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T882 0-170 Sentence denotes Resistance to IL-10 inhibition of interferon gamma production and expression of suppressor of cytokine signaling 1 in CD4+ T cells from patients with rheumatoid arthritis
T1 0-170 Sentence denotes Resistance to IL-10 inhibition of interferon gamma production and expression of suppressor of cytokine signaling 1 in CD4+ T cells from patients with rheumatoid arthritis
T2 172-180 Sentence denotes Abstract
T883 181-298 Sentence denotes IL-10 has been shown to block the antigen-specific T-cell cytokine response by inhibiting the CD28 signaling pathway.
T3 181-298 Sentence denotes IL-10 has been shown to block the antigen-specific T-cell cytokine response by inhibiting the CD28 signaling pathway.
T884 299-634 Sentence denotes We found that peripheral blood CD4+ T cells from patients with active rheumatoid arthritis (RA) were able to produce greater amounts of interferon gamma after CD3 and CD28 costimulation in the presence of 1 ng/ml IL-10 than were normal control CD4+ T cells, although their surface expression of the type 1 IL-10 receptor was increased.
T4 299-634 Sentence denotes We found that peripheral blood CD4+ T cells from patients with active rheumatoid arthritis (RA) were able to produce greater amounts of interferon gamma after CD3 and CD28 costimulation in the presence of 1 ng/ml IL-10 than were normal control CD4+ T cells, although their surface expression of the type 1 IL-10 receptor was increased.
T885 635-835 Sentence denotes The phosphorylation of signal transducer and activator of transcription 3 was sustained in both blood and synovial tissue CD4+ T cells of RA, but it was not augmented by the presence of 1 ng/ml IL-10.
T5 635-835 Sentence denotes The phosphorylation of signal transducer and activator of transcription 3 was sustained in both blood and synovial tissue CD4+ T cells of RA, but it was not augmented by the presence of 1 ng/ml IL-10.
T886 836-1019 Sentence denotes Sera from RA patients induced signal transducer and activator of transcription 3 phosphorylation in normal CD4+ T cells, which was mostly abolished by neutralizing anti-IL-6 antibody.
T6 836-1019 Sentence denotes Sera from RA patients induced signal transducer and activator of transcription 3 phosphorylation in normal CD4+ T cells, which was mostly abolished by neutralizing anti-IL-6 antibody.
T887 1020-1132 Sentence denotes Preincubation of normal CD4+ T cells with IL-6 reduced IL-10-mediated inhibition of interferon gamma production.
T7 1020-1132 Sentence denotes Preincubation of normal CD4+ T cells with IL-6 reduced IL-10-mediated inhibition of interferon gamma production.
T888 1133-1358 Sentence denotes Blood CD4+ T cells from RA patients contained higher levels of suppressor of cytokine signaling 1 but lower levels of suppressor of cytokine signaling 3 mRNA compared with control CD4+ T cells, as determined by real-time PCR.
T8 1133-1358 Sentence denotes Blood CD4+ T cells from RA patients contained higher levels of suppressor of cytokine signaling 1 but lower levels of suppressor of cytokine signaling 3 mRNA compared with control CD4+ T cells, as determined by real-time PCR.
T889 1359-1678 Sentence denotes These results indicate that RA CD4+ T cells become resistant to the immunosuppressive effect of IL-10 before migration into synovial tissue, and this impaired IL-10 signaling may be associated with sustained signal transducer and activator of transcription 3 activation and suppressor of cytokine signaling 1 induction.
T9 1359-1678 Sentence denotes These results indicate that RA CD4+ T cells become resistant to the immunosuppressive effect of IL-10 before migration into synovial tissue, and this impaired IL-10 signaling may be associated with sustained signal transducer and activator of transcription 3 activation and suppressor of cytokine signaling 1 induction.
T10 1680-1692 Sentence denotes Introduction
T3234 1693-1833 Sentence denotes IL-10 is a key cytokine in regulating inflammatory responses, mainly by inhibiting the production and function of proinflammatory cytokines.
T11 1693-1833 Sentence denotes IL-10 is a key cytokine in regulating inflammatory responses, mainly by inhibiting the production and function of proinflammatory cytokines.
T3235 1834-2026 Sentence denotes IL-10 binds to the IL-10 receptor (IL-10R) complex that is composed of two subunits, the primary ligand-binding component type 1 IL-10R (IL-10R1) and the accessory component type 2 IL-10R [1].
T12 1834-2026 Sentence denotes IL-10 binds to the IL-10 receptor (IL-10R) complex that is composed of two subunits, the primary ligand-binding component type 1 IL-10R (IL-10R1) and the accessory component type 2 IL-10R [1].
T3236 2027-2218 Sentence denotes The interaction of IL-10 and IL-10R engages the Janus kinase (JAK) family tyrosine kinases Jak1 and Tyk2, which are constitutively associated with IL-10R1 and type 2 IL-10R, respectively [2].
T13 2027-2218 Sentence denotes The interaction of IL-10 and IL-10R engages the Janus kinase (JAK) family tyrosine kinases Jak1 and Tyk2, which are constitutively associated with IL-10R1 and type 2 IL-10R, respectively [2].
T3237 2219-2383 Sentence denotes IL-10 induces tyrosine phosphorylation and activation of the latent transcriptional factors signal transducer and activator of transcription (STAT) 3 and STAT1 [3].
T14 2219-2383 Sentence denotes IL-10 induces tyrosine phosphorylation and activation of the latent transcriptional factors signal transducer and activator of transcription (STAT) 3 and STAT1 [3].
T3238 2384-2534 Sentence denotes Upon phosphorylation, STAT1 and STAT3 proteins form homodimers or heterodimers, rapidly translocate into the nucleus, and modulate gene transcription.
T15 2384-2534 Sentence denotes Upon phosphorylation, STAT1 and STAT3 proteins form homodimers or heterodimers, rapidly translocate into the nucleus, and modulate gene transcription.
T3239 2535-2660 Sentence denotes Intriguingly, STAT3 is indispensable for both IL-10-derived anti-inflammatory and IL-6-derived proinflammatory responses [4].
T16 2535-2660 Sentence denotes Intriguingly, STAT3 is indispensable for both IL-10-derived anti-inflammatory and IL-6-derived proinflammatory responses [4].
T3240 2661-2917 Sentence denotes Studies of cell-type-specific STAT3-deficient mice have shown that STAT3 activation is essential for IL-10-mediated anti-inflammatory reactions in macrophages and neutrophils [5], but is responsible for IL-6-mediated prevention of apoptosis in T cells [6].
T17 2661-2917 Sentence denotes Studies of cell-type-specific STAT3-deficient mice have shown that STAT3 activation is essential for IL-10-mediated anti-inflammatory reactions in macrophages and neutrophils [5], but is responsible for IL-6-mediated prevention of apoptosis in T cells [6].
T3241 2918-3212 Sentence denotes The suppressor of cytokine signaling (SOCS) proteins have been identified as a family of endogenous JAK kinase inhibitors that can act in classic feedback inhibition loops, but their roles as the mediators of crosstalk inhibition by opposing cytokine signaling pathways have been clarified [7].
T18 2918-3212 Sentence denotes The suppressor of cytokine signaling (SOCS) proteins have been identified as a family of endogenous JAK kinase inhibitors that can act in classic feedback inhibition loops, but their roles as the mediators of crosstalk inhibition by opposing cytokine signaling pathways have been clarified [7].
T3242 3213-3413 Sentence denotes Recent studies indicate that SOCS3 plays a key role in regulating the divergent action of IL-10 and IL-6, by specifically blocking STAT3 activation induced by IL-6 but not that induced by IL-10 [8,9].
T19 3213-3413 Sentence denotes Recent studies indicate that SOCS3 plays a key role in regulating the divergent action of IL-10 and IL-6, by specifically blocking STAT3 activation induced by IL-6 but not that induced by IL-10 [8,9].
T3243 3414-3652 Sentence denotes The synovial membrane of rheumatoid arthritis (RA) is characterized by an infiltrate of a variety of inflammatory cells, such as lymphocytes, macrophages, and dendritic cells, together with proliferation of synovial fibroblast-like cells.
T20 3414-3652 Sentence denotes The synovial membrane of rheumatoid arthritis (RA) is characterized by an infiltrate of a variety of inflammatory cells, such as lymphocytes, macrophages, and dendritic cells, together with proliferation of synovial fibroblast-like cells.
T3244 3653-3806 Sentence denotes Numerous cytokines are overproduced in the inflamed joint, and macrophages and synovial fibroblasts are an important source of proinflammatory cytokines.
T21 3653-3806 Sentence denotes Numerous cytokines are overproduced in the inflamed joint, and macrophages and synovial fibroblasts are an important source of proinflammatory cytokines.
T3245 3807-4134 Sentence denotes Tumor necrosis factor alpha (TNF-α) and IL-1, two major macrophage products, are crucial in the process of chronic inflammation and joint destruction, and they give rise to effector components, including other inflammatory cytokines, chemokines, growth factors, matrix proteases, nitric oxide, and reactive oxygen species [10].
T22 3807-4134 Sentence denotes Tumor necrosis factor alpha (TNF-α) and IL-1, two major macrophage products, are crucial in the process of chronic inflammation and joint destruction, and they give rise to effector components, including other inflammatory cytokines, chemokines, growth factors, matrix proteases, nitric oxide, and reactive oxygen species [10].
T3246 4135-4401 Sentence denotes IL-6 is a pleiotropic cytokine produced substantially by activated fibroblasts, and its proinflammatory actions include simulating the acute-phase response, B-cell maturation into plasma cells, T-cell functions, and hematopoietic precursor cell differentiation [11].
T23 4135-4401 Sentence denotes IL-6 is a pleiotropic cytokine produced substantially by activated fibroblasts, and its proinflammatory actions include simulating the acute-phase response, B-cell maturation into plasma cells, T-cell functions, and hematopoietic precursor cell differentiation [11].
T3247 4402-4513 Sentence denotes However, anti-inflammatory cytokines and cytokine inhibitors are also present in large quantities in RA joints.
T24 4402-4513 Sentence denotes However, anti-inflammatory cytokines and cytokine inhibitors are also present in large quantities in RA joints.
T3248 4514-4766 Sentence denotes IL-10, produced by macrophages and partly by T cells in the synovial tissue (ST), is best known as a negative regulator for macrophage and Th1 cells, but the expression level is insufficient to counterbalance the cascade of proinflammatory events [12].
T25 4514-4766 Sentence denotes IL-10, produced by macrophages and partly by T cells in the synovial tissue (ST), is best known as a negative regulator for macrophage and Th1 cells, but the expression level is insufficient to counterbalance the cascade of proinflammatory events [12].
T3249 4767-4906 Sentence denotes In addition, the anti-inflammatory action of IL-10 appears to be modulated at the level of signal transduction during chronic inflammation.
T26 4767-4906 Sentence denotes In addition, the anti-inflammatory action of IL-10 appears to be modulated at the level of signal transduction during chronic inflammation.
