Expression of Jak3, STAT1, STAT4, and STAT6 in inflammatory arthritis: unique Jak3 and STAT4 expression in dendritic cells in seropositive rheumatoid arthritis

BACKGROUND: Modulation of Jak-STAT signalling may provide an effective therapeutic strategy in inflammatory arthritis. OBJECTIVE: To document Jak-STAT expression in a cohort of patients with active rheumatoid arthritis (RA), spondyloarthritis (SpA), and osteoarthritis (OA) and compare these subsets with normal synovial tissue. METHODS: Synovial tissue biopsy specimens from patients with RA, OA, and SpA and histologically normal tissue (n = 10 in each arthritis group) were examined for the presence of Jak3, STAT1, STAT4, and STAT6 expression using immunohistochemistry. Phenotyping was performed using immunohistochemistry and immunofluorescence. Clinical and serological characteristics of patients with RA expressing Jak3-STAT4 were assessed. RESULTS: STAT1, STAT4, and Jak3 protein expression was generally increased in inflammatory arthritis. In contrast, STAT6 expression was relatively heterogeneous. A subpopulation of CD1a positive dendritic cells unique to seropositive patients with RA was detected. These cells showed intense protein expression for Jak3, STAT4, and STAT6. CONCLUSION: CD1a positive dendritic cells intensely express Jak3, STAT4, and STAT6 in seropositive RA tissue and may be an alternative marker for dendritic cells in their early stages of activation as well as providing a tool for identifying RA at the level of the synovium. Jak3 inhibition may be a potential therapeutic target to prevent dendritic cell maturation in RA. STAT1 expression is increased in inflammatory arthritis, suggesting that its pro-apoptotic and anti-inflammatory effects cannot effectively counteract inflammation. STAT6 expression is heterogeneous in synovium, suggesting a possible homoeostatic role in addition to any anti-inflammatory effects.     

Differentially regulated expression of growth differentiation factor 5 and bone morphogenetic protein 7 in articular cartilage and synovium in murine chronic arthritis: potential importance for cartilage breakdown and synovial hypertrophy

OBJECTIVE: To examine whether the endogenous expression of growth differentiation factor 5 (GDF-5) and bone morphogenetic protein 7 (BMP-7) is altered in the cartilage and synovium of human tumor necrosis factor alpha (TNFalpha)-transgenic (hTNFtg) mice with chronic arthritis, and to investigate the response of hTNFtg chondrocytes as well as fibroblast-like synoviocytes (FLS) to these morphogens in vitro. METHODS: Analyses were performed in hTNFtg mice with chronic destructive arthritis and in wild-type (WT) mice as controls. Expression of GDF-5 and BMP-7 in the articular cartilage and synovium was examined by real-time polymerase chain reaction and immunohistochemistry. Human TNFtg cartilage explants, chondrocytes, and FLS monolayer cultures were assessed for basal matrix biosynthesis as well as growth factor responsiveness, using (35)S-sulfate incorporation assays. In addition, the DNA content/cell proliferation rate was measured. RESULTS: The expression of GDF-5 and BMP-7 was decreased in articular cartilage from hTNFtg mice, whereas expression of both morphogens was increased in arthritic synovium from hTNFtg mice, as compared with the levels in WT controls. Isotope incorporation revealed a marked reduction of matrix synthesis in hTNFtg cartilage as well as a decrease in responsiveness to GDF-5 and BMP-7. The DNA content did not change in arthritic cartilage as compared with WT cartilage. In hTNFtg FLS, growth factor stimulation increased the rate of cell proliferation and the production of extracellular matrix. CONCLUSION: In this murine model of TNFalpha-mediated arthritis, the expression of GDF-5 and BMP-7 is regulated differentially in articular cartilage and synovium. In articular cartilage, the down-regulation of GDF-5 and BMP-7, which function to maintain matrix integrity, could potentially compromise tissue repair, whereas in synovium, the increased expression of GDF-5 and BMP-7 might contribute to synovial hypertrophy.     

