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.

     

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.

     

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.

     

Effector function of type II collagen–stimulated T cells from rheumatoid arthritis patients: Cross‐talk between T cells and synovial fibroblasts

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.

     

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.

     

Protein Tyrosine Phosphatase Expression Profile of Rheumatoid Arthritis Fibroblast‐like Synoviocytes: A Novel Role of SH2 Domain–Containing Phosphatase 2 as a Modulator of Invasion and Survival

Objective

The fibroblast‐like synoviocytes (FLS) in the synovial intimal lining of the joint are key mediators of inflammation and joint destruction in rheumatoid arthritis (RA). In RA, these cells aggressively invade the extracellular matrix, producing cartilage‐degrading proteases and inflammatory cytokines. The behavior of FLS is controlled by multiple interconnected signal transduction pathways involving reversible phosphorylation of proteins on tyrosine residues. However, little is known about the role of the protein tyrosine phosphatases (PTPs) in FLS function. This study was undertaken to explore the expression of all of the PTP genes (the PTPome) in FLS.

Methods

A comparative screening of the expression of the PTPome in FLS from patients with RA and patients with osteoarthritis (OA) was conducted. The functional effect on RA FLS of SH2 domain–containing phosphatase 2 (SHP‐2), a PTP that was up‐regulated in RA, was then analyzed by knockdown using cell‐permeable antisense oligonucleotides.

Results

PTPN11 was overexpressed in RA FLS compared to OA FLS. Knockdown of PTPN11, which encodes SHP‐2, reduced the invasion, migration, adhesion, spreading, and survival of RA FLS. Additionally, signaling in response to growth factors and inflammatory cytokines was impaired by SHP‐2 knockdown. RA FLS that were deficient in SHP‐2 exhibited decreased activation of focal adhesion kinase and mitogen‐activated protein kinases.

Conclusion

These findings indicate that SHP‐2 has a novel role in mediating human FLS function and suggest that it promotes the invasiveness and survival of RA FLS. Further investigation may reveal SHP‐2 to be a candidate therapeutic target for RA.

     

RANK and RANKL expression as markers of dendritic cell–t cell interactions in paired samples of rheumatoid synovium and lymph nodes

Objective

The RANK/RANKL pathway is critical in bone destruction in conditions such as rheumatoid arthritis (RA). Since RANK/RANKL‐deficient mice show major lymph node (LN) abnormalities, undertook this study to investigate the expression of RANK/RANKL in paired samples of synovium and LNs from RA patients.

Methods

Using immunohistochemistry, RANK/RANKL expression by dendritic cell (DC) and T cell subsets was studied in this unique set of samples and in RA synoviocytes stimulated with interleukin‐1β (IL‐1β), tumor necrosis factor α (TNFα), and IL‐17.

Results

In RA synovium, RANKL+ cells were detected in the lining layer and the lymphocytic infiltrates, whereas RANK expression was restricted to the perivascular infiltrates. In LNs, RANK+ and RANKL+ cells were diffusely expressed in the T cell zone and in germinal centers. Double staining of paired RA synovium and LN sections showed that some immature CD1a+ DCs expressed RANK and RANKL, while some mature DC‐LAMP+ DCs expressed only RANK. Some CD3+, CD4+, interferon‐γ+, and IL‐17+ cells expressed RANKL, while none expressed RANK. Treatment of synoviocytes with TNFα or IL‐1β in combination with IL‐17 was particularly potent at inducing RANKL expression.

Conclusion

This study shows the involvement of RANK/RANKL in DC–T cell interactions during an inflammatory process. RANK expression appears to be limited to the sites of immune reaction, both in the synovium and in LNs. Therapeutic control of these targets may have both positive and negative consequences for the immune system.

     

Association of the –2849 interleukin‐10 promoter polymorphism with autoantibody production and joint destruction in rheumatoid arthritis

Objective

To analyze the –2849 A/G interleukin‐10 (IL‐10) promoter polymorphism, which is associated with high (AG/GG) and low (AA) IL‐10 production, in a cohort of rheumatoid arthritis (RA) patients and controls in order to gain a better understanding of its role in the incidence and progression of RA.

Methods

Allele frequencies of the promoter polymorphism –2849 A/G and carriage rates were compared in 283 RA patients, 413 patients with other rheumatic diseases, and 1,220 healthy controls. The rate of joint damage and baseline levels of IgG and IgM rheumatoid factors and anti–citrullinated peptide antibodies were measured and were correlated with the IL‐10 gene polymorphism. Furthermore, the correlation between the invasiveness of fibroblast‐like synoviocytes (FLS) and the −2849 IL‐10 genotype was tested.