T3250 4907-5053 Sentence denotes IL-10 signaling is impaired in macrophages upon chronic exposure to proinflammatory cytokines such as TNF-α and IL-1 and immune complexes [13,14].
T27 4907-5053 Sentence denotes IL-10 signaling is impaired in macrophages upon chronic exposure to proinflammatory cytokines such as TNF-α and IL-1 and immune complexes [13,14].
T3251 5054-5230 Sentence denotes Cell surface expression of IL-10R1 is decreased in synovial fluid dendritic cells due to the presence of TNF-α, IL-1, and granulocyte–macrophage colony-stimulating factor [15].
T28 5054-5230 Sentence denotes Cell surface expression of IL-10R1 is decreased in synovial fluid dendritic cells due to the presence of TNF-α, IL-1, and granulocyte–macrophage colony-stimulating factor [15].
T3252 5231-5353 Sentence denotes CD4+ T cells may be activated by arthritogenic antigens, in conjunction with CD28-mediated costimulatory signaling, in RA.
T29 5231-5353 Sentence denotes CD4+ T cells may be activated by arthritogenic antigens, in conjunction with CD28-mediated costimulatory signaling, in RA.
T3253 5354-5667 Sentence denotes The significance of this autoimmune process has been supported by the linkage of the MHC class II antigens HLA-DRB1*0404 and HLA-DRB1*0401 with disease susceptibility and severity [16,17], and by the high-level expression of MHC class II molecules and both CD28 ligands, CD80 and CD86, in the inflamed ST [18-20].
T30 5354-5667 Sentence denotes The significance of this autoimmune process has been supported by the linkage of the MHC class II antigens HLA-DRB1*0404 and HLA-DRB1*0401 with disease susceptibility and severity [16,17], and by the high-level expression of MHC class II molecules and both CD28 ligands, CD80 and CD86, in the inflamed ST [18-20].
T3254 5668-6009 Sentence denotes The continuing emergence of activated CD4+ T cells, even though few in number, may be crucial in sustaining the activation of macrophages and synovial fibroblasts through cell surface signaling by means of cell surface CD69 and CD11, as well as the release of proinflammatory Th1 cytokines such as interferon gamma (IFN)-γ and IL-17 [21,22].
T31 5668-6009 Sentence denotes The continuing emergence of activated CD4+ T cells, even though few in number, may be crucial in sustaining the activation of macrophages and synovial fibroblasts through cell surface signaling by means of cell surface CD69 and CD11, as well as the release of proinflammatory Th1 cytokines such as interferon gamma (IFN)-γ and IL-17 [21,22].
T3255 6010-6155 Sentence denotes In addition, CD4+ T cells could stimulate B-cell production of autoantibodies such as rheumatoid factor and osteoclast-mediated bone destruction.
T32 6010-6155 Sentence denotes In addition, CD4+ T cells could stimulate B-cell production of autoantibodies such as rheumatoid factor and osteoclast-mediated bone destruction.
T3256 6156-6476 Sentence denotes Their obligatory role in RA synovitis was recently proved by successful treatment of active disease by selective inhibition of T-cell activation with fusion protein of cytotoxic T-cell-associated antigen 4 (CD152)-IgG, which can block the engagement of CD28 on T cells by binding to CD80 and CD86 with high avidity [23].
T33 6156-6476 Sentence denotes Their obligatory role in RA synovitis was recently proved by successful treatment of active disease by selective inhibition of T-cell activation with fusion protein of cytotoxic T-cell-associated antigen 4 (CD152)-IgG, which can block the engagement of CD28 on T cells by binding to CD80 and CD86 with high avidity [23].
T3257 6477-6676 Sentence denotes IL-10 efficiently blocks the antigen-specific T-cell cytokine response by inhibiting the CD28 signaling pathway [24], as well as indirectly by downregulating the function of antigen-presenting cells.
T34 6477-6676 Sentence denotes IL-10 efficiently blocks the antigen-specific T-cell cytokine response by inhibiting the CD28 signaling pathway [24], as well as indirectly by downregulating the function of antigen-presenting cells.
T3258 6677-7010 Sentence denotes To elucidate the resistance of CD4+ T cells to this direct inhibition in RA, we investigated the production of IFN-γ after CD3 and CD28 costimulation in the presence of IL-10, the induction of STAT1 and STAT3 phosphorylation by IL-10, and the expression of SOCS1 and SOCS3 mRNA in peripheral blood (PB) CD4+ T cells from RA patients.
T35 6677-7010 Sentence denotes To elucidate the resistance of CD4+ T cells to this direct inhibition in RA, we investigated the production of IFN-γ after CD3 and CD28 costimulation in the presence of IL-10, the induction of STAT1 and STAT3 phosphorylation by IL-10, and the expression of SOCS1 and SOCS3 mRNA in peripheral blood (PB) CD4+ T cells from RA patients.
T36 7012-7033 Sentence denotes Materials and methods
T6856 7035-7055 Sentence denotes Patients and samples
T37 7035-7055 Sentence denotes Patients and samples
T6857 7056-7335 Sentence denotes The total patient population consisted of 32 patients with RA (25 women and seven men; mean ± standard deviation age, 52.8 ± 12.4 years) diagnosed according to the revised 1987 criteria of the American College of Rheumatology (formally, the American Rheumatism Association) [25].
T38 7056-7335 Sentence denotes The total patient population consisted of 32 patients with RA (25 women and seven men; mean ± standard deviation age, 52.8 ± 12.4 years) diagnosed according to the revised 1987 criteria of the American College of Rheumatology (formally, the American Rheumatism Association) [25].
T6858 7336-7434 Sentence denotes All patients were receiving prednisolone (≤ 7.5 mg/day) and disease-modifying antirheumatic drugs.
T39 7336-7434 Sentence denotes All patients were receiving prednisolone (≤ 7.5 mg/day) and disease-modifying antirheumatic drugs.
T6859 7435-7685 Sentence denotes Clinical parameters in the study patients were as follows (mean ± standard deviation): erythrocyte sedimentation rate, 55.9 ± 35.4 mm/hour; serum C-reactive protein (CRP) level, 32.0 ± 32.0 mg/l; and IgM class rheumatoid factor titer, 142 ± 158 U/ml.
T40 7435-7685 Sentence denotes Clinical parameters in the study patients were as follows (mean ± standard deviation): erythrocyte sedimentation rate, 55.9 ± 35.4 mm/hour; serum C-reactive protein (CRP) level, 32.0 ± 32.0 mg/l; and IgM class rheumatoid factor titer, 142 ± 158 U/ml.
T6860 7686-7724 Sentence denotes Patients were divided into two groups:
T41 7686-7724 Sentence denotes Patients were divided into two groups:
T6861 7725-7988 Sentence denotes 24 patients with active disease, who had multiple tender and/or swollen joints and elevated serum CRP level (≥ 10 mg/l); and eight patients with inactive disease, who satisfied the American College of Rheumatology preliminary criteria for clinical remission [26].
T42 7725-7988 Sentence denotes 24 patients with active disease, who had multiple tender and/or swollen joints and elevated serum CRP level (≥ 10 mg/l); and eight patients with inactive disease, who satisfied the American College of Rheumatology preliminary criteria for clinical remission [26].
T6862 7989-8083 Sentence denotes Sixteen healthy volunteers (11 women and five men; age, 45.8 ± 11.2 years) served as controls.
T43 7989-8083 Sentence denotes Sixteen healthy volunteers (11 women and five men; age, 45.8 ± 11.2 years) served as controls.
T6863 8084-8166 Sentence denotes ST samples were obtained from three RA patients undergoing total knee replacement.
T44 8084-8166 Sentence denotes ST samples were obtained from three RA patients undergoing total knee replacement.
T6864 8167-8202 Sentence denotes All patients gave informed consent.
T45 8167-8202 Sentence denotes All patients gave informed consent.
T7467 8204-8229 Sentence denotes Isolation of CD4+ T cells
T46 8204-8229 Sentence denotes Isolation of CD4+ T cells
T7468 8230-8402 Sentence denotes Peripheral blood mononuclear cells (PBMC) were prepared from heparinized blood samples by centrifugation over Ficoll-Hypaque density gradients (Pharmacia, Uppsala, Sweden).
T47 8230-8402 Sentence denotes Peripheral blood mononuclear cells (PBMC) were prepared from heparinized blood samples by centrifugation over Ficoll-Hypaque density gradients (Pharmacia, Uppsala, Sweden).
T7469 8403-8586 Sentence denotes CD4+ T cells were purified from PBMC by positive selection using anti-CD4 mAb-coated magnetic beads (Miltenyi Biotec, Gladbach, Germany), according to the manufacturer's instructions.
T48 8403-8586 Sentence denotes CD4+ T cells were purified from PBMC by positive selection using anti-CD4 mAb-coated magnetic beads (Miltenyi Biotec, Gladbach, Germany), according to the manufacturer's instructions.
T7470 8587-8660 Sentence denotes CD4+ T cells were isolated from ST samples, as previously described [27].
T49 8587-8660 Sentence denotes CD4+ T cells were isolated from ST samples, as previously described [27].
T50 8661-8762 Sentence denotes Briefly, fresh ST samples were fragmented and digested with collagenase and DNase for 1 hour at 37°C.
T7471 8661-9171 Sentence denotes Briefly, fresh ST samples were fragmented and digested with collagenase and DNase for 1 hour at 37°C. After removing tissue debris, ST cell suspensions in culture medium (RPMI 1640 medium; Life Technologies, Gaithersburg, MD, USA) supplemented with 25 mM HEPES (2 mM L-glutamine, 2% nonessential amino acids, 100 IU/ml penicillin, and 100 mg/ml streptomycin; Life Technologies) with 10% heat-inactivated FCS (Life Technologies) were incubated at 37°C in six-well plates (Coster, Cambridge, MA, USA) for 45 min.
T51 8763-9171 Sentence denotes After removing tissue debris, ST cell suspensions in culture medium (RPMI 1640 medium; Life Technologies, Gaithersburg, MD, USA) supplemented with 25 mM HEPES (2 mM L-glutamine, 2% nonessential amino acids, 100 IU/ml penicillin, and 100 mg/ml streptomycin; Life Technologies) with 10% heat-inactivated FCS (Life Technologies) were incubated at 37°C in six-well plates (Coster, Cambridge, MA, USA) for 45 min.
T7472 9172-9280 Sentence denotes Non-adherent cells were harvested and CD4+ T cells were purified by positive selection as already described.
T52 9172-9280 Sentence denotes Non-adherent cells were harvested and CD4+ T cells were purified by positive selection as already described.
T8016 9282-9305 Sentence denotes Culture of CD4+ T cells
T53 9282-9305 Sentence denotes Culture of CD4+ T cells
T8017 9306-9576 Sentence denotes PB CD4+ T-cell populations were resuspended at a density of 1 × 106 cells/ml in culture medium with 10% FCS, and 0.5 ml cell suspensions were dispensed into the wells of 24-well microtiter plates (Coster) coated with 1 μg/ml anti-CD3 mAb (Immunotech, Marseille, France).
