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Katsuya Nagatani Kenji Itoh Kyoichi Nakajima Hirohumi Kuroki Yozo Katsuragawa Makoto Mochizuki Shinichi Aotsuka Akio Mimori 《Arthritis \u0026amp; Rheumatology》2007,56(11):3554-3563
Objective
Fibroblast‐like synoviocytes (FLS) are among the principal effector cells in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to examine the variety of stimulating effects of APRIL and its specific effect on FLS in the affected RA synovium.Methods
Synovium and serum samples were obtained from patients with RA, patients with osteoarthritis (OA), and healthy subjects. Soluble APRIL proteins were assayed by enzyme‐linked immunosorbent assay. The relative gene expression of APRIL, BCMA, interleukin‐6 (IL‐6), tumor necrosis factor α (TNFα), IL‐1β, and RANKL was assessed in RA and OA FLS by polymerase chain reaction. Effects of APRIL on the production of proinflammatory cytokines and RANKL in RA FLS were investigated by flow cytometry and with the use of a BCMA‐Fc fusion protein.Results
A significantly higher level of soluble APRIL was detected in RA serum compared with normal serum. Among the 3 receptors of APRIL tested, RA FLS expressed only BCMA, whereas OA FLS expressed none of the receptors. APRIL stimulated RA FLS, but not OA FLS, to produce IL‐6, TNFα, IL‐1β, and APRIL itself. In addition, APRIL increased RA FLS expression of RANKL and also enhanced progression of the cell cycle of RA FLS. Neutralization of APRIL by the BCMA‐Fc fusion protein attenuated all of these stimulating effects of APRIL on RA FLS.Conclusion
RA FLS are stimulated by APRIL and express the APRIL receptor BCMA. These results provide evidence that APRIL is one of the main regulators in the pathogenesis of RA.2.
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Derek Lacey Annaleise Sampey Robert Mitchell Richard Bucala Lanie Santos Michelle Leech Eric Morand 《Arthritis \u0026amp; Rheumatology》2003,48(1):103-109
Objective
The hyperplasia of fibroblast‐like synoviocytes (FLS) is considered essential to the evolution of joint destruction in rheumatoid arthritis (RA), but the mechanisms underlying FLS proliferation remain poorly understood. Macrophage migration inhibitory factor (MIF) is a cytokine that has recently been shown to exert proinflammatory effects on RA FLS. This study sought to identify the mechanisms of activation of FLS by MIF, and to assess the effects of MIF on synovial cell proliferation.Methods
Human RA FLS were treated with recombinant MIF, interleukin‐1β (IL‐1β), tumor necrosis factor α (TNFα), and/or anti‐MIF monoclonal antibodies (mAb). Proliferation was measured with tritiated thymidine incorporation. Nuclear factor κB (NF‐κB) and mitogen‐activated protein (MAP) kinase activation were measured with immunohistochemistry and Western blotting, respectively.Results
FLS proliferation was significantly increased by MIF. IL‐1β and TNFα also induced proliferation, but these effects were prevented by neutralization with anti‐MIF mAb. Activation of NF‐κB was induced by IL‐1β, but not by MIF. Anti‐MIF mAb had no effect on IL‐1β–induced NF‐κB nuclear translocation. By contrast, MIF induced phosphorylation of extracellular signal–regulated kinase (ERK) MAP kinase. ERK antagonism, but not NF‐κB antagonism, prevented the effect of MIF on FLS proliferation.Conclusion
These data suggest that MIF may regulate RA synovial hyperplasia by acting directly and via involvement in the effects of IL‐1β and TNFα. In addition, the effects of MIF on FLS activation are independent of NF‐κB, and dependent on ERK MAP kinase. These data suggest an important therapeutic potential for MIF antagonism in RA.6.
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Yasuhiro Take Ken Nakata Jun Hashimoto Hideki Tsuboi Norihiro Nishimoto Takahiro Ochi Hideki Yoshikawa 《Arthritis \u0026amp; Rheumatology》2009,60(12):3591-3601
Objective
Osteopontin (OPN) is expressed by fibroblast‐like synoviocytes (FLS) in rheumatoid arthritis (RA), but its pathologic role is still obscure. The present study was undertaken to analyze the role of OPN in RA by focusing on its effects on cell–cell interactions between FLS and B lymphocytes.Methods
FLS obtained from 10 patients with RA and 10 non‐RA subjects and a B lymphocyte cell line were studied. The characteristics of OPN expression by FLS were analyzed by Western blotting, immunoprecipitation, and immunofluorescence studies. In cocultures of FLS and B lymphocytes, the effects of OPN on adhesion of B lymphocytes to FLS and the consequent production of interleukin‐6 (IL‐6) were analyzed in experiments involving overexpression and knockdown of OPN and inhibitory studies with an OPN‐blocking antibody. In vivo, the expression of OPN in RA synovium was examined by immunohistochemistry.Results
A specifically modified 75‐kd form of OPN was predominantly expressed in RA FLS, and this was associated with expression of >200‐kd thrombin‐cleaved OPN that was crosslinked with fibronectin and localized on the surface of the FLS. In FLS–B lymphocyte cocultures, 75‐kd OPN–positive FLS produced a significantly higher amount of IL‐6 than did 75‐kd OPN–negative FLS. When the FLS were separated from B lymphocytes or cultured alone, the production of IL‐6 was low and was not significantly different between these 2 culture conditions. Moreover, OPN overexpression enhanced production of IL‐6 in 75‐kd OPN–positive FLS–B lymphocyte cocultures. Addition of the OPN‐blocking antibody inhibited the adhesion of B lymphocytes to FLS. Immunohistochemical analyses revealed that localization of IL‐6–positive cells coincided with the sites at which OPN and B lymphocytes were colocalized.Conclusion
Specifically modified 75‐kd OPN was expressed by RA FLS. This form of OPN affected FLS–B lymphocyte interactions by supporting the adhesion of B lymphocytes to FLS and enhancing the production of IL‐6.10.
