Up‐regulation of stromal cell–derived factor 1 (CXCL12) production in rheumatoid synovial fibroblasts through interactions with T lymphocytes: Role of interleukin‐17 and CD40L–CD40 interaction

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.

     

Rheumatoid arthritis fibroblast‐like synoviocytes express BCMA and are stimulated by APRIL

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.

     

Down‐regulation of FLIP sensitizes rheumatoid synovial fibroblasts to Fas‐mediated apoptosis

Objective

Hyperplasia of fibroblast‐like synoviocytes (FLS) contributes to chronic inflammation and joint destruction in rheumatoid arthritis (RA). FLICE‐inhibitory protein (FLIP) is an antiapoptotic protein that might prevent apoptotic elimination of FLS in response to death ligands such as tumor necrosis factor α (TNFα) or Fas ligand, which are present in RA synovium. Previous studies on FLIP expression by osteoarthritis (OA) and RA FLS have shown variable results, and the specific role of FLIP as an apoptosis inhibitor in these cells remains unclear. We undertook this study to investigate the expression and antiapoptotic function of FLIP in FLS.

Methods

We studied the expression of FLIP by immunohistochemistry and immunoblotting in synovial tissues or cultured FLS from RA and OA patients. FLS apoptosis was induced by an agonistic anti‐Fas monoclonal antibody and FLS were then quantified. We studied the effects of cycloheximide (CHX), TNFα, and FLIP antisense oligonucleotide on FLIP expression and FLS apoptotic susceptibility.

Results

FLIP_L was the isoform mainly expressed in lining synoviocytes and cultured FLS. Synovial tissues and cultured FLS from OA and RA tissues displayed similar patterns and levels of expression of FLIP. Fas‐induced apoptosis was variable in different FLS lines, but differences between OA and RA groups were not detected. TNFα induced increases in FLIP_L and FLIP_S expression and protected RA FLS from apoptosis, while CHX induced the opposite effects. Down‐regulation of FLIP by antisense oligonucleotide strongly sensitized RA FLS to Fas‐mediated apoptosis.

Conclusion

Apoptosis susceptibility and FLIP expression are similar in OA and RA FLS. Down‐regulation of FLIP sensitizes RA FLS to Fas‐mediated apoptosis and may be a valuable tool for targeting RA FLS hyperplasia.

     

Specifically modified osteopontin in rheumatoid arthritis fibroblast‐like synoviocytes supports interaction with B cells and enhances production of interleukin‐6

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.

     

Control of fibroblast‐like synoviocyte proliferation by macrophage migration inhibitory factor

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.

     

Expression of the MAPK kinases MKK‐4 and MKK‐7 in rheumatoid arthritis and their role as key regulators of JNK

Objective

The mitogen‐activated protein (MAP) kinase JNK is a key regulator of interleukin‐1 (IL‐1)–induced collagenase gene expression and joint destruction in arthritis. Two upstream kinases, MKK‐4 and MKK‐7, have been identified as potential activators of JNK. However, the role of MAP kinase kinases (MAPKKs) and their functional organization within fibroblast‐like synoviocytes (FLS) have not been defined. We therefore evaluated the interactions between the various MAP kinase components and determined their subcellular localization.

Methods

MKKs were identified by immunohistochemistry of rheumatoid arthritis (RA) and osteoarthritis (OA) synovium. Western blotting was used to determine the expression of FLS. Immunoprecipitation experiments using antibodies specific for MKK‐4, MKK‐7, and JNK were performed. Phosphospecific antibodies and immunohistochemistry were used to evaluate the activation state of synovial MKK‐4 and MKK‐7. Confocal microscopy was used to determine the subcellular location of the kinases.

Results

Immunohistochemistry studies demonstrated abundant MKK‐4 and MKK‐7 in RA and OA synovium, but the levels of phosphorylated kinases were significantly higher in RA synovium. MKK‐4 and MKK‐7 were constitutively expressed by cultured RA and OA FLS, and IL‐1 stimulation resulted in rapid phosphorylation of both kinases. JNK was detected in MKK‐4 and MKK‐7 immunoprecipitates. Furthermore, MKK‐4 coprecipitated with MKK‐7 and vice versa, indicating that the 3 kinases form a stable complex in FLS. Confocal microscopy confirmed that JNK, MKK‐4, and MKK‐7 colocalized in the cytoplasm, with JNK migrating to the nucleus after IL‐1 stimulation. The signal complex containing MKK‐4, MKK‐7, and JNK was functionally active and able to phosphorylate c‐Jun after IL‐1 stimulation of FLS.

Conclusion

These studies demonstrate that JNK, MKK‐4, and MKK‐7 form an active signaling complex in FLS. This novel JNK signalsome is activated in response to IL‐1 and migrates to the nucleus. The JNK signalsome represents a new target for therapeutic interventions designed to prevent joint destruction.

     

Modulation of interleukin‐6 and matrix metalloproteinase 2 expression in human fibroblast‐like synoviocytes by functional ionotropic glutamate receptors

Objective

Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology.

Methods

Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast‐like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin‐6 (IL‐6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP‐2] and MMP‐9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP‐1] and TIMP‐2). IL‐6 concentrations were determined by enzyme‐linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL‐6 or MMP‐2.

Results

Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N‐methyl‐D ‐aspartate (NMDA) receptors in RA FLS increased proMMP‐2 release, whereas non‐NMDA ionotropic glutamate receptor antagonists reduced IL‐6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors.

Conclusion

Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL‐6 expression.