Proinflammatory role of fractalkine (CX3CL1) in rheumatoid arthritis

OBJECTIVE: Fractalkine (CX3CL1) represents the sole member of the so-called CX3C chemokines. In rheumatoid arthritis (RA), functional studies suggest a role for this chemokine in monocyte chemotaxis and angiogenesis in the rheumatoid synovium. We analyzed the expression of fractalkine within different T cell subsets of the peripheral blood and expression of its receptor CX3CR1 within the rheumatoid synovium to further characterize its pathogenic role in RA. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 17 patients with RA and analyzed by flow cytometry in comparison to healthy blood donors. To identify the T helper cell cytokine profile of fractalkine-expressing cells, flow cytometric analysis of PBMC was performed after stimulation with PMA and ionomycin. Expression of fractalkine and its receptor was characterized in RA synovium by immunohistochemistry and laser capture microdissection microscopy. RESULTS: Flow cytometric analysis of fractalkine-expressing T cell subsets revealed a low proportion of fractalkine-expressing CD4+ and CD8+ T cells in both RA patients and controls. In addition, fractalkine was predominantly expressed in CD4+ T cells with a Th1-type cytokine expression profile. In RA synovium, fractalkine was detected in synovial macrophages, dendritic cells, endothelial cells, and a small proportion of T cells. The fractalkine receptor CX3CR1 was found in synovial macrophages, dendritic cells, and T cells as well as in synovial fibroblasts. Fractalkine stimulation of cultured synovial fibroblasts resulted in a marked upregulation of matrix metalloproteinase-2 (MMP-2) production. CONCLUSION: The results suggest that fractalkine may represent a Th1-type chemokine. Upregulation of MMP-2 production in synovial fibroblasts upon fractalkine stimulation in vitro supports the hypothesis of a proinflammatory role of this chemokine in RA.     

The TNF superfamily member LIGHT contributes to survival and activation of synovial fibroblasts in rheumatoid arthritis

OBJECTIVES: The TNF superfamily member LIGHT has a T-cell co-stimulatory role and has previously been associated with inflammation and autoimmunity. To investigate its role in rheumatoid arthritis (RA), a disease where activated T cells contribute in a prominent way, we have analysed the expression of LIGHT and its receptors in RA and analysed its effects on synovial fibroblasts in vitro. METHODS: The expression of LIGHT was measured in synovial tissues and fluids and the receptors of LIGHT were detected on synovial fibroblasts derived from patients with RA and osteoarthritis (OA). The effects of recombinant LIGHT on the production of proinflammatory cytokines and proteases and on the apoptosis of synovial fibroblasts was assessed. RESULTS: LIGHT mRNA was present in synovial tissues of patients with RA but not with OA. Correspondingly, soluble LIGHT protein could be detected in RA synovial fluid samples at much higher levels than in synovial fluid from patients with OA. Immunohistochemical detection of LIGHT and analysis of synovial fluid cells by flow cytometry revealed CD4 T cells as the major source of LIGHT in the rheumatoid joint. Synovial fibroblasts from RA patients were found to express the LIGHT receptors HVEM and LTbetaR. Recombinant LIGHT induced RA synovial fibroblasts to upregulate MMP-9 mRNA, CD54 and IL-6 in an NF-kappaB-dependent fashion. In vitro, exposure of cultured synovial fibroblasts to LIGHT reduced FAS-mediated apoptosis significantly, without affecting the rate of spontaneous apoptosis. CONCLUSIONS: The results provide evidence for a novel T-cell-dependent activation of synovial fibroblasts by LIGHT in joints of patients with RA, contributing to an inflammatory and destructive phenotype.     