Results

The IL‐10 genotype was not associated with the incidence of RA, but instead, correlated with disease progression, as determined by the extent of joint destruction. A higher rate of joint destruction was observed in patients with the genotype associated with high IL‐10 production. Since FLS are thought to be involved in joint destruction, we analyzed IL‐10 genotypes in conjunction with FLS invasiveness. Although adenoviral gene transfer of IL‐10 to FLS inhibited their invasiveness, no differences were observed in vitro in the FLS from RA patients who were –2849 non‐G carriers compared with those who were G carriers. Instead, patients with the –2849 AG/GG genotype, which is associated with high IL‐10 production, had higher autoantibody titers at baseline.

Conclusion

The –2849 IL‐10 promoter polymorphism is associated with autoantibody production and subsequent joint damage in RA.

     

PUMA regulation and proapoptotic effects in fibroblast‐like synoviocytes

Objective

Although p53 is overexpressed in rheumatoid arthritis (RA) synovial tissue (ST), few synoviocytes undergo apoptosis. This could be partly due to low expression of proapoptotic genes. Deficient p53 up‐regulated modulator of apoptosis (PUMA), which is a major effector of p53‐mediated cell death, could contribute to this phenomenon. To evaluate a method to induce apoptosis, the expression and function of PUMA was investigated in ST and cultured fibroblast‐like synoviocytes (FLS).

Methods

PUMA expression in ST was measured by immunohistochemistry, Western blot analysis, and quantitative polymerase chain reaction analysis. Ad‐p53 and plasmids encoding hemagglutinin‐tagged, full‐length PUMA expression vector (HA‐PUMA), PUMA lacking the Bcl‐2 homology 3 domain, or pCEP4 were used to transfect FLS. Apoptosis was quantified by trypan blue exclusion, DNA fragmentation, and caspase 3 activation.

Results

PUMA protein was detected in RA ST, although most of the immunoreactive protein was localized to sublining cells rather than the intimal lining synoviocytes. Western blot analysis showed no difference between RA ST and osteoarthritis (OA) ST. PUMA messenger RNA was detected in RA and OA ST, although the amounts were markedly lower than in the spleen and FLS. To determine if PUMA was inducible, FLS were transduced with Ad‐p53. Even though p53 protein was produced and p21 expression was increased, PUMA expression was not enhanced. Consistent with this observation, Ad‐p53 did not induce apoptosis in FLS. However, HA‐PUMA transfection into FLS resulted in rapid apoptosis with the activation of caspase 3.

Conclusion

PUMA can induce apoptosis by FLS and represents a potential target in RA.

     

Fractalkine is a novel chemoattractant for rheumatoid arthritis fibroblast‐like synoviocyte signaling through MAP kinases and Akt

Objective

Fibroblast‐like synoviocytes (FLS) are a major constituent of the hyperplastic synovial pannus that aggressively invades cartilage and bone during the course of rheumatoid arthritis (RA). Fractalkine (FKN/CX₃CL1) expression is up‐regulated in RA synovium and RA synovial fluid. While RA FLS express the FKN receptor, CX₃CR1, the pathophysiologic relevance of FKN stimulation of RA FLS is not understood. This study was undertaken to better characterize the relationship between FKN and the RA FLS that both produce it and express its receptor.

Methods

RA FLS were subjected to chemotaxis and proliferation assays, Western blotting, enzyme‐linked immunosorbent assays, and filamentous actin staining to characterize the relationship between FKN and RA FLS.

Results

FKN secretion by RA FLS was regulated mainly by tumor necrosis factor α. Stimulation of RA FLS with FKN led to significant cytoskeletal rearrangement but no proliferation. Chemotaxis assays revealed that FKN was a novel chemoattractant for RA FLS. Stimulation of RA FLS with FKN resulted in activation of MAP kinases and Akt. JNK, ERK‐1/2, and Akt (at both Ser‐473 and Thr‐308) were each up‐regulated in a time‐dependent manner. Inhibition of ERK‐1/2–mediated signaling, but not JNK or Akt, significantly repressed FKN‐induced RA FLS migration.

Conclusion

These findings indicate a novel role of FKN in regulating RA FLS cytoskeletal structure and migration. FKN specifically induces RA FLS phosphorylation of the MAP kinases JNK and ERK‐1/2, as well as full activation of Akt.