Introduction
Activated SFB play a major role in the crippling destruction of cartilage and bone in the joints of patients with RA [1,2,12,13,14,15], either autonomously [1] or in concert with T cells and macrophages [16,17,18,19]. RA-SFB are a major part of the invasive pannus, a vascular and fibrous granulation tissue arising from the joint recessus and extending onto the surface of cartilage [20]. Several studies indicate that RA-SFB are morphologically and functionally altered [4,5,12], and strongly express markers of activation, including surface molecules (eg MHC-II, CD13, or VCAM-1 and other integrins) [1,2,13,21,22] and proto-oncogenes [23,24,25].
Activation of SFB in vitro generates several functional responses that may considerably contribute to joint pathology in RA; that is, the production of matrix components [11,26], soluble mediators [17,27], or matrix degrading enzymes [28,29,30]. Because of the capability to act as antigen-presenting cells, activated SFB can also potentially fuel local antigen-driven processes relevant to the immunopathogenesis of RA [19,31]. Finally, stimulated RA-SFB elicit erosive arthritis upon intra-articular injection or engraftment into severe combined immunodeficiency mice [6,7].
To investigate ex vivo the pathophysiological properties of activated FB from the RASM, it is desirable to obtain freshly isolated SFB with phenotypic features as close as possible to the configuration observed in vivo. Because the generation of cell lines requires a number of passages in vitro to eliminate contaminating cells (especially macrophages), the risk of substantial in vitro growth selection exists [32]. Indeed, increasing the number of passages modifies the surface phenotype of synoviocytes [19] and increases the frequency of genotype changes [33]. We attempted to isolate FB from primary cultures of synovial cells to minimize these artifacts, with careful characterization of each preparation step and comparison of the phenotypic and functional features of primary-culture SFB with those of repeated-passage cells. Both positive and negative isolation approaches were pursued.
Positive identification/isolation of SFB was attempted using the mAb AS02, an antibody initially described as FB-specific [9] and subsequently shown to recognize the Thy-1 molecule (CD90, highly glycosylated membrane-bound protein; molecular weight, 30–35 kDa; core protein, 17 kDa; anchored via glycosylphosphatidyl-inositol [34]).
Negative isolation was performed using magnetobead-conjugated anti-CD14 mAbs. The choice of CD14 was based on the consideration that this is regarded as a reliable surface marker for RA synovial macrophages [23,35]; CD14 is also the macrophage marker most clearly correlated with disease activity and radiologic progression of joint destruction in RA [36]. The use of the most valid alternative (ie anti-CD68 mAbs) is limited by predominant expression of the CD68 antigen in the lysosomal compartment (requiring permeabilization of all cells for negative selection), and by the fact that CD68 epitopes recognized by certain mAbs are not specific for macrophages, but are also expressed by SFB [37,38]. The possibility of using anti-CD11b mAbs was also discarded, because CD11b is not specific for monocytes/macrophages and is also found on granulocytes and NK cells. CD11b expression on subpopulations of RA synovial macrophages may also be variable [39], possibly due to the local influence of cytokine stimulation [40,41]. Therefore, although the expression of CD14 on RA synovial macrophages may also be influenced by cytokines [36,42,43], the anti-CD14 mAb appeared the most appropriate choice for the purpose of this study.
Negative isolation from primary culture with magnetobead-conjugated anti-CD14 mAbs yielded highly enriched RA-SFB, positive for the known FB marker prolyl-4-hydroxylase (85%) and the recently suggested FB marker Thy-1 (74% [9]), with <2% macrophage contamination and <1% T cells, B cells, plasma cells, NK cells, dendritic cells, endothelial cells, or PMN. These cells bore surface and intracellular characteristics typical of the FB lineage when compared with primary-culture normal skin-FB (lineage control) or OA-SFB (disease control).