T54 9306-9576 Sentence denotes PB CD4+ T-cell populations were resuspended at a density of 1 × 106 cells/ml in culture medium with 10% FCS, and 0.5 ml cell suspensions were dispensed into the wells of 24-well microtiter plates (Coster) coated with 1 μg/ml anti-CD3 mAb (Immunotech, Marseille, France).
T8018 9577-9806 Sentence denotes The cells were incubated with 1 μg/ml anti-CD28 mAb (Immunotech) in the presence or absence of the indicated concentrations of IL-10 (Becton Dickinson, San Jose, CA, USA) at 37°C in a humidified atmosphere containing 5% CO2 [28].
T55 9577-9806 Sentence denotes The cells were incubated with 1 μg/ml anti-CD28 mAb (Immunotech) in the presence or absence of the indicated concentrations of IL-10 (Becton Dickinson, San Jose, CA, USA) at 37°C in a humidified atmosphere containing 5% CO2 [28].
T8019 9807-9922 Sentence denotes Culture supernatants were collected 36 hours later and cell-free samples were stored at -30°C until cytokine assay.
T56 9807-9922 Sentence denotes Culture supernatants were collected 36 hours later and cell-free samples were stored at -30°C until cytokine assay.
T8020 9923-10145 Sentence denotes To examine the effect of IL-6 on T-cell responsiveness to IL-10, CD4+ T cells from healthy controls were incubated in culture medium with 10% FCS in the presence or absence of 10 ng/ml IL-6 (Becton Dickinson) for 36 hours.
T57 9923-10145 Sentence denotes To examine the effect of IL-6 on T-cell responsiveness to IL-10, CD4+ T cells from healthy controls were incubated in culture medium with 10% FCS in the presence or absence of 10 ng/ml IL-6 (Becton Dickinson) for 36 hours.
T8021 10146-10266 Sentence denotes Cells were then stimulated for 36 hours with anti-CD3 mAb and anti-CD28 mAb in the presence or absence of 1 ng/ml IL-10.
T58 10146-10266 Sentence denotes Cells were then stimulated for 36 hours with anti-CD3 mAb and anti-CD28 mAb in the presence or absence of 1 ng/ml IL-10.
T8022 10267-10327 Sentence denotes Culture supernatants were measured for IFN-γ concentrations.
T59 10267-10327 Sentence denotes Culture supernatants were measured for IFN-γ concentrations.
T8693 10329-10376 Sentence denotes Flow cytometric analysis for IL-10R1 expression
T60 10329-10376 Sentence denotes Flow cytometric analysis for IL-10R1 expression
T8694 10377-10446 Sentence denotes A sample of 5 × 105 cells of PBMC was resuspended in PBS with 1% FCS.
T61 10377-10446 Sentence denotes A sample of 5 × 105 cells of PBMC was resuspended in PBS with 1% FCS.
T8695 10447-10745 Sentence denotes PBMC were incubated with saturating concentrations of anti-IL-10R1 mAb (IgG1; R&D systems, Minneapolis, MN, USA) or with isotype-matched control mAb (Immunotech), followed by incubation with FITC-conjugated goat anti-mouse IgG1 polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA).
T62 10447-10745 Sentence denotes PBMC were incubated with saturating concentrations of anti-IL-10R1 mAb (IgG1; R&D systems, Minneapolis, MN, USA) or with isotype-matched control mAb (Immunotech), followed by incubation with FITC-conjugated goat anti-mouse IgG1 polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA).
T8696 10746-10834 Sentence denotes Cells were then incubated with phycoerythrin-conjugated anti-CD4 mAb (Becton Dickinson).
T63 10746-10834 Sentence denotes Cells were then incubated with phycoerythrin-conjugated anti-CD4 mAb (Becton Dickinson).
T8697 10835-10894 Sentence denotes Cells were washed well with 1% FCS/PBS between incubations.
T64 10835-10894 Sentence denotes Cells were washed well with 1% FCS/PBS between incubations.
T8698 10895-10965 Sentence denotes Analysis was performed on a FACScan flow cytometer (Becton Dickinson).
T65 10895-10965 Sentence denotes Analysis was performed on a FACScan flow cytometer (Becton Dickinson).
T9068 10967-10997 Sentence denotes Immunoassay for IFN-γ and IL-2
T66 10967-10997 Sentence denotes Immunoassay for IFN-γ and IL-2
T9069 10998-11301 Sentence denotes Concentrations of IFN-γ and IL-2 in culture supernatants of CD4+ T cells were measured in duplicate by the quantitative sandwich ELISA using cytokine-specific capture with biotinylated detection mAb and recombinant cytokine proteins (all from Becton Dickinson), according to the manufacturer's protocol.
T67 10998-11301 Sentence denotes Concentrations of IFN-γ and IL-2 in culture supernatants of CD4+ T cells were measured in duplicate by the quantitative sandwich ELISA using cytokine-specific capture with biotinylated detection mAb and recombinant cytokine proteins (all from Becton Dickinson), according to the manufacturer's protocol.
T9070 11302-11356 Sentence denotes The detection limits for IFN-γ and IL-2 were 15 pg/ml.
T68 11302-11356 Sentence denotes The detection limits for IFN-γ and IL-2 were 15 pg/ml.
T9723 11358-11393 Sentence denotes Isolation of mRNA and real-time PCR
T69 11358-11393 Sentence denotes Isolation of mRNA and real-time PCR
T9724 11394-11749 Sentence denotes Total cellular RNA was extracted from PB CD4+ T cells using an RNA isolation kit (RNeasy Mini kit; Qiagen, Valencia, CA, USA), according to the manufacturer's instructions. cDNA was synthesized from total RNA with Molony murine leukemia virus reverse transcriptase (US Biochemical, Cleveland, OH, USA) and oligo-(dT)15 primers (Promega, Madison, WI, USA).
T70 11394-11749 Sentence denotes Total cellular RNA was extracted from PB CD4+ T cells using an RNA isolation kit (RNeasy Mini kit; Qiagen, Valencia, CA, USA), according to the manufacturer's instructions. cDNA was synthesized from total RNA with Molony murine leukemia virus reverse transcriptase (US Biochemical, Cleveland, OH, USA) and oligo-(dT)15 primers (Promega, Madison, WI, USA).
T9725 11750-11870 Sentence denotes Real-time PCR was performed with the LightCycler Instrument (Roche Diagnostics, Penzberg, Germany) in glass capillaries.
T71 11750-11870 Sentence denotes Real-time PCR was performed with the LightCycler Instrument (Roche Diagnostics, Penzberg, Germany) in glass capillaries.
T9726 11871-12083 Sentence denotes The reaction mix containing Taq DNA polymerase and DNA double-strand-specific SYBR Green I dye (Lightcycler FastStart DNA Master SYBR Green I; Roche Diagnostics) and specific primers were added to cDNA dilutions.
T72 11871-12083 Sentence denotes The reaction mix containing Taq DNA polymerase and DNA double-strand-specific SYBR Green I dye (Lightcycler FastStart DNA Master SYBR Green I; Roche Diagnostics) and specific primers were added to cDNA dilutions.
T9727 12084-12376 Sentence denotes The cDNA samples were denatured at 95° C for 10 min, and were then amplified for 40–50 cycles: at 95° C (10 s), at 65° C (15 s), and 72° C (22 s) for β-actin; at 95° C (10 s), at 62° C (15 s), and at 72° C (10 s) for SOCS1; and at 96° C (10 s), at 68° C (15 s), and at 72° C (15 s) for SOCS3.
T73 12084-12376 Sentence denotes The cDNA samples were denatured at 95° C for 10 min, and were then amplified for 40–50 cycles: at 95° C (10 s), at 65° C (15 s), and 72° C (22 s) for β-actin; at 95° C (10 s), at 62° C (15 s), and at 72° C (10 s) for SOCS1; and at 96° C (10 s), at 68° C (15 s), and at 72° C (15 s) for SOCS3.
T9728 12377-12508 Sentence denotes Amplification curves of the fluorescence values versus cycle number were obtained, and a melting curve analysis was then performed.
T74 12377-12508 Sentence denotes Amplification curves of the fluorescence values versus cycle number were obtained, and a melting curve analysis was then performed.
T9729 12509-12612 Sentence denotes The levels of SOCS1 and SOCS3 expression were determined by normalizing relative to β-actin expression.
T75 12509-12612 Sentence denotes The levels of SOCS1 and SOCS3 expression were determined by normalizing relative to β-actin expression.
T9730 12613-12879 Sentence denotes The forward and reverse primers were as follows: for β-actin, 5'-GTGGGGCGCCCCAGGCACCA-3' and 5'-CTCCTTAATGTCACGCACGATTTC-3' ; for SOCS1, 5'-AGACCCCTTCTCACCTCTTG-3' and 5'-GCACAGCAGAAAAATAAAGC-3' ; and for SOCS3, 5'-CCCGCCGGCACCTTTCTG-3' and 5'-AGGGGCCGGCTCAACACC-3'.
T76 12613-12879 Sentence denotes The forward and reverse primers were as follows: for β-actin, 5'-GTGGGGCGCCCCAGGCACCA-3' and 5'-CTCCTTAATGTCACGCACGATTTC-3' ; for SOCS1, 5'-AGACCCCTTCTCACCTCTTG-3' and 5'-GCACAGCAGAAAAATAAAGC-3' ; and for SOCS3, 5'-CCCGCCGGCACCTTTCTG-3' and 5'-AGGGGCCGGCTCAACACC-3'.
T10927 12881-12902 Sentence denotes Western blot analysis
T77 12881-12902 Sentence denotes Western blot analysis
T10928 12903-13061 Sentence denotes CD4+ T cells were stimulated for 20 min by the indicated concentrations of IL-10 and IL-6 at a density of 5 × 105 cells in 0.5 ml culture medium with 10% FCS.
T78 12903-13061 Sentence denotes CD4+ T cells were stimulated for 20 min by the indicated concentrations of IL-10 and IL-6 at a density of 5 × 105 cells in 0.5 ml culture medium with 10% FCS.
T10929 13062-13338 Sentence denotes To examine the effect of serum IL-6 on STAT phosphorylation, normal CD4+ T cells were stimulated for 20 min with 30% active RA serum in culture medium with 40 μg/ml neutralizing goat anti-IL-6 polyclonal antibody (IgG; Techne, Princeton, NJ, USA) or control goat IgG (Techne).
T79 13062-13338 Sentence denotes To examine the effect of serum IL-6 on STAT phosphorylation, normal CD4+ T cells were stimulated for 20 min with 30% active RA serum in culture medium with 40 μg/ml neutralizing goat anti-IL-6 polyclonal antibody (IgG; Techne, Princeton, NJ, USA) or control goat IgG (Techne).