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Kyoung‐Woon Kim Mi‐La Cho Hae‐Rim Kim Ji‐Hyeon Ju Mi‐Kyung Park Hye‐Jwa Oh Joon‐Seok Kim Sung‐Hwan Park Sang‐Heon Lee Ho‐Youn Kim 《Arthritis \u0026amp; Rheumatology》2007,56(4):1076-1086
Objective
Stromal cell–derived factor 1 (SDF‐1) is a potent chemoattractant for memory T cells in inflamed rheumatoid arthritis (RA) synovium. This study was undertaken to investigate the effect of interleukin‐17 (IL‐17) and CD40–CD40L interaction on SDF‐1 production in RA fibroblast‐like synoviocytes (FLS).Methods
Synovial fluid (SF) and serum levels of SDF‐1 in RA patients were measured by enzyme‐linked immunosorbent assay (ELISA). The SDF‐1 produced by cultured RA FLS was evaluated by real‐time polymerase chain reaction and ELISA after FLS were treated with IL‐17 and inhibitors of intracellular signal molecules. The SDF‐1 level was also determined after FLS were cocultured with T cells in the presence and absence of IL‐17.Results
Concentrations of SDF‐1 in the sera and SF were higher in RA patients than in osteoarthritis patients, although the increase in the serum levels did not reach statistical significance. The production of SDF‐1 in RA FLS was enhanced by IL‐17 stimulation. This effect of IL‐17 was blocked by inhibitors of phosphatidylinositol 3‐kinase (PI 3‐kinase), NF‐κB, and activator protein 1 (AP‐1). When FLS were cocultured with T cells, SDF‐1 production was up‐regulated, especially in the presence of IL‐17, but FLS were inhibited by neutralizing anti–IL‐17 and anti‐CD40L antibodies. Addition of RA SF to cultured RA FLS significantly up‐regulated SDF‐1 messenger RNA expression, which was hampered by pretreatment with anti–IL‐17 antibody.Conclusion
SDF‐1 is overproduced in RA FLS, and IL‐17 could up‐regulate the expression of SDF‐1 in RA FLS via pathways mediated by PI 3‐kinase, NF‐κB, and AP‐1. Our findings suggest that inhibition of the interaction between IL‐17 from T cells and SDF‐1 in FLS may provide a new therapeutic approach in RA.13.
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Seung‐Ah Yoo Hyung‐Ju Yoon Hyun‐Sook Kim Chi‐Bom Chae Sandro De Falco Chul‐Soo Cho Wan‐Uk Kim 《Arthritis \u0026amp; Rheumatology》2009,60(2):345-354
Objective
To investigate the direct effects of placenta growth factor (PlGF) and its specific receptor, flt‐1, which are known to mediate angiogenesis, on the inflammatory process of rheumatoid arthritis (RA).Methods
Expression of PlGF and flt‐1 in the synovial tissue of RA patients was examined using immunohistochemistry. Enzyme‐linked immunosorbent assay was used to determine the concentrations of PlGF, tumor necrosis factor α (TNFα), and interleukin‐6 (IL‐6) in culture supernatants of either mononuclear cells or synoviocytes. The flt‐1 expression level in mononuclear cells was analyzed by flow cytometry. Experimental arthritis was induced in mice either by immunization with type II collagen (CII) or by injection of anti‐CII antibody.Results
PlGF was highly expressed in the synovium of RA patients, and its primary source was fibroblast‐like synoviocytes (FLS). When stimulated with IL‐1β, FLS from RA patients produced higher amounts of PlGF than did FLS from patients with osteoarthritis. Exogenous PlGF specifically increased the production of TNFα and IL‐6 in mononuclear cells from RA patients (but not those from healthy controls) via a calcineurin‐dependent pathway. The response to PlGF was associated with increased expression of flt‐1 on RA monocytes, which could be induced by IL‐1β and TNFα. A novel anti–flt‐1 hexapeptide, GNQWFI, abrogated the PlGF‐induced increase in TNFα and IL‐6 production, and also suppressed CII‐induced arthritis and serum IL‐6 concentrations in mice. Moreover, genetic ablation of PlGF prevented the development of anti‐CII antibody–induced arthritis in mice.Conclusion
Our data suggest that enhanced expression of PlGF and flt‐1 may contribute to rheumatoid inflammation by triggering production of proinflammatory cytokines. The use of the novel anti–flt‐1 peptide, GNQWFI, may be an effective strategy for the treatment of RA.16.
