Rheumatoid synovial fluid interleukin-17-producing CD4 T cells have abundant tumor necrosis factor-alpha co-expression, but little interleukin-22 and interleukin-23R expression.

INTRODUCTION: Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to systematically analyse the phenotype, cytokine profile and frequency of interleukin-17 (IL-17) producing CD4-positive T cells in mononuclear cells isolated from peripheral blood, synovial fluid and synovial tissue of RA patients with established disease, and to correlate cell frequencies with disease activity. METHODS: Flow cytometry was used to analyse the phenotype and cytokine production of mononuclear cells isolated from peripheral blood (PBMC) (n = 44), synovial fluid (SFMC) (n = 14) and synovium (SVMC) (n = 10) of RA patients and PBMC of healthy controls (n = 13). RESULTS: The frequency of IL-17-producing CD4 T cells was elevated in RA SFMC compared with RA PBMC (P = 0.04). However, the frequency of this population in RA SVMC was comparable to that in paired RA PBMC. The percentage of IL-17-producing CD4 T cells coexpressing tumor necrosis factor alpha (TNFα) was significantly increased in SFMC (P = 0.0068). The frequency of IFNγ-producing CD4 T cells was also significantly higher in SFMC than paired PBMC (P = 0.042). The majority of IL-17-producing CD4 T cells coexpressed IFNγ. IL-17-producing CD4 T cells in RA PBMC and SFMC exhibited very little IL-22 or IL-23R coexpression. CONCLUSIONS: These findings demonstrate a modest enrichment of IL-17-producing CD4 T cells in RA SFMC compared to PBMC. Th17 cells in SFMC produce more TNFα than their PBMC counterparts, but are not a significant source of IL-22 and do not express IL-23R. However, the percentage of CD4 T cells which produce IL-17 in the rheumatoid joint is low, suggesting that other cells may be alternative sources of IL-17 within the joints of RA patients.

Galectin 3 induces a distinctive pattern of cytokine and chemokine production in rheumatoid synovial fibroblasts via selective signaling pathways.

OBJECTIVE: High expression of galectin 3 at sites of joint destruction in rheumatoid arthritis (RA) suggests that galectin 3 plays a role in RA pathogenesis. Previous studies have demonstrated the effects of galectins on immune cells, such as lymphocytes and macrophages. This study was undertaken to investigate the hypothesis that galectin 3 induces proinflammatory effects in RA by modulating the pattern of cytokine and chemokine production in synovial fibroblasts. METHODS: Matched samples of RA synovial and skin fibroblasts were pretreated with galectin 3 or tumor necrosis factor alpha (TNFalpha), and the levels of a panel of cytokines, chemokines, and matrix metalloproteinases (MMPs) were determined using enzyme-linked immunosorbent assays and multiplex assays. Specific inhibitors were used to dissect signaling pathways, which were confirmed by Western blotting and NF-kappaB activation assay. RESULTS: Galectin 3 induced secretion of interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor, CXCL8, and MMP-3 in both synovial and skin fibroblasts. By contrast, galectin 3-induced secretion of TNFalpha, CCL2, CCL3, and CCL5 was significantly greater in synovial fibroblasts than in skin fibroblasts. TNFalpha blockade ruled out autocrine TNFalpha-stimulated induction of chemokines. The MAPKs p38, JNK, and ERK were necessary for IL-6 production, but phosphatidylinositol 3-kinase (PI 3-kinase) was required for selective CCL5 induction. NF-kappaB activation was required for production of both IL-6 and CCL5. CONCLUSION: Our findings indicate that galectin 3 promotes proinflammatory cytokine secretion by tissue fibroblasts. However, galectin 3 induces the production of mononuclear cell-recruiting chemokines uniquely from synovial fibroblasts, but not matched skin fibroblasts, via a PI 3-kinase signaling pathway. These data provide further evidence of the role of synovial fibroblasts in regulating the pattern and persistence of the inflammatory infiltrate in RA and suggest a new and important functional consequence of the observed high expression of galectin 3 in the rheumatoid synovium.

Prolonged, granulocyte-macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha.

INTRODUCTION: A surprising feature of the inflammatory infiltrate in rheumatoid arthritis is the accumulation of neutrophils within synovial fluid and at the pannus cartilage boundary. Recent findings suggest that a distinct subset of IL-17-secreting T-helper cells (TH17 cells) plays a key role in connecting the adaptive and innate arms of the immune response and in regulating neutrophil homeostasis. We therefore tested the hypothesis that synovial fibroblasts bridge the biological responses that connect TH17 cells to neutrophils by producing neutrophil survival factors following their activation with IL-17. METHODS: IL-17-expressing cells in the rheumatoid synovium, and IL-17-expressing cells in the peripheral blood, and synovial fluid were examined by confocal microscopy and flow cytometry, respectively. Peripheral blood neutrophils were cocultured either with rheumatoid arthritis synovial fibroblasts (RASF) or with conditioned medium from RASF that had been pre-exposed to recombinant human IL-17, TNFalpha or a combination of the two cytokines. Neutrophils were harvested and stained with the vital mitochondrial dye 3,3'-dihexyloxacarbocyanine iodide before being enumerated by flow cytometry. RESULTS: TH17-expressing CD4+ cells were found to accumulate within rheumatoid synovial tissue and in rheumatoid arthritis synovial fluid. RASF treated with IL-17 and TNFalpha (RASFIL-17/TNF) effectively doubled the functional lifespan of neutrophils in coculture. This was entirely due to soluble factors secreted from the fibroblasts. Specific depletion of granulocyte-macrophage colony-stimulating factor from RASFIL-17/TNF-conditioned medium demonstrated that this cytokine accounted for approximately one-half of the neutrophil survival activity. Inhibition of phosphatidylinositol-3-kinase and NF-kappaB pathways showed a requirement for both signalling pathways in RASFIL-17/TNF-mediated neutrophil rescue. CONCLUSION: The increased number of neutrophils with an extended lifespan found in the rheumatoid synovial microenvironment is partly accounted for by IL-17 and TNFalpha activation of synovial fibroblasts. TH17-expressing T cells within the rheumatoid synovium are likely to contribute significantly to this effect.