T10930 13339-13511 Sentence denotes Whole cell lysates were prepared by placing cells in 100 μl SDS lysing buffer (62.5 mM Tris–HCl [pH 6.8], 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromphenol blue).
T80 13339-13511 Sentence denotes Whole cell lysates were prepared by placing cells in 100 μl SDS lysing buffer (62.5 mM Tris–HCl [pH 6.8], 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromphenol blue).
T10931 13512-13760 Sentence denotes Then 20 μl protein samples were fractionated on 10% SDS-polyacrylamide gels and were transferred to nitrocellulose membranes (Amersham, Buckinghamshire, UK), and the membrane was blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20.
T81 13512-13760 Sentence denotes Then 20 μl protein samples were fractionated on 10% SDS-polyacrylamide gels and were transferred to nitrocellulose membranes (Amersham, Buckinghamshire, UK), and the membrane was blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20.
T10932 13761-13941 Sentence denotes Tyrosine phosphorylation of STAT1 and STAT3 was detected using commercial available kits (Cell Signaling Technology, Beverly, MA, USA) according to the manufacturer's instructions.
T82 13761-13941 Sentence denotes Tyrosine phosphorylation of STAT1 and STAT3 was detected using commercial available kits (Cell Signaling Technology, Beverly, MA, USA) according to the manufacturer's instructions.
T10933 13942-14251 Sentence denotes Briefly, the membrane was incubated with the antibodies (rabbit IgG) anti-STAT1 antibody, anti-phosphorylated tyrosine 701 of STAT1 antibody, anti-STAT3 antibody, and anti-phosphorylated tyrosine 705 of STAT3 antibody, diluted as recommended at 1/2000 with Tris-buffered saline with 0.1% Tween 20 with 5% BSA.
T83 13942-14251 Sentence denotes Briefly, the membrane was incubated with the antibodies (rabbit IgG) anti-STAT1 antibody, anti-phosphorylated tyrosine 701 of STAT1 antibody, anti-STAT3 antibody, and anti-phosphorylated tyrosine 705 of STAT3 antibody, diluted as recommended at 1/2000 with Tris-buffered saline with 0.1% Tween 20 with 5% BSA.
T10934 14252-14472 Sentence denotes Antibody binding was detected by horseradish peroxidase-conjugated anti-rabbit IgG antibody diluted at 1/4000 with Tris-buffered saline with 0.1% Tween 20 with 5% BSA, and was revealed using the chemiluminescence system.
T84 14252-14472 Sentence denotes Antibody binding was detected by horseradish peroxidase-conjugated anti-rabbit IgG antibody diluted at 1/4000 with Tris-buffered saline with 0.1% Tween 20 with 5% BSA, and was revealed using the chemiluminescence system.
T10935 14473-14621 Sentence denotes Protein bands were quantified by densitometry using NIH-Image analysis, and STAT phosphorylation was compared with the total amount of STAT protein.
T85 14473-14621 Sentence denotes Protein bands were quantified by densitometry using NIH-Image analysis, and STAT phosphorylation was compared with the total amount of STAT protein.
T10936 14622-14709 Sentence denotes IFN-γ-stimulated Hela cells were used as a positive control for STAT1 phosophorylation.
T86 14622-14709 Sentence denotes IFN-γ-stimulated Hela cells were used as a positive control for STAT1 phosophorylation.
T11973 14711-14731 Sentence denotes Statistical analysis
T87 14711-14731 Sentence denotes Statistical analysis
T11974 14732-14811 Sentence denotes Data are expressed as the mean value ± standard error of the mean or box plots.
T88 14732-14811 Sentence denotes Data are expressed as the mean value ± standard error of the mean or box plots.
T11975 14812-14950 Sentence denotes The statistical significance of differences between two groups was determined by the Mann–Whitney U test or the Wilcoxon signed rank test.
T89 14812-14950 Sentence denotes The statistical significance of differences between two groups was determined by the Mann–Whitney U test or the Wilcoxon signed rank test.
T11976 14951-14987 Sentence denotes P < 0.05 was considered significant.
T90 14951-14987 Sentence denotes P < 0.05 was considered significant.
T91 14989-14996 Sentence denotes Results
T12796 14998-15067 Sentence denotes Resistance to IL-10 inhibition of IFN-γ production in RA CD4+ T cells
T92 14998-15067 Sentence denotes Resistance to IL-10 inhibition of IFN-γ production in RA CD4+ T cells
T12797 15068-15309 Sentence denotes The CD28 costimulatory pathway is crucial for effective antigen-specific T-cell cytokine production, and IL-10 can directly suppress this response by inhibiting CD28 tyrosine phosphorylation and binding of phosphatidylinositol 3-kinase [24].
T93 15068-15309 Sentence denotes The CD28 costimulatory pathway is crucial for effective antigen-specific T-cell cytokine production, and IL-10 can directly suppress this response by inhibiting CD28 tyrosine phosphorylation and binding of phosphatidylinositol 3-kinase [24].
T12798 15310-15639 Sentence denotes To evaluate the responsiveness of RA CD4+ T cells to IL-10, purified PB CD4+ T cells from three patients with active RA and from three healthy controls were stimulated by immobilized anti-CD3 antibody and anti-CD28 antibody with or without diluted concentrations of IL-10 for 36 hours, and IFN-γ production was measured by ELISA.
T94 15310-15639 Sentence denotes To evaluate the responsiveness of RA CD4+ T cells to IL-10, purified PB CD4+ T cells from three patients with active RA and from three healthy controls were stimulated by immobilized anti-CD3 antibody and anti-CD28 antibody with or without diluted concentrations of IL-10 for 36 hours, and IFN-γ production was measured by ELISA.
T12799 15640-15773 Sentence denotes As shown in Fig. 1, IFN-γ production by activated normal CD4+ T cells was mostly inhibited at concentrations as low as 1 ng/ml IL-10.
T95 15640-15773 Sentence denotes As shown in Fig. 1, IFN-γ production by activated normal CD4+ T cells was mostly inhibited at concentrations as low as 1 ng/ml IL-10.
T12800 15774-15965 Sentence denotes However, RA CD4+ T cells were able to produce significant amounts of IFN-γ in the presence of 1 ng/ml IL-10, and the maximal but not complete inhibition by IL-10 was obtained at 10–100 ng/ml.
T96 15774-15965 Sentence denotes However, RA CD4+ T cells were able to produce significant amounts of IFN-γ in the presence of 1 ng/ml IL-10, and the maximal but not complete inhibition by IL-10 was obtained at 10–100 ng/ml.
T12801 15966-16269 Sentence denotes We thus compared the levels of IFN-γ production by CD4+ T cells after CD3 and CD28 costimulation in the presence of 1 ng/ml IL-10 in RA patients with active disease (multiple inflammatory joints, CRP level ≥ 10 mg/l) and inactive disease (in remission, CRP level < 10 mg/l) [26] and in healthy controls.
T97 15966-16269 Sentence denotes We thus compared the levels of IFN-γ production by CD4+ T cells after CD3 and CD28 costimulation in the presence of 1 ng/ml IL-10 in RA patients with active disease (multiple inflammatory joints, CRP level ≥ 10 mg/l) and inactive disease (in remission, CRP level < 10 mg/l) [26] and in healthy controls.
T12802 16270-16662 Sentence denotes There were no statistically significant differences in IFN-γ production without IL-10 among these three groups (Fig. 2a), but the inhibitory effect of IL-10 on IFN-γ production was significantly limited in the active RA group as compared with the inactive RA group and healthy controls (percentage decrease: active RA, 2.9 ± 14.4%; inactive RA, 45.6 ± 14.4%; controls, 65.8 ± 7.9%) (Fig. 2b).
T98 16270-16662 Sentence denotes There were no statistically significant differences in IFN-γ production without IL-10 among these three groups (Fig. 2a), but the inhibitory effect of IL-10 on IFN-γ production was significantly limited in the active RA group as compared with the inactive RA group and healthy controls (percentage decrease: active RA, 2.9 ± 14.4%; inactive RA, 45.6 ± 14.4%; controls, 65.8 ± 7.9%) (Fig. 2b).
T12803 16663-16822 Sentence denotes As a consequence, CD4+ T cells from active RA patients produced higher levels of IFN-γ in the presence of 1 ng/ml IL-10 than did normal CD4+ T cells (Fig. 2a).
T99 16663-16822 Sentence denotes As a consequence, CD4+ T cells from active RA patients produced higher levels of IFN-γ in the presence of 1 ng/ml IL-10 than did normal CD4+ T cells (Fig. 2a).
T12804 16823-16984 Sentence denotes In addition, we compared IL-2 production by CD4+ T cells after CD3 and CD28 costimulation in the presence of IL-10 in active RA patients and in healthy controls.
T100 16823-16984 Sentence denotes In addition, we compared IL-2 production by CD4+ T cells after CD3 and CD28 costimulation in the presence of IL-10 in active RA patients and in healthy controls.
T12805 16985-17178 Sentence denotes Similarly, IL-2 production was not affected by 1 ng/ml IL-10 in RA patients (percentage decrease, -2.1 ± 13.8%), while it was significantly reduced in healthy controls (61.1 ± 13.7%; P < 0.05).
T101 16985-17178 Sentence denotes Similarly, IL-2 production was not affected by 1 ng/ml IL-10 in RA patients (percentage decrease, -2.1 ± 13.8%), while it was significantly reduced in healthy controls (61.1 ± 13.7%; P < 0.05).
T12806 17179-17327 Sentence denotes Taken together, these results indicate that RA CD4+ T cells become less susceptible to the immunoregulatory effect of IL-10 during the active phase.
T102 17179-17327 Sentence denotes Taken together, these results indicate that RA CD4+ T cells become less susceptible to the immunoregulatory effect of IL-10 during the active phase.
T14713 17329-17392 Sentence denotes Increased expression of cell surface IL-10R1 on RA CD4+ T cells
T103 17329-17392 Sentence denotes Increased expression of cell surface IL-10R1 on RA CD4+ T cells
T14714 17393-17551 Sentence denotes The functional receptor complex of IL-10 consists of two subunits, the primary ligand-binding component IL-10R1 and the accessory component type 2 IL-10R [1].
T104 17393-17551 Sentence denotes The functional receptor complex of IL-10 consists of two subunits, the primary ligand-binding component IL-10R1 and the accessory component type 2 IL-10R [1].
T14715 17552-17629 Sentence denotes IL-10R1 expression plays a critical role in cellular responses to IL-10 [29].
T105 17552-17629 Sentence denotes IL-10R1 expression plays a critical role in cellular responses to IL-10 [29].
T14716 17630-17894 Sentence denotes To examine whether the resistance to IL-10 inhibition in RA CD4+ T cells was due to limited receptor expression, the cell surface expression of IL-10R1 on PB CD4+ T cells from active RA patients and from healthy controls was determined by flow cytometric analysis.
T106 17630-17894 Sentence denotes To examine whether the resistance to IL-10 inhibition in RA CD4+ T cells was due to limited receptor expression, the cell surface expression of IL-10R1 on PB CD4+ T cells from active RA patients and from healthy controls was determined by flow cytometric analysis.