Expression of interferon beta in synovial tissue from patients with rheumatoid arthritis: comparison with patients with osteoarthritis and reactive arthritis

BACKGROUND: IFNbeta may have immunomodulatory effects in rheumatoid arthritis (RA) and its increased production in RA synovium may be a reactive attempt to inhibit inflammation. OBJECTIVE: To determine the expression of IFNbeta in the synovial tissue of patients with RA, osteoarthritis, and reactive arthritis. METHODS: Synovial biopsy specimens were obtained by arthroscopy from patients with RA and disease controls for immunohistological analysis using a monoclonal antibody specific for IFNbeta. Bound antibody was detected by an immunoperoxidase method. Stained sections were evaluated by computer assisted image analysis. Double stainings were performed with antibodies to detect CD55 positive fibroblast-like synoviocytes (FLS), CD68 positive macrophages, and CD83 positive dendritic cells (DCs) co-expressing IFNbeta. RESULTS: IFNbeta protein was abundantly expressed in the synovium of patients with RA. Digital image analysis showed a significant increase in the mean integrated optical density for IFNbeta expression in RA synovial tissue compared with disease controls. Specific up regulation of IFNbeta expression was also seen when the results were controlled for cell numbers. Phenotypic analysis showed that FLS, especially, but also macrophages and DCs may express IFNbeta in RA synovial tissue. CONCLUSIONS: The increased expression of IFNbeta in RA synovium suggests activation of an immunomodulatory mechanism that could inhibit synovial inflammation.     

Detection of the p53 regulator murine double-minute protein 2 in rheumatoid arthritis

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by hyperplasia of synovial lining tissue, which is involved directly in the damage of cartilage and bone. One of the factors thought to contribute to this synovial lining hyperplasia is dysregulation of, or functional abnormality in, the tumor suppressor protein p53. The protein known as murine double-minute protein 2 (MDM2) is the major negative regulator of p53, and in tumors contributes to increased cell proliferation. The detection of MDM2 in rheumatoid synovium has not previously been described. We investigated whether this protein is detectable in cells and tissues derived from patients with RA. METHODS: Expression of MDM2 protein was examined in fibroblast-like synoviocytes (FLS) by methods including permeabilization flow cytometry, immunofluorescence, and Western blotting, and in synovial tissues using immunohistochemistry. The proliferative capacity of these cells was also examined using 3H/thymidine incorporation. Cell cycle analysis was performed by propidium iodide incorporation. RESULTS: MDM2 was detected in RA FLS and synovial tissues. MDM2 protein was identified in CD14-positive and CD14-negative synovial lining cells and CD14-positive sublining cells. RA FLS exhibited faster proliferative rates and higher levels of MDM2 expression than FLS derived from patients with osteoarthritis (OA). Both OA and RA FLS were found to be in similar phases of the cell cycle at the time of MDM2 protein analysis. CONCLUSION: The abundant expression of MDM2 in RA may be a contributing factor to the hypoapoptotic phenotype of lining tissue through its capacity to downregulate p53 levels and effects. Further studies are required to determine the relationship between this cell-cycle protein profile, tissue hyperplasia, and the functional abnormality of p53 in RA.     

Wnt1 inducible signaling pathway protein-3 regulation and microsatellite structure in arthritis

OBJECTIVE: Rheumatoid arthritis (RA) synovial tissue expresses several embryonic gene families, including wingless (wnt) and their receptors, frizzled (fz). The Wnt proteins, including Wnt-1, activate the Wnt inducible signaling pathway proteins (WISP), which are members of the CCN family that regulate cell growth and differentiation. WISP3 is of particular interest because it contains a microsatellite region in its coding region that is susceptible to frameshift mutations and leads to a truncated protein. To investigate the contribution of WISP3 to synovial inflammation, we evaluated its expression and regulation in arthritis. METHODS: mRNA and protein expression of WISP3 were determined by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. For mutation analysis, PCR product amplified from genomic DNA of synovial tissue and cultured fibroblast-like synoviocytes (FLS) was subcloned and sequenced. RESULTS: WISP3 mRNA is expressed in synovial tissue, but is 11-fold higher in RA than osteoarthritis (OA) or normal samples. Surprisingly, WISP3 protein levels are similar in RA, OA, and normal synovium samples. Immunohistochemistry of synovial tissue reveals that WISP3 protein is located primarily in the synovial intimal lining. WISP3 mRNA expression is also 6-fold higher in RA FLS compared with OA FLS and 50-fold higher in RA than in normal FLS. When RA FLS are stimulated with interleukin 1 or tumor necrosis factor-a, WISP3 mRNA is significantly increased. The cytokines also increase WISP3 mRNA in OA FLS, but the maximal level in stimulated OA FLS is still less than medium-treated RA FLS. Mutation analysis in the coding region microsatellite of the WISP3 gene in RA and OA synovium and FLS shows a limited number of insertion and deletion mutations. CONCLUSION: WISP3 gene expression is higher in RA synovium and FLS compared with OA and normal synovial tissue and is further induced by proinflammatory cytokines in vitro. Protein levels are not increased, indicating discoordinate regulation of WISP3 protein and mRNA. Although functionally relevant mutations were observed in genomic DNA, they were noted in both OA and RA samples.     