Galectin 3 and its binding protein in rheumatoid arthritis

OBJECTIVE: To characterize the expression pattern and role of galectin 3 and galectin 3 binding protein (G3BP) in rheumatoid arthritis (RA), in comparison with galectin 1, and to explore whether soluble galectin 3 and G3BP, investigated in serum, synovial fluid, or cell culture supernatant, are associated with disease. METHODS: Synovial tissues from patients with RA or osteoarthritis (OA), as well as from healthy controls, were analyzed for galectins 1 and 3 and G3BP by in situ hybridization and immunohistochemistry. Levels of galectin 3 and G3BP in serum and synovial fluid from patients with RA and OA and controls, as well as in cell culture supernatants, were determined by enzyme-linked immunosorbent assay (ELISA). In vitro, the intracellular expression of galectin 3 in RA and OA synovial fibroblasts after modulation with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and anti-CD40 monoclonal antibodies was measured by flow cytometry. RESULTS: In RA, galectin 3 messenger RNA and protein stained throughout the synovial membrane, whereas G3BP was particularly expressed at sites of bone destruction. In contrast, the expression of galectin 1 was not uniform in different RA specimens, and was never found at sites of invasion. In OA and normal synovial tissues, only a small number of cells were positive for galectins and/or G3BP. Galectin 3 was elevated in RA sera and synovial fluids, whereas G3BP was increased in RA synovial fluids only. In RA, serum galectin 3 correlated with C-reactive protein levels, whereas G3BP was associated with joint destruction and/or synovial cell activation as measured by the levels of cartilage oligomeric matrix protein. In vitro, RA synovial fibroblasts showed an increased release of galectin 3 into culture medium, as measured by ELISA, but decreased secretion of G3BP. In RA synovial fibroblasts with low basal expression of galectin 3, TNFalpha increased its intracellular level in a dose-dependent manner. In contrast, IL-1beta or anti-CD40 monoclonal antibodies showed no effect. CONCLUSION: Our data indicate that galectin 3 and G3BP are not only involved in inflammation, but also contribute to the activation of synovial fibroblasts. The intracellular accumulation of galectin 3 can be enhanced by TNFalpha. Thus, galectin 3 and G3BP represent novel markers of disease activity in RA.     

Pathological significance of elevated soluble CD14 production in rheumatoid arthritis: in the presence of soluble CD14, lipopolysaccharides at low concentrations activate RA synovial fibroblasts

In order to establish what contributes to elevated levels of soluble CD14 (sCD14) in rheumatoid arthritis (RA) plasma, levels of sCD14 were compared in RA-paired plasma and synovial fluids and, further, in the culture supernatants of monocyte-rich fractions from patients with RA and healthy donors, and macrophage-rich fractions from RA synovial tissues. The results showed elevated sCD14 in RA synovial fluid in 9 of 16 paired samples and in RA macrophage-rich fractions, suggesting that elevated sCD14 in RA plasma might be due to the sCD14 production by RA synovial macrophages. From the molecular analysis of elevated sCD14, the proteolytic cleavage of membranous CD14 (mCD14) was important in accelerated sCD14 production. Lipopolysaccharides (LPS) at low concentrations and sCD14 increased the ICAM-1 expression on RA synovial fibroblasts. This result implies that in vivo RA synovial fibroblasts may be sensitive to LPS in the presence of sCD14 and LPS-binding protein (LBP). Received: 4 October 1997 / Accepted: 20 January 1998     

Hepatocyte growth factor (HGF), HGF activator, and c-Met in synovial tissues in rheumatoid arthritis and osteoarthritis

OBJECTIVE: Hepatocyte growth factor (HGF) is a multifunctional polypeptide that has been implicated in cancer growth, tissue development, and wound repair. Its actions are dependent on activation by HGF activator (HGFA) and its binding to a specific HGF receptor (c-Met). We examined the role of HGF, HGFA, and c-Met in synovial tissues in rheumatoid arthritis (RA) and osteoarthritis (OA), and their localization and mRNA expression. METHODS: Immunohistochemical staining, Western blotting, RT-PCR, and in situ hybridization (ISH) for HGF, HGFA, and c-Met were performed on synovial tissue specimens from 10 patients with RA and 4 with OA, and 2 healthy controls. RESULTS: Immunohistochemical staining revealed that HGFA and c-Met were strongly expressed in fibroblasts, macrophages, endothelial cells, and synovial lining cells. HGF was expressed only faintly in macrophages and fibroblasts, and not at all in the endothelial cells of RA and OA synovial tissue. HGFA was detected near 73 and 34 kDa on Western blot analysis, corresponding to inactive and active HGFA, respectively. RT-PCR showed HGF, HGFA, and c-Met mRNA in RA, OA, and control synovial tissue. ISH and immunohistochemistry revealed mRNA expression for HGF, HGFA, and c-Met in the cell types mentioned above. CONCLUSION: HGFA, HGF, and c-Met mRNA are expressed in synovial tissue in RA and OA, and HGF is activated by HGFA and binds to c-Met on endothelial cells, inducing angiogenesis.     