T14717 17895-18048 Sentence denotes As shown in Fig. 3a,3b, the intensity of IL-10R1 expression on CD4+ T cells was significantly increased in RA patients compared with in healthy controls.
T107 17895-18048 Sentence denotes As shown in Fig. 3a,3b, the intensity of IL-10R1 expression on CD4+ T cells was significantly increased in RA patients compared with in healthy controls.
T14718 18049-18176 Sentence denotes These results suggest that the intracellular signal transduction pathway of IL-10 may be impaired in CD4+ T cells of active RA.
T108 18049-18176 Sentence denotes These results suggest that the intracellular signal transduction pathway of IL-10 may be impaired in CD4+ T cells of active RA.
T15852 18178-18243 Sentence denotes Defective IL-10-mediated STAT3 phosphorylation in RA CD4+ T cells
T109 18178-18243 Sentence denotes Defective IL-10-mediated STAT3 phosphorylation in RA CD4+ T cells
T15853 18244-18385 Sentence denotes The interaction of IL-10R with IL-10 induces tyrosine phosphorylation and activation of the latent transcription factors STAT1 and STAT3 [3].
T110 18244-18385 Sentence denotes The interaction of IL-10R with IL-10 induces tyrosine phosphorylation and activation of the latent transcription factors STAT1 and STAT3 [3].
T15854 18386-18637 Sentence denotes Macrophage-specific STAT3-deficient mice demonstrated that STAT3 plays a dominant role in IL-10-mediated anti-inflammatory responses [5], which has recently been confirmed in human macrophages by studies of dominant-negative STAT3 overexpression [30].
T111 18386-18637 Sentence denotes Macrophage-specific STAT3-deficient mice demonstrated that STAT3 plays a dominant role in IL-10-mediated anti-inflammatory responses [5], which has recently been confirmed in human macrophages by studies of dominant-negative STAT3 overexpression [30].
T15855 18638-18801 Sentence denotes The induction of STAT1 and STAT3 phosphorylation by IL-10 in PB CD4+ T cells from active RA patients and from healthy controls was examined using western blotting.
T112 18638-18801 Sentence denotes The induction of STAT1 and STAT3 phosphorylation by IL-10 in PB CD4+ T cells from active RA patients and from healthy controls was examined using western blotting.
T15856 18802-18923 Sentence denotes STAT3 phosphorylation was dose-dependently induced after IL-10 activation for 20 min in normal CD4+ T cells (Fig. 4a,4b).
T113 18802-18923 Sentence denotes STAT3 phosphorylation was dose-dependently induced after IL-10 activation for 20 min in normal CD4+ T cells (Fig. 4a,4b).
T15857 18924-19077 Sentence denotes In contrast, STAT3 was phosphorylated in freshly isolated PB CD4+ cells from RA patients and this STAT3 phosphorylation was detectable for up to 6 hours.
T114 18924-19077 Sentence denotes In contrast, STAT3 was phosphorylated in freshly isolated PB CD4+ cells from RA patients and this STAT3 phosphorylation was detectable for up to 6 hours.
T15858 19078-19163 Sentence denotes STAT3 phosphorylation was augmented only when activated by as much as 10 ng/ml IL-10.
T115 19078-19163 Sentence denotes STAT3 phosphorylation was augmented only when activated by as much as 10 ng/ml IL-10.
T15859 19164-19294 Sentence denotes Both sustained STAT3 phosphorylation and defective IL-10-induced STAT3 phosphorylation were found in RA ST CD4+ T cells (Fig. 4c).
T116 19164-19294 Sentence denotes Both sustained STAT3 phosphorylation and defective IL-10-induced STAT3 phosphorylation were found in RA ST CD4+ T cells (Fig. 4c).
T15860 19295-19426 Sentence denotes On the other hand, IL-10-induced STAT1 phosphorylation was not detected in either RA CD4+ T cells or normal CD4+ T cells (Fig. 4a).
T117 19295-19426 Sentence denotes On the other hand, IL-10-induced STAT1 phosphorylation was not detected in either RA CD4+ T cells or normal CD4+ T cells (Fig. 4a).
T15861 19427-19631 Sentence denotes These results indicate that STAT3 is the major IL-10-activated STAT in CD4+ T cells, and IL-10-induced STAT3 activation may be diminished in active RA, in association with sustained STAT3 phosphorylation.
T118 19427-19631 Sentence denotes These results indicate that STAT3 is the major IL-10-activated STAT in CD4+ T cells, and IL-10-induced STAT3 activation may be diminished in active RA, in association with sustained STAT3 phosphorylation.
T18008 19633-19722 Sentence denotes IL-6-mediated STAT3 phosphorylation and inhibition of IL-10 effect in normal CD4+ T cells
T119 19633-19722 Sentence denotes IL-6-mediated STAT3 phosphorylation and inhibition of IL-10 effect in normal CD4+ T cells
T18009 19723-20126 Sentence denotes STAT3 is activated by many cytokines and growth factors such as the IL-6 family of cytokines (IL-6, IL-11, leukemia inhibitory factor, and oncostatin M), platelet-derived growth factor, and epidermal growth factor, in addition to IL-10 [4], but previous studies have demonstrated that IL-6 is the major factor in RA synovial fluid that induces constitutive activation of STAT3 in mononuclear cells [31].
T120 19723-20126 Sentence denotes STAT3 is activated by many cytokines and growth factors such as the IL-6 family of cytokines (IL-6, IL-11, leukemia inhibitory factor, and oncostatin M), platelet-derived growth factor, and epidermal growth factor, in addition to IL-10 [4], but previous studies have demonstrated that IL-6 is the major factor in RA synovial fluid that induces constitutive activation of STAT3 in mononuclear cells [31].
T18010 20127-20438 Sentence denotes Since IL-6 is also abundant in sera of active RA patients, frequently detected at > 1 ng/ml [27], we examined whether persistent exposure of CD4+ T cells to high concentrations of IL-6 in the blood circulation was responsible for their sustained STAT3 activation and resistance to IL-10 inhibition in active RA.
T121 20127-20438 Sentence denotes Since IL-6 is also abundant in sera of active RA patients, frequently detected at > 1 ng/ml [27], we examined whether persistent exposure of CD4+ T cells to high concentrations of IL-6 in the blood circulation was responsible for their sustained STAT3 activation and resistance to IL-10 inhibition in active RA.
T18011 20439-20583 Sentence denotes Both STAT1 and STAT3 phosphorylation was activated by IL-6 in normal CD4+ T cells (data not shown), in agreement with previous observations [4].
T122 20439-20583 Sentence denotes Both STAT1 and STAT3 phosphorylation was activated by IL-6 in normal CD4+ T cells (data not shown), in agreement with previous observations [4].
T18012 20584-20820 Sentence denotes Normal CD4+ T cells were thus incubated for 20 min with culture medium containing 30% serum from active RA patients and neutralizing anti-IL-6 antibody or control antibody, and STAT phosphorylation was examined by western blot analysis.
T123 20584-20820 Sentence denotes Normal CD4+ T cells were thus incubated for 20 min with culture medium containing 30% serum from active RA patients and neutralizing anti-IL-6 antibody or control antibody, and STAT phosphorylation was examined by western blot analysis.
T18013 20821-20990 Sentence denotes RA serum was able to induce tyrosine phosphorylation of STAT3 but not STAT1, and this STAT3 activation was mostly abolished by neutralization of IL-6 activity (Fig. 5a).
T124 20821-20990 Sentence denotes RA serum was able to induce tyrosine phosphorylation of STAT3 but not STAT1, and this STAT3 activation was mostly abolished by neutralization of IL-6 activity (Fig. 5a).
T18014 20991-21104 Sentence denotes These results indicate that IL-6 is the dominant STAT3-activating factor contained in sera of active RA patients.
T125 20991-21104 Sentence denotes These results indicate that IL-6 is the dominant STAT3-activating factor contained in sera of active RA patients.
T18015 21105-21332 Sentence denotes The lack of STAT1 activation by RA serum suggests that much higher concentrations of IL-6 may be required for STAT1 activation as compared with STAT3 activation, or that inhibitors of STAT1 signaling may be present in RA serum.
T126 21105-21332 Sentence denotes The lack of STAT1 activation by RA serum suggests that much higher concentrations of IL-6 may be required for STAT1 activation as compared with STAT3 activation, or that inhibitors of STAT1 signaling may be present in RA serum.
T18016 21333-21442 Sentence denotes We next examined whether IL-6 could suppress the effect of IL-10 to inhibit IFN-γ production by CD4+ T cells.
T127 21333-21442 Sentence denotes We next examined whether IL-6 could suppress the effect of IL-10 to inhibit IFN-γ production by CD4+ T cells.
T18017 21443-21678 Sentence denotes After preincubation with or without 10 ng/ml IL-6 for 36 hours, normal CD4+ T cells were stimulated by CD3 and CD28 costimulation in the presence or absence of 1 ng/ml IL-10 for 36 hours, and the IFN-γ production was measured by ELISA.
T128 21443-21678 Sentence denotes After preincubation with or without 10 ng/ml IL-6 for 36 hours, normal CD4+ T cells were stimulated by CD3 and CD28 costimulation in the presence or absence of 1 ng/ml IL-10 for 36 hours, and the IFN-γ production was measured by ELISA.
T18018 21679-21868 Sentence denotes IL-6 pretreatment of normal cells reduced IL-10-mediated inhibition of IFN-γ production (Fig. 5b), indicating that high concentrations of IL-6 could modulate T-cell responsiveness to IL-10.
T129 21679-21868 Sentence denotes IL-6 pretreatment of normal cells reduced IL-10-mediated inhibition of IFN-γ production (Fig. 5b), indicating that high concentrations of IL-6 could modulate T-cell responsiveness to IL-10.
T18019 21869-22077 Sentence denotes Taken together, these findings suggest that persistent exposure to serum IL-6 may have a role in both the induction of STAT3 activation and the resistance to the inhibitory effect of IL-10 in RA CD4+ T cells.
T130 21869-22077 Sentence denotes Taken together, these findings suggest that persistent exposure to serum IL-6 may have a role in both the induction of STAT3 activation and the resistance to the inhibitory effect of IL-10 in RA CD4+ T cells.
T20392 22079-22127 Sentence denotes High expression of SOCS1 mRNA in RA CD4+ T cells
T131 22079-22127 Sentence denotes High expression of SOCS1 mRNA in RA CD4+ T cells
T20393 22128-22363 Sentence denotes IL-6 induces two potent inhibitors of JAKs (SOCS1 and SOCS3 proteins) that not only act as mediators of negative feedback inhibition, but also play a major role in crosstalk inhibition by opposing other cytokine-signaling pathways [7].
T132 22128-22363 Sentence denotes IL-6 induces two potent inhibitors of JAKs (SOCS1 and SOCS3 proteins) that not only act as mediators of negative feedback inhibition, but also play a major role in crosstalk inhibition by opposing other cytokine-signaling pathways [7].
