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.

     

Simvastatin inhibits production of interleukin 6 (IL-6) and IL-8 and cell proliferation induced by tumor necrosis factor-alpha in fibroblast-like synoviocytes from patients with rheumatoid arthritis

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory disease in which the synovial environment is characterized by intense immunological activity. Evidence suggests that statins modulate immune functions and may have a beneficial effect on patients with RA. We investigated whether simvastatin could inhibit the expression of interleukin 6 (IL-6) and IL-8 and cell proliferation induced by tumor necrosis factor-alpha (TNF-alpha) in fibroblast-like synoviocytes (FLS) obtained from RA patients undergoing joint replacement therapy. METHODS: RA FLS were cultured with or without 0.05-10 microM simvastatin for 12 h. Cytokine mRNA expression and secretion levels were detected using real-time PCR and ELISA, respectively. Cell proliferation of FLS induced by TNF-alpha was determined by MTT assay. RESULTS: Real-time PCR analysis revealed that the levels of IL-6 and IL-8 mRNA expressed by FLS were reduced by simvastatin in a dose-dependent manner. Levels of IL-6 and IL-8 in FLS culture supernatants were decreased by simvastatin in a time-dependent and dose-dependent manner. MTT assay revealed that simvastatin could inhibit proliferation of FLS induced by TNF-alpha. These effects of simvastatin on IL-6 and IL-8 production and cell proliferation were reversed in the presence of mevalonic acid or geranylgeranyl-pyrophosphate, but not with farnesyl-pyrophosphate. CONCLUSION: Our results suggest that the beneficial effect of simvastatin in RA patients may involve inhibition of IL-6 and IL-8 production, as well as reduction of cell proliferation.     

Role of placenta growth factor and its receptor flt‐1 in rheumatoid inflammation: A link between angiogenesis and inflammation

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.

     

Fractalkine mediates T cell–dependent proliferation of synovial fibroblasts in rheumatoid arthritis

Objective

In rheumatoid arthritis (RA), synovial fibroblasts proliferate excessively, eventually eroding bone and cartilage. The aim of this study was to examine the mechanisms through which CD4 T cells, the dominant lymphocyte population in patients with rheumatoid synovitis, regulate synoviocyte proliferation.

Methods

Fibroblast‐like synoviocyte (FLS) lines were established from rheumatoid synovium. CD4 T cells from patients with RA and age‐matched control subjects were cultured on FLS monolayers. FLS proliferation was quantified by cytometry, using carboxyfluorescein succinimidyl ester staining or microscopic enumeration of PKH26‐stained FLS. Surface expression of the fractalkine (FKN) receptor CX₃CR1 was monitored by fluorescence‐activated cell sorting. The induction of CX₃CR1 and its ligand FKN in FLS was quantified by real‐time polymerase chain reaction.

Results

The proliferation of FLS was significantly increased in the presence of CD4 T cells from patients with RA compared with control T cells. CD4+,CD28– T cells were particularly effective in supporting FLS growth, inducing a 25‐fold expansion compared with a 5‐fold expansion induced by CD4+,CD28+ T cells. The growth‐promoting activity of CD4+,CD28– T cells was mediated through CX₃CR1, a chemokine receptor expressed on both T cells and FLS. Anti‐CX₃CR1 antibodies inhibited T cell production of tumor necrosis factor α (TNFα) and suppressed FLS proliferation. TNFα amplified the expansion of FLS by enhancing their expression of CX₃CR1 and FKN.

Conclusion

FKN–CX₃CR1 receptor–ligand interactions regulate FLS growth and FLS‐dependent T cell function. FLS stimulate autocrine growth by releasing FKN and triggering the activity of their own CX₃CR1. This growth‐promotion loop is amplified by TNFα produced by CX₃CR1‐expressing T cells upon stimulation by FKN‐expressing FLS. These data assign a critical role to FKN and its receptor in fibroblast proliferation and pannus formation in RA.

     

Lysophosphatidic acid receptor 1 suppression sensitizes rheumatoid fibroblast‐like synoviocytes to tumor necrosis factor–induced apoptosis

Objective

To investigate the role of lysophosphatidic acid (LPA) receptors in the proliferation and apoptosis of fibroblast‐like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).

Methods

Expression of LPA receptors 1–3 was analyzed by real‐time polymerase chain reaction (PCR). LPAR1 and LPAR2 were suppressed in RA FLS by small interfering RNA (siRNA) transfection. Proliferation of RA FLS after tumor necrosis factor (TNF) and LPA stimulation was determined with a luminescent cell viability assay. Apoptosis was analyzed by quantification of nucleosome release and measurement of activated caspase 3/7. Genes involved in the apoptotic response were identified with a human apoptosis PCR array and validated with Western blot assays. The requirement of these genes for apoptosis sensitization was assessed by siRNA transfection. Secretion of mediators of inflammation was analyzed by enzyme‐linked immunosorbent assay.