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 alpha (TNFalpha) 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), TNFalpha, 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. TNFalpha 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.     

Regulation of p53 by macrophage migration inhibitory factor in inflammatory arthritis

OBJECTIVE: To study the capacity of macrophage migration inhibitory factor (MIF) to regulate proliferation, apoptosis, and p53 in an animal model of rheumatoid arthritis (RA) and in fibroblast-like synoviocytes (FLS) from humans with RA. METHODS: Antigen-induced arthritis (AIA) was induced in MIF(-/-) mice and littermate controls. FLS were obtained from patients with RA. Western blotting and immunohistochemistry were used to measure p53 in cells and tissues. Apoptosis was detected in cells by flow cytometry using TUNEL and annexin V/propidium iodide labeling. Apoptosis in tissue was detected using TUNEL. Proliferation was assessed in cultured cells and tissue by (3)H-thymidine incorporation and Ki-67 immunostaining, respectively. RESULTS: MIF inhibited p53 expression in human RA FLS. Levels of p53 were correspondingly increased in MIF(-/-) mouse tissues and cells. Spontaneous and sodium nitroprusside-induced apoptosis were significantly increased in MIF(-/-) cells. In vitro exposure of FLS to MIF reduced apoptosis and significantly induced FLS proliferation. Synoviocyte proliferation in MIF(-/-) mice was correspondingly reduced. A decrease in the severity of AIA in MIF(-/-) mice was associated with an increase in p53 and apoptosis in synovium. Evidence of in situ proliferation was scant in this model, and no difference in in situ proliferation was detectable in MIF(-/-) mice compared with wild-type mice. CONCLUSION: These results indicate a role for MIF in the regulation of p53 expression and p53-mediated events in the inflamed synovium and support the hypothesis that MIF is of critical importance in the pathogenesis of RA.     

Regulation of apoptosis in fibroblast-like synoviocytes by the hypoxia-induced Bcl-2 family member Bcl-2/adenovirus E1B 19-kd protein-interacting protein 3

OBJECTIVE: Rheumatoid arthritis (RA) synovial hyperplasia is related in part to a resistance to apoptosis exhibited by fibroblast-like synoviocytes (FLS). Since hypoxia is a regulator of apoptosis, and since RA synovium is hypoxic, we conducted this study to examine the effects of hypoxia on the Bcl-2 pathway and the role this may play in regulating apoptosis in FLS. METHODS: Synovium samples from RA patients, osteoarthritis (OA) patients, and normal subjects were used for immunohistologic assessments and for generating FLS lines in vitro. FLS were stimulated under conditions of hypoxia (1% O(2)) and using 100 microM CoCl(2) to simulate the effects of severe hypoxia. Changes in the gene expression profile of FLS were evaluated using microarrays and were confirmed by quantitative polymerase chain reaction (PCR). Changes in protein expression were detected by Western blotting. The effect of transient transfection with a BNIP3 plasmid on the apoptosis of FLS was evaluated in the presence and absence of cytokines. RESULTS: Gene expression profiling demonstrated that BNIP3 was unique among the BCL2 family, in that it was induced by hypoxia in FLS. Quantitative PCR indicated a 2-3-fold induction of BNIP3 messenger RNA, and Western blotting showed a 3-5-fold increase in the 30-kd Bcl-2/adenovirus E1B 19-kd protein-interacting protein 3 (BNIP-3) monomer. BNIP-3 was widely expressed in RA synovium and was prominent in FLS from the lining layer. Overexpression of BNIP3 increased FLS apoptosis under hypoxic conditions, an effect that was inhibited by tumor necrosis factor alpha and interleukin-1beta. CONCLUSION: The proapoptotic protein BNIP-3 is induced in FLS by hypoxia and is widely expressed in RA synovium, but its proapoptotic effects may be inhibited in vivo by proinflammatory cytokines. Since overexpression of BNIP3 in FLS increases apoptosis, this may provide a novel approach for controlling synovial hyperplasia in RA.     