Up-regulation of metastasis-promoting S100A4 (Mts-1) in rheumatoid arthritis: putative involvement in the pathogenesis of rheumatoid arthritis

OBJECTIVE: To examine the involvement of the metastasis-inducing protein S100A4 (Mts-1) in the pathogenesis of rheumatoid arthritis (RA). METHODS: Synovial tissue, synovial fluid, and plasma were obtained from RA and osteoarthritis (OA) patients who were undergoing joint surgery. Immunohistochemical and immunofluorescence analyses and enzyme-linked immunosorbent assays were used to determine the locations and concentrations of S100A4. The conformational structure of S100A4 in plasma and synovial fluid was determined after fractionation by size-exclusion chromatography, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot analysis. Expression of various S100 proteins in RA synovium was determined by immunofluorescence and double-staining using specific anti-S100 antibodies. RESULTS: We found an up-regulation of S100A4 in cells infiltrating RA synovial tissue. Most cell types identified by cell-specific markers (fibroblasts, immune cells, and vascular cells) contributed to the production of S100A4 in RA synovial tissue. The pattern of S100A4 expression differed significantly from that of the proinflammatory proteins S100A9 and S100A12, which were restricted to phagocytes and granulocytes. The up-regulation of S100A4 in RA synovial tissue was consistent with the high concentrations of the protein in RA versus OA plasma (mean 1,100 versus 211 ng/ml) and synovial fluid (mean 1,980 versus 247 ng/ml). Moreover, we found that S100A4 in RA plasma and synovial fluid was present in bioactive multimeric (M-S100A4) conformations, whereas in OA, the majority of extracellular S100A4 was detected as the less active dimeric form. Consistent with our observations in tumor models, extracellular S100A4 stabilized the p53 tumor suppressor in RA synovial fibroblast-like cells and affected the regulation of p53 target genes, including Bcl-2, p21(WAF), and Hdm-2, as well as matrix metalloproteinases. CONCLUSION: Overexpression of S100A4 in RA synovial tissue and its release as M-S100A4 can influence p53 function and modulate the expression of several genes that are potentially implicated in the disease process. Thus, S100A4 might play an important role in the pathogenesis of RA and might represent a new target for the treatment of RA.     

Involvement of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor in osteoclastogenesis from synoviocytes in rheumatoid arthritis

OBJECTIVE: To clarify the mechanism by which osteoclasts are formed in culture of rheumatoid synoviocytes by exploring the involvement of receptor activator of nuclear factor kappaB ligand (RANKL)/osteoclast differentiation factor (ODF). METHODS: Osteoclast formation was evaluated in cocultures of rheumatoid synovial fibroblasts and peripheral blood mononuclear cells (PBMC) in the presence of macrophage colony stimulating factor and 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) utilizing separating membrane filters. RANKL/ODF expression was examined by Northern blotting in synovial tissues from 5 rheumatoid arthritis (RA) patients and tissues from patients with giant cell tumor (GCT), osteosarcoma (OS), and osteoarthritis (OA). RANKL/ODF expression and the ability of synovial fibroblasts to support osteoclastogenesis were investigated in coculture with PBMC in the presence or absence of 1,25(OH)2D3, and soluble RANKL/ODF and osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) were measured by enzyme-linked immunosorbent assay. The effects of OPG/OCIF on the osteoclastogenesis in the primary culture of rheumatoid synoviocytes and the coculture system were determined. RESULTS: Synovial fibroblasts did not induce osteoclastogenesis when separately cocultured with PBMC. Northern blotting revealed that RANKL/ODF was highly expressed in all tissues from RA and GCT patients, but not from OA or OS patients. Cultured rheumatoid synovial fibroblasts efficiently induced osteoclastogenesis in the presence of 1,25(OH)2D3, which was accompanied by up-regulated expression of RANKL/ODF and decreased production of OPG/OCIF. Osteoclastogenesis from synoviocytes was dose-dependently inhibited by OPG/OCIF. CONCLUSION: RANKL/ODF expressed on synovial fibroblasts is involved in rheumatoid bone destruction by inducing osteoclastogenesis and would therefore be a good therapeutic target.     