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Myew‐Ling Toh Yuan Yang Michelle Leech Lanie Santos Eric F. Morand 《Arthritis \u0026amp; Rheumatology》2004,50(10):3118-3128
Objective
Mitogen‐activated protein kinases (MAPKs) are activated by proinflammatory stimuli. MAPK phosphatases (MKPs), in particular MKP‐1, have been identified as endogenous negative regulators of MAPK activation. Since MAPKs are known to be important in rheumatoid arthritis (RA) synoviocyte activation, this study assessed the expression, regulation, and function of MKP‐1 in RA.Methods
MKP‐1 expression was measured by Western blotting (WB) and real‐time polymerase chain reaction (PCR). RA fibroblast‐like synoviocytes (FLS) were treated with interleukin‐1β (IL‐1β), tumor necrosis factor α, fetal calf serum, and dexamethasone. Expression of MAPKs in RA FLS was analyzed by WB using phosphospecific antibodies, while IL‐6 expression was assessed by real‐time PCR.Results
MKP‐1 protein and messenger RNA were detected in cultured RA FLS. IL‐1β rapidly up‐regulated MKP‐1, coinciding with reciprocal down‐regulation of ERK, JNK, and p38 MAPK phosphorylation. Dexamethasone rapidly and sustainably up‐regulated MKP‐1, and this also coincided with down‐regulation of ERK, JNK, and p38 MAPK phosphorylation. In addition, dexamethasone augmented IL‐1β–induced up‐regulation of MKP‐1, and this was associated with inhibition of ERK, JNK, and p38 MAPK phosphorylation and IL‐6 expression. Dexamethasone had no effect on the phosphorylation of upstream kinases such as MEKK‐3/6. In the presence of glucocorticoid (GC) receptor antagonist RU 486, the dexamethasone‐mediated up‐regulation of MKP‐1 was impaired. Moreover, inhibition of MKP‐1 expression impaired dexamethasone‐mediated inhibition of MAPK phosphorylation.Conclusion
This study demonstrates the expression of MKP‐1 in RA FLS. Cytokine and GC regulation of MKP‐1 may be important in determining the magnitude of the inflammatory response in RA that is mediated via MAPKs. The effects of GCs in RA may be mediated, in part, via GC receptor–dependent up‐regulation of MKP‐1.19.
Mi‐La Cho Chong‐Hyeon Yoon Sue‐Yun Hwang Mi‐Kyung Park So‐Youn Min Sang‐Heon Lee Sung‐Hwan Park Ho‐Youn Kim 《Arthritis \u0026amp; Rheumatology》2004,50(3):776-784
Objective
To investigate the effector function exerted by type II collagen (CII)–stimulated T cells on rheumatoid arthritis (RA) fibroblast‐like synoviocytes (FLS), and to determine their contribution to RA pathogenesis.Methods
We used enzyme‐linked immunosorbent assays to measure the levels of interleukin‐15 (IL‐15), tumor necrosis factor α (TNFα), and IL‐18 production by FLS that were cocultured with antigen‐activated T cells. Likewise, we analyzed the levels of interferon‐γ (IFNγ) and IL‐17 production by RA T cells coincubated with FLS. To investigate the cross‐talk between CII‐stimulated T cells and RA FLS, we examined the effect of using a transwell membrane to separate T cells and FLS in a culture chamber, as well as the effect of adding an antibody to block CD40 ligation.Results
The levels of IL‐15, TNFα, IFNγ, and IL‐17 were all significantly increased in the serum of RA patients compared with normal control serum. Among the patients, the group with a stronger T cell proliferation response to CII showed higher levels of these inflammatory mediators. When coincubated with RA FLS, these T cells induced the production of IL‐15, TNFα, and IL‐18 by FLS with an intensity that increased in proportion to the duration of CII stimulation. T cells, in turn, responded to FLS stimulation by secreting higher amounts of IL‐17 and IFNγ in coculture. Interestingly, T cells that were activated by CII for longer periods of time showed stronger induction of these cytokines. The cross‐talk between T cells and FLS appeared to require direct cell–cell contact as well as CD40 ligation, at least in part.Conclusion
Through repeated stimulation by CII, RA synovial T cells became trained effector cells that induced the production of proinflammatory mediators by FLS, while in the process the T cells becoming more sensitized to the activation signal from FLS.20.
Diego Kyburz Janine Rethage Reinhart Seibl Roger Lauener Renate E. Gay Dennis A. Carson Steffen Gay 《Arthritis \u0026amp; Rheumatology》2003,48(3):642-650