T20394 22364-22533 Sentence denotes SOCS3 has recently been shown to specifically inhibit STAT3 activation induced by IL-6 but not by IL-10, thereby regulating the divergent action of IL-6 and IL-10 [8,9].
T133 22364-22533 Sentence denotes SOCS3 has recently been shown to specifically inhibit STAT3 activation induced by IL-6 but not by IL-10, thereby regulating the divergent action of IL-6 and IL-10 [8,9].
T20395 22534-22690 Sentence denotes On the contrary, SOCS1 is able to partially inhibit IL-10-mediated STAT3 activation and cellular responses, as well as IFN-γ-mediated STAT1 activation [32].
T134 22534-22690 Sentence denotes On the contrary, SOCS1 is able to partially inhibit IL-10-mediated STAT3 activation and cellular responses, as well as IFN-γ-mediated STAT1 activation [32].
T20396 22691-22963 Sentence denotes To determine whether SOCSs were involved in the defective IL-10-induced STAT3 activation of RA CD4+ T cells, the levels of SOCS1 and SOCS3 mRNA expression in PB CD4+ T cells from active RA patients and from healthy controls were compared by semiquantitative real-time PCR.
T135 22691-22963 Sentence denotes To determine whether SOCSs were involved in the defective IL-10-induced STAT3 activation of RA CD4+ T cells, the levels of SOCS1 and SOCS3 mRNA expression in PB CD4+ T cells from active RA patients and from healthy controls were compared by semiquantitative real-time PCR.
T20397 22964-23095 Sentence denotes The RA CD4+ T cells contained higher levels of SOCS1 but lower levels of SOCS3 transcripts than did control CD4+ T cells (Fig. 6a).
T136 22964-23095 Sentence denotes The RA CD4+ T cells contained higher levels of SOCS1 but lower levels of SOCS3 transcripts than did control CD4+ T cells (Fig. 6a).
T20398 23096-23289 Sentence denotes Constitutive expression of SOCS1 mRNA in RA CD4+ T cells was comparable with the expression in normal CD4+ T cells stimulated by 10 ng/ml IL-6 (Fig. 6b), supporting its functional significance.
T137 23096-23289 Sentence denotes Constitutive expression of SOCS1 mRNA in RA CD4+ T cells was comparable with the expression in normal CD4+ T cells stimulated by 10 ng/ml IL-6 (Fig. 6b), supporting its functional significance.
T20399 23290-23420 Sentence denotes Defective IL-10-induced STAT3 activation therefore appears to be due at least in part to an abundance of SOCS1 in RA CD4+ T cells.
T138 23290-23420 Sentence denotes Defective IL-10-induced STAT3 activation therefore appears to be due at least in part to an abundance of SOCS1 in RA CD4+ T cells.
T139 23422-23432 Sentence denotes Discussion
T22797 23433-23512 Sentence denotes CD4+ T cells orchestrate the Th1-type cell-mediated immune response in RA [22].
T140 23433-23512 Sentence denotes CD4+ T cells orchestrate the Th1-type cell-mediated immune response in RA [22].
T22798 23513-23754 Sentence denotes Activated CD4+ T cells stimulate macrophages, synovial fibroblasts, B cells, and osteoclasts through the expression of cell surface molecules and Th1 cytokines, thereby contributing to both the chronic inflammation and the joint destruction.
T141 23513-23754 Sentence denotes Activated CD4+ T cells stimulate macrophages, synovial fibroblasts, B cells, and osteoclasts through the expression of cell surface molecules and Th1 cytokines, thereby contributing to both the chronic inflammation and the joint destruction.
T22799 23755-23864 Sentence denotes CD4+ T cells require two signals to be activated; antigen receptor occupancy and CD28-mediated costimulation.
T142 23755-23864 Sentence denotes CD4+ T cells require two signals to be activated; antigen receptor occupancy and CD28-mediated costimulation.
T22800 23865-24062 Sentence denotes In the ST lesion, the CD28 ligands, both CD80 and CD86, together with MHC class II antigens, are substantially expressed by antigen-presenting cells such as macrophages and dendritic cells [18-20].
T143 23865-24062 Sentence denotes In the ST lesion, the CD28 ligands, both CD80 and CD86, together with MHC class II antigens, are substantially expressed by antigen-presenting cells such as macrophages and dendritic cells [18-20].
T22801 24063-24273 Sentence denotes The significance of CD28 engagement in the T-cell-mediated disease process has recently been proven by the clinical efficacy of its blocker cytotoxic T-cell-associated antigen 4 (CD152)-IgG in RA patients [23].
T144 24063-24273 Sentence denotes The significance of CD28 engagement in the T-cell-mediated disease process has recently been proven by the clinical efficacy of its blocker cytotoxic T-cell-associated antigen 4 (CD152)-IgG in RA patients [23].
T22802 24274-24416 Sentence denotes IL-10 plays a predominant role in limiting immune and inflammatory responses by regulating the function of both macrophages and Th1 cells [1].
T145 24274-24416 Sentence denotes IL-10 plays a predominant role in limiting immune and inflammatory responses by regulating the function of both macrophages and Th1 cells [1].
T22803 24417-24593 Sentence denotes IL-10 inhibits the tyrosine phosphorylation of the CD28 molecule and the subsequent phosphatidylinositol 3-kinase binding in T cells, and thereby directly acts on T cells [24].
T146 24417-24593 Sentence denotes IL-10 inhibits the tyrosine phosphorylation of the CD28 molecule and the subsequent phosphatidylinositol 3-kinase binding in T cells, and thereby directly acts on T cells [24].
T22804 24594-24803 Sentence denotes In the present study, we found that PB CD4+ T cells from patients with active RA, in the presence of IL-10, are able to produce higher levels of IFN-γ after CD3 and CD28 costimulation than normal CD4+ T cells.
T147 24594-24803 Sentence denotes In the present study, we found that PB CD4+ T cells from patients with active RA, in the presence of IL-10, are able to produce higher levels of IFN-γ after CD3 and CD28 costimulation than normal CD4+ T cells.
T22805 24804-25062 Sentence denotes Despite high-level IL-10R1 expression and constitutive STAT3 activation, IL-10-induced tyrosine phosphorylation of STAT3 is suppressed in RA CD4+ T cells, in contrast to normal CD4+ T cells, where STAT3 phosphorylation is dose-dependently inducible by IL-10.
T148 24804-25062 Sentence denotes Despite high-level IL-10R1 expression and constitutive STAT3 activation, IL-10-induced tyrosine phosphorylation of STAT3 is suppressed in RA CD4+ T cells, in contrast to normal CD4+ T cells, where STAT3 phosphorylation is dose-dependently inducible by IL-10.
T22806 25063-25241 Sentence denotes Serum IL-6 from RA patients induces STAT3 but not STAT1 phosphorylation in normal CD4+ T cells, and exogenous IL-6 induces the resistance to IL-10 inhibition of IFN-γ production.
T149 25063-25241 Sentence denotes Serum IL-6 from RA patients induces STAT3 but not STAT1 phosphorylation in normal CD4+ T cells, and exogenous IL-6 induces the resistance to IL-10 inhibition of IFN-γ production.
T22807 25242-25374 Sentence denotes RA CD4+ T cells contain higher levels of SOCS1 but contain lower levels of SOCS3 transcripts in comparison with normal CD4+ T cells.
T150 25242-25374 Sentence denotes RA CD4+ T cells contain higher levels of SOCS1 but contain lower levels of SOCS3 transcripts in comparison with normal CD4+ T cells.
T22808 25375-25678 Sentence denotes These findings indicate that CD4+ T cells become resistant to the inhibitory effect of IL-10 before migration into the inflamed ST, and suggest that this resistance may be attributable to impaired IL-10-dependent STAT3 activation, in association with sustained STAT3 phosphorylation and SOCS1 induction.
T151 25375-25678 Sentence denotes These findings indicate that CD4+ T cells become resistant to the inhibitory effect of IL-10 before migration into the inflamed ST, and suggest that this resistance may be attributable to impaired IL-10-dependent STAT3 activation, in association with sustained STAT3 phosphorylation and SOCS1 induction.
T22809 25679-25834 Sentence denotes IL-10-mediated inhibition of CD4+ T-cell cytokine production is principally dependent on its inhibition of macrophage antigen-presenting cell function [1].
T152 25679-25834 Sentence denotes IL-10-mediated inhibition of CD4+ T-cell cytokine production is principally dependent on its inhibition of macrophage antigen-presenting cell function [1].
T22810 25835-26070 Sentence denotes However, this indirect inhibitory effect is thought to be restricted at the site of T-cell activation in RA, because macrophages in the ST express high levels of cytokines, CD80 and CD86 molecules, and MHC class II antigens [10,18-20].
T153 25835-26070 Sentence denotes However, this indirect inhibitory effect is thought to be restricted at the site of T-cell activation in RA, because macrophages in the ST express high levels of cytokines, CD80 and CD86 molecules, and MHC class II antigens [10,18-20].
T22811 26071-26211 Sentence denotes More recently, IL-10 has been shown to induce the antigen-specific T-cell unresponsiveness by inhibiting CD28 tyrosine phosphorylation [33].
T154 26071-26211 Sentence denotes More recently, IL-10 has been shown to induce the antigen-specific T-cell unresponsiveness by inhibiting CD28 tyrosine phosphorylation [33].
T22812 26212-26395 Sentence denotes This direct effect also may be limited in active RA patients, because their PB CD4+ T cells showed a defective IL-10 inhibition of CD28-costimulated production of both IFN-γ and IL-2.
T155 26212-26395 Sentence denotes This direct effect also may be limited in active RA patients, because their PB CD4+ T cells showed a defective IL-10 inhibition of CD28-costimulated production of both IFN-γ and IL-2.
T22813 26396-26587 Sentence denotes Numerous cytokines, both proinflammatory and anti-inflammatory, have been detected in the ST of RA, and the balance between these opposing cytokine activities regulates disease severity [10].
T156 26396-26587 Sentence denotes Numerous cytokines, both proinflammatory and anti-inflammatory, have been detected in the ST of RA, and the balance between these opposing cytokine activities regulates disease severity [10].
T22814 26588-26712 Sentence denotes Endogenous IL-10, produced mainly by macrophages and T cells, inhibits proinflammatory cytokine production by ST cells [12].
T157 26588-26712 Sentence denotes Endogenous IL-10, produced mainly by macrophages and T cells, inhibits proinflammatory cytokine production by ST cells [12].
T22815 26713-26798 Sentence denotes However, this regulatory activity seems to be restricted during chronic inflammation.
T158 26713-26798 Sentence denotes However, this regulatory activity seems to be restricted during chronic inflammation.
T22816 26799-27013 Sentence denotes The activation of both the extracellular stimulus-regulated kinase and p38 kinase pathways, induced by TNF-α and IL-1, inhibits the Jak1–STAT3 signaling pathway shared by IL-10 and IL-6 in adhered macrophages [13].