Angiopoietin-1 is expressed in the synovium of patients with rheumatoid arthritis and is induced by tumour necrosis factor alpha

OBJECTIVES: To examine the potential role of the angiogenic growth factor angiopoietin-1 (Ang-1) in inflammatory arthritis. METHODS: Eighteen synovial tissue samples were obtained from 17 patients with a clinical diagnosis of rheumatoid arthritis (RA) and compared with six synovial tissue samples from six patients with osteoarthritis (OA). Ang-1 expression in synovial tissues was determined by immunohistochemistry and in situ hybridisation. Ang-1 mRNA and protein expression were also examined by northern blot analysis and enzyme linked immunosorbent assay (ELISA) in cultured synovial fibroblasts and human umbilical vein endothelial cells (HUVECs) before and after treatment with tumour necrosis factor (TNF)alpha. RESULTS: Ang-1 protein expression was detected by immunohistochemistry in 16/18 RA synovial tissue samples. Ang-1 protein was frequently observed in the synovial lining layer and in cells within the sublining synovial tissue, in both perivascular areas and in areas remote from vessels. In contrast, Ang-1 was only weakly detected in these sites in OA samples. Ang-1 mRNA and protein were also expressed in cultured synovial fibroblasts derived from patients with RA. In addition, induction of Ang-1 mRNA and protein was observed by northern blot analysis and ELISA after stimulation of RA synovial fibroblasts, but not HUVECs, with the proinflammatory cytokine TNF alpha. CONCLUSIONS: Ang-1 mRNA and protein are expressed in the synovium of patients with RA, and are up regulated in synovial fibroblasts by TNF alpha. Ang-1 may therefore be an important regulator of angiogenesis in inflammatory arthritis.     

RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via Toll-like receptor 3

OBJECTIVE: To assess the expression of Toll-like receptor 3 (TLR-3) protein in synovial tissues and cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to investigate the consequences of stimulation of cultured synovial fibroblasts with TLR-3 ligands. METHODS: TLR-3 expression in synovial tissues was determined by immunohistochemistry and immunofluorescence, and expression in cultured RA synovial fibroblasts (RASFs) was determined by fluorescence-activated cell sorting and real-time polymerase chain reaction techniques. TLR-3 signaling was assessed by incubating RASFs with poly(I-C), lipopolysaccharide, palmitoyl-3-cysteine-serine-lysine-4, or necrotic synovial fluid cells from RA patients in the presence or absence of hydroxychloroquine or Benzonase. Subsequent determination of interferon-beta (IFNbeta), CXCL10, CCL5, and interleukin-6 (IL-6) protein production in the culture supernatants was performed by enzyme-linked immunosorbent assays. RESULTS: TLR-3 protein expression was found to be higher in RA synovial tissues than in OA synovial tissues. TLR-3 expression was localized predominantly in the synovial lining, with a majority of the TLR-3-expressing cells coexpressing fibroblast markers. Stimulation of cultured RASFs with the TLR-3 ligand poly(I-C) resulted in the production of high levels of IFNbeta, CXCL10, CCL5, and IL-6 protein. Similarly, coincubation of RASFs with necrotic synovial fluid cells from patients with RA resulted in up-regulation of these cytokines and chemokines in a TLR-3-dependent manner. CONCLUSION: Our findings demonstrate the expression of TLR-3 in RA synovial tissue and the activation of RASFs in vitro by the TLR-3 ligand poly(I-C) as well as by necrotic RA synovial fluid cells, and indicate that RNA released from necrotic cells might act as an endogenous TLR-3 ligand for the stimulation of proinflammatory gene expression in RASFs.