokines such as IL-4 and IL-10. Experimental autoimmune encephalomyelitis (EAE) of Lewis rats is a prototypic CD4+ Th1 cell-mediated disease. It is unknown whether the reported effects of cannabinoids on Th profile are relevant in EAE. An encephalitogenic T cell line derived from Lewis rats inoculated with guinea pig MBP, was cultured in presence of the synthetic cannabinoid R(+)-WIN 55,212-2 and analyzed by flow cytometry on a FACScanTM for detection of intracellular cytokines. Compared to control encephalitogenic cells, the cannabinoid reduced IFN gamma production and increased the concentration of IL-10, suggesting a shift towards the Th2 profile. Taking into account the crucial role played by the nuclear factor-kappa B (NF-kappa B) in the control of key genes involved in activation and differentiation of autoreactive T cells in vivo, we also investigated possible effects of the cannabinoid on activation of this nuclear factor. Using a construct containing NF-kappa B binding sequences, we observed that R(+)-WIN 55,212-2 decreased markedly promoter activity. These coordinated effects suggest a potential role of cannabinoids in autoimmune diseases. The Notch receptor, its ligands of the Delta/Jagged families and downstream signaling mediators form a highly conserved functional pathway playing a central role in cellular differentiation and proliferation events. Notch signalling is characterised by ligand-induced proteolytic cleavage events that release the Notch intracellular domain, which translocates to the nucleus and binds CBF1, converting it from repressor to transcriptional activator. Although transcriptional regulation via CBF1 remains the bestcharacterised outcome of Notch signalling, alternative CBF1-independent pathways have also been proposed. We show that Notch signalling via Delta promotes dose-dependent inhibition of murine T-cell effector cytokine production (IL-2, IL-5, IL-13, IFNgamma, TNFalpha) and upregulation of IL-4 and IL-10. Using a gamma-secretase inhibitor to block Notch cleavage, we show that Delta1-induced cytokine upregulation is CBF1-dependent. By contrast, Delta-mediated cytokine inhibition occurs rapidly and involves a different, cleavage-independent signalling mechanism. These activities are associated with Delta1-modified differentiation of cells: inhibited Th1 development and acquisition of a shared Th2/Treg profile. These effects occur even under conditions for promoting Th1 or Th2 differentiation. Notch signalling may, therefore, provide a unique therapeutic approach to the treatment of autoimmune disorders such as multiple sclerosis.
Targeting JAK-STAT pathway by nuclear receptor agonists in the treatment of Th1 cell-mediated autoimmune diseases J.J. Bright, G. Muthian, H.P. Raikwar, R. Johnson and C. Johnson Vanderbilt University Medical Center, Nashville, TN, USA Experimental allergic encephalomyelitis (EAE) is a CD4+ Th1 cellmediated autoimmune disease model of multiple sclerosis. IL-12 is a proinflammatory cytokine that plays a crucial role in the differentiation of neural antigen-specific Th1 cells and pathogenesis of EAE/MS. Signaling through its receptor, IL-12 induces the activation of JAK-STAT pathway in T cells and targeted disruption of this pathway inhibits Th1 differentiation. In this study, we have tested the hypothesis that JAK-STAT pathway is a novel therapeutic target in the treatment of Th1 cellmediated autoimmune diseases by nuclear receptor agonists. We found that in vivo treatment with specific agonists for nuclear receptors, PPARgamma, vitamin D receptor or estrogen receptor, ameliorated the clinical and pathological symptoms of EAE in SJL/J and C57BL/6 mice. Treatment of neural antigen-specific T cells with nuclear receptor agonists, 15d-PGJ2, Ciglitazone, vitamin D, estrogen or quercetin inhibited antigeninduced T cell proliferation and IFN-gamma production. The nuclear receptor agonists also inhibited IL-12-induced T cell proliferation and Th1 differentiation by blocking the tyrosine phosphorylation and activation of JAK2, TYK2, STAT3 and STAT4 proteins in T cells. These results suggest that nuclear receptor agonists modulate JAK-STAT signaling pathway in T cells and be useful in the treatment of MS and other Th1 cell mediated autoimmune diseases.