T159 26799-27013 Sentence denotes The activation of both the extracellular stimulus-regulated kinase and p38 kinase pathways, induced by TNF-α and IL-1, inhibits the Jak1–STAT3 signaling pathway shared by IL-10 and IL-6 in adhered macrophages [13].
T22817 27014-27114 Sentence denotes More importantly, IL-10-mediated STAT3 activation is mostly undetectable in RA synovial macrophages.
T160 27014-27114 Sentence denotes More importantly, IL-10-mediated STAT3 activation is mostly undetectable in RA synovial macrophages.
T22818 27115-27429 Sentence denotes This impaired IL-10 signaling is probably induced by chronic exposure to immune complexes in vivo, because both cell surface IL-10R1 expression and IL-10-induced Jak1 activation are suppressed in IFN-γ-primed macrophages by a protein kinase C-dependent pathway following ligation of the IgG Fc gamma receptor [14].
T161 27115-27429 Sentence denotes This impaired IL-10 signaling is probably induced by chronic exposure to immune complexes in vivo, because both cell surface IL-10R1 expression and IL-10-induced Jak1 activation are suppressed in IFN-γ-primed macrophages by a protein kinase C-dependent pathway following ligation of the IgG Fc gamma receptor [14].
T22819 27430-27720 Sentence denotes Furthermore, dendritic cells from RA synovial fluids are resistant to the immunoregulatory effect of IL-10 due to decreased transport of intracellular IL-10R1 in the presence of proinflammatory cytokine stimuli such as TNF-α, IL-1, and granulocyte–macrophage colony-stimulating factor [15].
T162 27430-27720 Sentence denotes Furthermore, dendritic cells from RA synovial fluids are resistant to the immunoregulatory effect of IL-10 due to decreased transport of intracellular IL-10R1 in the presence of proinflammatory cytokine stimuli such as TNF-α, IL-1, and granulocyte–macrophage colony-stimulating factor [15].
T22820 27721-27889 Sentence denotes We have demonstrated that the resistance of RA CD4+ T cells to IL-10 may be associated with defective IL-10-dependent STAT3 activation, but not with IL-10R1 expression.
T163 27721-27889 Sentence denotes We have demonstrated that the resistance of RA CD4+ T cells to IL-10 may be associated with defective IL-10-dependent STAT3 activation, but not with IL-10R1 expression.
T22821 27890-28064 Sentence denotes Inhibitory effects of IL-10 on these inflammatory cell types are therefore differentially modulated at the signal transduction level under the inflammatory environment in RA.
T164 27890-28064 Sentence denotes Inhibitory effects of IL-10 on these inflammatory cell types are therefore differentially modulated at the signal transduction level under the inflammatory environment in RA.
T22822 28065-28262 Sentence denotes In association with impaired IL-10-mediated STAT3 activation, STAT3 was found to be tyrosine phosphorylated persistently (up to 6 hours) in freshly isolated PB and ST CD4+ T cells from RA patients.
T165 28065-28262 Sentence denotes In association with impaired IL-10-mediated STAT3 activation, STAT3 was found to be tyrosine phosphorylated persistently (up to 6 hours) in freshly isolated PB and ST CD4+ T cells from RA patients.
T22823 28263-28456 Sentence denotes STAT3 is activated by a variety of cytokines, notably the IL-6 family of cytokines (e.g. IL-6, IL-11, leukemia inhibitory factor, and oncostatin M) and growth factors, in addition to IL-10 [4].
T166 28263-28456 Sentence denotes STAT3 is activated by a variety of cytokines, notably the IL-6 family of cytokines (e.g. IL-6, IL-11, leukemia inhibitory factor, and oncostatin M) and growth factors, in addition to IL-10 [4].
T22824 28457-28645 Sentence denotes Of these cytokines, IL-6 plays a predominant role in eliciting a systemic reaction such as the acute phase response in active RA, due mainly to its abundance in the blood circulation [27].
T167 28457-28645 Sentence denotes Of these cytokines, IL-6 plays a predominant role in eliciting a systemic reaction such as the acute phase response in active RA, due mainly to its abundance in the blood circulation [27].
T22825 28646-28873 Sentence denotes Consistent with this notion, IL-6 was the major STAT3-activating factor contained in the serum of active RA patients, and the responsiveness to IL-10 was suppressed in normal CD4+ T cells after 36 hours of incubation with IL-6.
T168 28646-28873 Sentence denotes Consistent with this notion, IL-6 was the major STAT3-activating factor contained in the serum of active RA patients, and the responsiveness to IL-10 was suppressed in normal CD4+ T cells after 36 hours of incubation with IL-6.
T22826 28874-29084 Sentence denotes These results suggest that both the sustained STAT3 activation and the resistance to IL-10 inhibition found in RA CD4+ T cells may be induced after chronic exposure in vivo to high concentrations of serum IL-6.
T169 28874-29084 Sentence denotes These results suggest that both the sustained STAT3 activation and the resistance to IL-10 inhibition found in RA CD4+ T cells may be induced after chronic exposure in vivo to high concentrations of serum IL-6.
T22827 29085-29359 Sentence denotes However, it is also possible that STAT3 activity could be constitutively induced in CD4+ T cells by their own IL-10 secretion, leading to the loss of sensitivity to exogenous IL-10, because RA CD4+ T cells in the ST are capable of producing significant levels of IL-10 [34].
T170 29085-29359 Sentence denotes However, it is also possible that STAT3 activity could be constitutively induced in CD4+ T cells by their own IL-10 secretion, leading to the loss of sensitivity to exogenous IL-10, because RA CD4+ T cells in the ST are capable of producing significant levels of IL-10 [34].
T22828 29360-29466 Sentence denotes CD4+ T cells isolated from the ST of RA also showed a defect in the IL-10-induced STAT3 signaling pathway.
T171 29360-29466 Sentence denotes CD4+ T cells isolated from the ST of RA also showed a defect in the IL-10-induced STAT3 signaling pathway.
T22829 29467-29663 Sentence denotes It is most probable that the resistance of CD4+ T cells to IL-10 can be even augmented after migration into the inflamed ST, because IL-6 is highly concentrated compared with the blood level [27].
T172 29467-29663 Sentence denotes It is most probable that the resistance of CD4+ T cells to IL-10 can be even augmented after migration into the inflamed ST, because IL-6 is highly concentrated compared with the blood level [27].
T22830 29664-29972 Sentence denotes In addition, the involvement of other essential proinflammatory cytokines in this process was suggested by our preliminary experiments demonstrating that IL-10-mediated IFN-γ inhibition in CD4+ T cells was reduced by pretreatment with IL-1β and TNF-α, although less effectively than by IL-6 (data not shown).
T173 29664-29972 Sentence denotes In addition, the involvement of other essential proinflammatory cytokines in this process was suggested by our preliminary experiments demonstrating that IL-10-mediated IFN-γ inhibition in CD4+ T cells was reduced by pretreatment with IL-1β and TNF-α, although less effectively than by IL-6 (data not shown).
T22831 29973-30157 Sentence denotes Furthermore, IFN-γ and IL-10 produced by CD4+ T cells themselves could be responsible for impaired IL-10 signaling in the ST, because T-cell infiltrates produce both cytokines [34,35].
T174 29973-30157 Sentence denotes Furthermore, IFN-γ and IL-10 produced by CD4+ T cells themselves could be responsible for impaired IL-10 signaling in the ST, because T-cell infiltrates produce both cytokines [34,35].
T22832 30158-30317 Sentence denotes In an autocrine fashion, IL-10 may persistently stimulate STAT3 activation and IFN-γ can induce SOCS1 protein as a crosstalk inhibitor of IL-10 signaling [32].
T175 30158-30317 Sentence denotes In an autocrine fashion, IL-10 may persistently stimulate STAT3 activation and IFN-γ can induce SOCS1 protein as a crosstalk inhibitor of IL-10 signaling [32].
T22833 30318-30508 Sentence denotes The T-cell-inhibitory effect of IL-10 may therefore be modulated complicatedly upon exposure to an inflammatory environment in RA joints, where many cytokines are present substantially [10].
T176 30318-30508 Sentence denotes The T-cell-inhibitory effect of IL-10 may therefore be modulated complicatedly upon exposure to an inflammatory environment in RA joints, where many cytokines are present substantially [10].
T22834 30509-30572 Sentence denotes STAT3 activation has been implicated in the pathogenesis of RA.
T177 30509-30572 Sentence denotes STAT3 activation has been implicated in the pathogenesis of RA.
T22835 30573-30672 Sentence denotes Active STAT3 is constitutively expressed in synovial fluid mononuclear cells from RA patients [36].
T178 30573-30672 Sentence denotes Active STAT3 is constitutively expressed in synovial fluid mononuclear cells from RA patients [36].
T22836 30673-30895 Sentence denotes IL-6 is the major STAT3-activating factor present in synovial fluid, which has a crucial role in the activation of monocyte functions such as gene expression of the Fc gamma receptor type I and type III and of HLA-DR [31].
T179 30673-30895 Sentence denotes IL-6 is the major STAT3-activating factor present in synovial fluid, which has a crucial role in the activation of monocyte functions such as gene expression of the Fc gamma receptor type I and type III and of HLA-DR [31].
T22837 30896-31124 Sentence denotes More recently, high levels of activated STAT3, thought to be induced mainly by IL-6, have been detected in the ST, and STAT3 activation has been shown to be involved in synovial fibroblast proliferation and IL-6 production [37].
T180 30896-31124 Sentence denotes More recently, high levels of activated STAT3, thought to be induced mainly by IL-6, have been detected in the ST, and STAT3 activation has been shown to be involved in synovial fibroblast proliferation and IL-6 production [37].
T22838 31125-31242 Sentence denotes In this regard, STAT3 is critical in the survival and expansion of growth factor-dependent synovial fibroblasts [38].
T181 31125-31242 Sentence denotes In this regard, STAT3 is critical in the survival and expansion of growth factor-dependent synovial fibroblasts [38].
T22839 31243-31595 Sentence denotes Furthermore, the significance of persistent STAT3 signaling in Th1-cell-dominated autoimmune arthritis has been suggested by studies of the gp130F759/F759 mice, in which the Src homology phosphatase-2 binding site of gp130 (the transmembrane glycoprotein β subunit of the IL-6 family cytokine receptor), tyrosine 759, was mutated to phenylalanine [39].
T182 31243-31595 Sentence denotes Furthermore, the significance of persistent STAT3 signaling in Th1-cell-dominated autoimmune arthritis has been suggested by studies of the gp130F759/F759 mice, in which the Src homology phosphatase-2 binding site of gp130 (the transmembrane glycoprotein β subunit of the IL-6 family cytokine receptor), tyrosine 759, was mutated to phenylalanine [39].
T22840 31596-31783 Sentence denotes In the gp130F759/F759 mice, T cells, particularly the CD4+ T-cell subset, are chronically activated and resistant to activation-induced cell death through gp130-mediated STAT3 activation.