Results

Only LPAR1 and LPAR2 were expressed by RA FLS, and their levels were higher than those in osteoarthritis (OA) FLS. Suppression of LPAR1 abrogated TNF‐induced proliferation and sensitized the RA FLS, but not the OA FLS, to TNF‐induced apoptosis. These changes occurred despite an increased early inflammatory response to TNF. Sensitization to apoptosis was associated with changes in expression of multiple apoptosis‐related genes. Three of the up‐regulated proapoptotic genes were further studied to confirm their involvement. In contrast, suppression of LPAR2 showed no effect in any of these analyses.

Conclusion

LPA₁ is an important receptor in RA FLS. Its suppression is accompanied by a global increase in the response to TNF that is ultimately dominated by sensitization to apoptosis.

     

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.     

Combination analgesia in 2005—a rational approach: focus on paracetamol–tramadol

Interleukin-17 (IL-17) is a proinflammatory cytokine that is expressed in the synovium T cells of rheumatoid arthritis (RA). This cytokine is implicated in the inflammation and destruction of the joint. However, the role of IL-17 on the production of vascular endothelial factor (VEGF) important to synovial proliferation has still not been identified. In this study, we investigated the effect on cultured rheumatoid fibroblast-like synoviocytes (FLS) of the IL-17 on the production and expression of VEGF, which play an important role in angiogenesis in rheumatoid synovium. IL-17 increased the production of VEGF dose dependently and the mRNA expression of VEGF. These results suggest that IL-17 might influence angiogenesis in RA by up-regulating the expression of VEGF in rheumatoid FLS.     

成纤维滑膜细胞p53基因表达对类风湿关节炎患者CD4+T淋巴细胞的调节

目的 研究人成纤维滑膜细胞(FLS)p53基因表达对类风湿关节炎(RA)患者CD4+T淋巴细胞的调节作用.方法 采用小分子干扰RNA(siRNA)转染抑制FKS内p53基因表达,并与RA患者外周血CD4+T淋巴细胞共同培养.检测转染后FLS中骨保护素(OPG)表达及白细胞介素(IL)-6分泌.并对与之共培养的CD4+细胞膜胞质内蛋白干扰素(IFN)-γ、IL-17、IL-4和CD25以及IFN-γ孤儿核受体γt(RORγt)、IL-17、Foxp3 RNA水平进行测定.结果 p53基因被抑制后,FLS分泌IL-6减少,但OPG表达未受影响.p53基因被抑制的FLS使共培养的CD4+T淋巴细胞内IL-17及IFN-γ蛋白和RNA表达上调,但对CD4+T淋巴细胞RORγt RNA影响不大.虽可上调Foxp3表达,但CD4+CD25high细胞阳性率并无明显变化.结论 FKS内D53表达对RA外周血Th1、Th17有调节作用.     

Circulating soluble tumor necrosis factor receptors,interleukin-2 receptors,tumor necrosis factor α, and interleukin-6 levels in rheumatoid arthritis.

Objective. To assess whether circulating concentrations of soluble tumor necrosis factor receptors (sTNFR; p55 and p75), soluble interleukin-2 receptors (sIL-2R), tumor necrosis factor α (TNFα), and interleukin-6 (IL-6) reflect clinical response and whether changes are dependent on the drug used in rheumatoid arthritis (RA) patients taking methotrexate (MTX) or azathioprine (AZA). Methods. These cytokines and soluble receptors were assessed in 20 control subjects and serially for up to 48 weeks in 61 RA patients, by bioassay (IL-6) and immunoassays (sTNFR, sIL-2R, TNFα, and IL-6). Results. Concentrations of p55 and p75, sIL-2R, and TNFα (but not IL-6) were significantly higher in RA patients than in controls. Significant decreases in sIL-2R and p55 concentrations were associated with clinical improvement and were observed in patients treated with MTX, but not AZA. Both treatments induced decreases in IL-6 concentrations, but circulating AZA (or its metabolites) appears to interfere with the measurement of IL-6 bioactivity. TNFα and p75 levels did not show significant changes. Conclusion. Measurement of circulating sIL-2R, p55, and IL-6 may be useful in the evaluation of RA disease activity and response to therapy. Interference by circulating levels of drugs must be ruled out when bioassays are used to evaluate cytokine levels.     

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.