Computer modeling of lymphocyte behavior in inflamed brain venules by using stochastic Pi-calculus P. Lecca a , C. Priami a , C. Laudanna b and G. Constantin b a University of Trento, Trento, Italy; b University of Verona, Verona, Italy
The objective of this study was to use new mathematical languages to integrate quantitative and qualitative biological data and create a predictive computer modeling of lymphocyte recruitment in brain venules. Methods: Intravital microscopy was performed in inflamed brain microcirculation. A stochastic extension of the Pi-calculus, an ion based on the notion of naming, was used to model and simulate biological data. Results: Integration of interactions between PSGL-1-E-/P-selectin, VLA-4-VCAM-1 and LFA-1-ICAM-1 was required for efficient rolling of autoreactive lymphocytes, while Giprotein-linked signaling and interactions between LFA-1-ICAM-1 and VLA-4-VCAM-1 led to lymphocyte arrest. These results together with the quantification of hemodynamical parameters and adhesion molecule density allowed us to model the biological data as a set of concurrent processes. We created a computer model based on stochastic process algebras for mobile systems able to simulate the processes involved in lymphocyte recruitment in inflamed brain microvessels. Moreover, we showed that the model predicts the percentage of rolling lymphocytes in relation to hemodynamical parameters such as vessel diameter reproducing the functional exponential behavior of the in vivo results. Conclusions: The usage of mathematical languages may increase our ability to predict, and thus pharmacologically manipulate, complex key phenomena in the pathogenesis of autoimmune diseases.
Visualising T cell activation in situ in the nervous system K.R. Williams a , E.K. Mathey a , A. Flugel b and C. Linington a a Department of Medicine and Therapeutics, Institute of Medical Sciences, University of Aberdeen, Foresterhill Aberdeen, Scotland, UK; b Department of Neuroimmunology, Max Planck Institute of Neurobiology, Martinsried Munich, Germany
Recent studies using central nervous system antigen (CNS) specific T cell lines engineered to express green fluorescent protein (GFP) have demonstrated that cell activation is crucial in determining disease severity. In adoptive transfer experimental autoimmune encephalomyelitis differences in activation levels of infiltrated T cells are antigen specific and determine their ability to recruit macrophages, explaining why some T cell lines are weakly pathogenic (S100beta) while others are strongly pathogenic (myelin basic protein). However, very little is known about reactivation of T cells in the CNS, in particular when does it occur, where does it take place and what cell types act as the antigen presenting cells? To answer these questions we have developed a retroviral construct that tags GFP to CD3zeta, a component of the T cell receptor (TCR), which is recruited to the immune synapse formed between a T cell and stimulating antigen-presenting cell. We demonstrate that antigen-specific stimulation of transduced T cells in vitro results in the translocation of CD3zetaGFP to the cell surface where it co-localises with other components of the TCR complex. This immune synapse forms adjacent to class II MHC expression on the surface of antigen presenting cells. Analysis of autoantigen-specific CD3zetaGFP T cells after adoptive transfer reveals that immune synapses can be visualised in the CNS, enabling the identification of antigen-presenting cells in experimental autoimmune disease models.
VIP induces tolerogenic differentiation of immune cells J. Leceta, C. Martinez, M.G. Juarranz, C. Abad, A. Arranz, F. Rosignoli, M. Garcia and R.P. Gomariz Departamento de Biología Celular, Facultad de Biología, Universidad Complutense, Madrid, Spain
Vasoactive intestinal peptide (VIP) is a neuropeptide that modulates immunity through cellular signalling mediated by G protein-coupled receptors in immune cells. It is a potent anti-inflammatory factor, inducing differentiation toward a Th2 response. Several autoimmune diseases present an imbalance in Th1/Th2 responses, with a prevalence of Th1. VIP has a protective effect in a murine experimental model of collagen type II (CII)induced arthritis (CIA), an autoimmune process similar to human rheumatoid arthritis. The aim of this study was to determine the expression of cytokines, chemokines, and their receptors in joints of arthritic mice treated with VIP, as well as to check the immune response against CII of cells from treated mice. Gene expression for cytokines, chemokines and their receptors were performed using mouse-targeted DNA microarrays. Proliferative response and secretion of cytokines in cultures of synovial and spleen cells stimulated with CII were used to study the immune response. Th1 and Th2 cytokines are expressed in the joints of CIA mice. However, VIP reduces their expression more than 80%, but different cytokines are affected selectively. IL-10 is the more expressed cytokine and IL-2Ra is little affected. Local expression of mediators after VIP treatment indicates a shift in the cellular infiltration favouring the homing of mature dendritic cells, CD25+ cells, naive T cells, and Th2 cells. VIP treatment inhibits proliferation of CII stimulated cells. In conclusion, VIP biases immune response toward a Th2/T regulatory reactivity.
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