T183 31596-31783 Sentence denotes In the gp130F759/F759 mice, T cells, particularly the CD4+ T-cell subset, are chronically activated and resistant to activation-induced cell death through gp130-mediated STAT3 activation.
T22841 31784-31898 Sentence denotes The longevity of cytokine signals transduced by the JAK–STAT pathway is regulated by the SOCS family proteins [7].
T184 31784-31898 Sentence denotes The longevity of cytokine signals transduced by the JAK–STAT pathway is regulated by the SOCS family proteins [7].
T22842 31899-32050 Sentence denotes We found that CD4+ T cells from patients with active RA expressed higher levels of SOCS1, but lower levels of SOCS3, compared with normal CD4+ T cells.
T185 31899-32050 Sentence denotes We found that CD4+ T cells from patients with active RA expressed higher levels of SOCS1, but lower levels of SOCS3, compared with normal CD4+ T cells.
T22843 32051-32241 Sentence denotes SOCS1 prevents activation of JAK by directly binding to JAK, and SOCS3 inhibits the action of JAK by binding to the Src homology phosphatase-2-binding domain of receptors such as gp130 [40].
T186 32051-32241 Sentence denotes SOCS1 prevents activation of JAK by directly binding to JAK, and SOCS3 inhibits the action of JAK by binding to the Src homology phosphatase-2-binding domain of receptors such as gp130 [40].
T22844 32242-32381 Sentence denotes SOCS1 and SOCS3 are induced by various cytokines, including IL-6 and IL-10, as mediators of negative feedback and crosstalk inhibition [7].
T187 32242-32381 Sentence denotes SOCS1 and SOCS3 are induced by various cytokines, including IL-6 and IL-10, as mediators of negative feedback and crosstalk inhibition [7].
T22845 32382-32519 Sentence denotes Recent studies with mice lacking SOCS3 or SOCS1 revealed that SOCS3 is a negative regulator of IL-6 signaling but not of IL-10 signaling.
T188 32382-32519 Sentence denotes Recent studies with mice lacking SOCS3 or SOCS1 revealed that SOCS3 is a negative regulator of IL-6 signaling but not of IL-10 signaling.
T22846 32520-32692 Sentence denotes Studies of conditional SOCS3-deficient mice have shown that SOCS3 deficiency, but not SOCS1 deficiency, results in sustained activation of STAT3 in response to IL-6 [8,41].
T189 32520-32692 Sentence denotes Studies of conditional SOCS3-deficient mice have shown that SOCS3 deficiency, but not SOCS1 deficiency, results in sustained activation of STAT3 in response to IL-6 [8,41].
T22847 32693-32835 Sentence denotes The analysis of SOCS3-deficient macrophages has indicated that SOCS3 is a crucial inhibitor of the IL-6-induced transcriptional response [42].
T190 32693-32835 Sentence denotes The analysis of SOCS3-deficient macrophages has indicated that SOCS3 is a crucial inhibitor of the IL-6-induced transcriptional response [42].
T22848 32836-32969 Sentence denotes However, SOCS3 is dispensable for both the negative feedback inhibition and the immunoregulatory action of IL-10 in macrophages [41].
T191 32836-32969 Sentence denotes However, SOCS3 is dispensable for both the negative feedback inhibition and the immunoregulatory action of IL-10 in macrophages [41].
T22849 32970-33053 Sentence denotes On the contrary, SOCS1 was found to directly inhibit IL-10-mediated signaling [43].
T192 32970-33053 Sentence denotes On the contrary, SOCS1 was found to directly inhibit IL-10-mediated signaling [43].
T22850 33054-33323 Sentence denotes Increased SOCS1 expression in RA CD4+ T cells may therefore be associated with both the impaired responsiveness to IL-10 and to IL-10-mediated STAT3 activation, and defective SOCS3 expression may be responsible for persistent STAT3 activation in response to serum IL-6.
T193 33054-33323 Sentence denotes Increased SOCS1 expression in RA CD4+ T cells may therefore be associated with both the impaired responsiveness to IL-10 and to IL-10-mediated STAT3 activation, and defective SOCS3 expression may be responsible for persistent STAT3 activation in response to serum IL-6.
T22851 33324-33601 Sentence denotes There is a possibility that SOCS1 induction may be associated with the ability of CD4+ T cells to produce IFN-γ, because CD4+ T cells from active RA could produce high levels of IFN-γ in the presence of IL-10, and because IFN-γ has been known as a potent inducer of SOCS1 [32].
T194 33324-33601 Sentence denotes There is a possibility that SOCS1 induction may be associated with the ability of CD4+ T cells to produce IFN-γ, because CD4+ T cells from active RA could produce high levels of IFN-γ in the presence of IL-10, and because IFN-γ has been known as a potent inducer of SOCS1 [32].
T22852 33602-33743 Sentence denotes It is of interest in this regard to indicate that polarized Th1 and Th2 cells express high levels of SOCS1 and SOCS3 mRNA, respectively [44].
T195 33602-33743 Sentence denotes It is of interest in this regard to indicate that polarized Th1 and Th2 cells express high levels of SOCS1 and SOCS3 mRNA, respectively [44].
T22853 33744-33904 Sentence denotes IL-12-induced STAT4 activation is inhibited by SOCS3 induction in Th2 cells, whereas IL-4-induced STAT6 signaling is diminished by SOCS1 induction in Th1 cells.
T196 33744-33904 Sentence denotes IL-12-induced STAT4 activation is inhibited by SOCS3 induction in Th2 cells, whereas IL-4-induced STAT6 signaling is diminished by SOCS1 induction in Th1 cells.
T22854 33905-34098 Sentence denotes SOCS1 and SOCS3 may thus have important roles as Th1-specific and Th2-specific, mutually exclusive, cross-talk repressors of the IL-4–STAT6 and the IL-12–STAT4 signaling pathways, respectively.
T197 33905-34098 Sentence denotes SOCS1 and SOCS3 may thus have important roles as Th1-specific and Th2-specific, mutually exclusive, cross-talk repressors of the IL-4–STAT6 and the IL-12–STAT4 signaling pathways, respectively.
T22855 34099-34322 Sentence denotes Consistent with this notion, PB T cells from patients with allergic diseases significantly express high levels of SOCS3 transcripts, and the SOCS3 expression correlates well with serum IgE levels and disease pathology [45].
T198 34099-34322 Sentence denotes Consistent with this notion, PB T cells from patients with allergic diseases significantly express high levels of SOCS3 transcripts, and the SOCS3 expression correlates well with serum IgE levels and disease pathology [45].
T22856 34323-34571 Sentence denotes Higher SOCS1 expression with lower SOCS3 expression in PB CD4+ T cells from RA patients, compared with healthy controls, is therefore probably consistent with their systemic bias towards a Th1 phenotype, as has previously been demonstrated [46-49].
T199 34323-34571 Sentence denotes Higher SOCS1 expression with lower SOCS3 expression in PB CD4+ T cells from RA patients, compared with healthy controls, is therefore probably consistent with their systemic bias towards a Th1 phenotype, as has previously been demonstrated [46-49].
T200 34573-34583 Sentence denotes Conclusion
T30932 34584-34787 Sentence denotes CD4+ T cells from active RA patients are characterized by their resistance to IL-10 inhibition of IFN-γ production, due to constitutive STAT3 phosphorylation and impaired IL-10-mediated STAT3 activation.
T201 34584-34787 Sentence denotes CD4+ T cells from active RA patients are characterized by their resistance to IL-10 inhibition of IFN-γ production, due to constitutive STAT3 phosphorylation and impaired IL-10-mediated STAT3 activation.
T30933 34788-34876 Sentence denotes The defective STAT3 signaling is possibly associated with SOCS1 predominance over SOCS3.
T202 34788-34876 Sentence denotes The defective STAT3 signaling is possibly associated with SOCS1 predominance over SOCS3.
T30934 34877-34997 Sentence denotes These abnormalities in active RA are thought to be induced mainly after chronic exposure to high concentrations of IL-6.
T203 34877-34997 Sentence denotes These abnormalities in active RA are thought to be induced mainly after chronic exposure to high concentrations of IL-6.
T30935 34998-35157 Sentence denotes The limited efficacy of IL-10 treatment of RA patients [50] may be explained in part by the unresponsiveness to IL-10 of inflammatory cells, including T cells.
T204 34998-35157 Sentence denotes The limited efficacy of IL-10 treatment of RA patients [50] may be explained in part by the unresponsiveness to IL-10 of inflammatory cells, including T cells.
T30936 35158-35395 Sentence denotes On the contrary, the therapeutic efficacy of anti-IL-6 receptor antibody has been reported in RA patients [51], and one of the effects of this therapy may be to normalize T cells through the inhibition of IL-6-dependent STAT3 activation.
T205 35158-35395 Sentence denotes On the contrary, the therapeutic efficacy of anti-IL-6 receptor antibody has been reported in RA patients [51], and one of the effects of this therapy may be to normalize T cells through the inhibition of IL-6-dependent STAT3 activation.
T30937 35396-35574 Sentence denotes More specific therapy targeting STAT3 activation will be awaited; for example, the induction of the SOCS3 gene, the efficacy of which has been demonstrated in animal models [37].
T206 35396-35574 Sentence denotes More specific therapy targeting STAT3 activation will be awaited; for example, the induction of the SOCS3 gene, the efficacy of which has been demonstrated in animal models [37].
T207 35576-35589 Sentence denotes Abbreviations
T208 35590-36318 Sentence denotes BSA = bovine serum albumin; CRP = C-reactive protein; ELISA = enzyme-linked immunosorbent assay; Fc = crystallazibe fragment; FCS = fetal calf serum; FITC = fluorescein isothiocyanate; IFN-γ = interferon gamma; IL = interleukin; IL-10R = interleukin-10 receptor; IL-10R1 = type 1 interleukin-10 receptor; JAK = Janus kinase; mAb = monoclonal antibody; MHC = major histocompatibility complex; PB = peripheral blood; PBMC = peripheral blood mononuclear cells; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; RA = rheumatoid arthritis; SOXS = suppressor of cytokine signaling; ST = synovial tissue; STAT = signal transducer and activator of transcription; Th = T helper cells; TNF-α = tumor necrosis factor alpha.
T209 36320-36339 Sentence denotes Competing interests
T210 36340-36400 Sentence denotes The author(s) declare that they have no competing interests.
T211 36402-36424 Sentence denotes Authors' contributions
T212 36425-36512 Sentence denotes Jiro Yamana was responsible for the experiments and data analysis and wrote the report.
T213 36513-36608 Sentence denotes Masahiro Yamamura was responsible for the planning of the research and wrote up the manuscript.
T214 36609-36704 Sentence denotes Akira Okamoto, Tetsushi Aita, Mitsuhiro Iwahashi, and Katsue Sunahori assisted the experiments.
T215 36705-36752 Sentence denotes Hirofumi Makino critically read the manuscript.