Localisation of apoptosis and expression of apoptosis related proteins in the synovium of patients with rheumatoid arthritis.

OBJECTIVES: To investigate whether apoptosis occurs in the synovium of rheumatoid arthritis (RA), and the intermediate molecules operating in this process. METHODS: DNA fragmentation was detected by in situ nick end labelling (ISNEL) in the synovium of patients with RA (n = 11) and control patients with femoral neck fracture (n = 5). The expression of proteins p53, p21WAFI/CIPI, c-myc, proliferating cell nuclear antigen (PCNA), and Bcl-2 was also examined by immunohistochemistry. RESULTS: ISNEL positive synovial cells with apoptosis specific morphology were detected in extremely limited areas in only two RA synovial tissue specimens. Proteins p53, p21WAFI/CIPI, and c-myc, known inducers of apoptosis or cell cycle arrest or both, were expressed in the sublining cells independent of ISNEL positive cells. PCNA, a marker for cell proliferation, was observed in the synovial lining cells. Bcl-2, an inhibitor of apoptosis, was expressed mainly in infiltrated lymphocytes and in parts of the sublining layer cells of RA; it also did not correspond with ISNEL staining. CONCLUSIONS: Our findings indicate that RA synovial cells undergo apoptosis in addition to cell proliferation, but the frequency of apoptosis was very low. We suspect that the apoptotic process in the RA synovium may be suppressed by over-expression of Bcl-2. Although expressed proteins p53, p21WAFI/CIPI, and c-myc were present in the RA synovium, these protooncogenes are probably not implicated in the apoptotic process.     

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.     

Mechanisms and clinical relevance of TRAIL‐triggered responses in the synovial fibroblasts of patients with rheumatoid arthritis

Objective

Results of studies in mice suggest a protective role for TRAIL in arthritis. The aim of this study was to investigate the role of TRAIL in patients with rheumatoid arthritis (RA).

Methods

In the present study, we compared RA fibroblast‐like synoviocytes (FLS) that were resistant or sensitive to TRAIL‐induced apoptosis and the expression of TRAIL receptors in these cells, and also investigated the clinical features of the patients from whom the FLS were derived. Furthermore, we evaluated the levels of TRAIL and its soluble decoy receptor osteoprotegerin (OPG) in patients with RA, patients with osteoarthritis (OA), and patients with spondylarthritis (SpA).

Results

Sensitivity to TRAIL‐induced apoptosis varied in FLS from different patients, and the severity of disease in patients with RA was inversely correlated with the susceptibility of their FLS to TRAIL‐induced apoptosis. TRAIL‐sensitive cells expressed significantly lower levels of TRAILR‐1, and silencing of TRAILR‐1 increased TRAIL‐induced apoptosis in RA FLS. TRAIL levels were elevated in the arthritic joints of patients with established RA, and TRAIL levels in the synovial fluid of these patients were elevated compared with levels in the synovial fluid of patients with OA or SpA. At baseline, a low OPG‐to‐TRAIL ratio in the sera of patients with early RA was associated with a better evolution of disease activity, but high serum levels of TRAIL at followup were associated with joint damage.

Conclusion

These findings suggest that TRAIL has a dual role in RA, and that the resistance of RA FLS to TRAIL‐induced apoptosis is associated with a disease‐promoting activity of TRAIL in RA.

     

Comparative analysis of cathepsin l,cathepsin d,and collagenase messenger rna expression in synovial tissues of patients with rheumatoid arthritis and osteoarthritis,by in situ hybridization

Objective. To compare the expression of cathepsin L, cathepsin D, and collagenase messenger RNA (mRNA) in synovial specimens from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Methods. The expression of cathepsins L and D as well as collagenase mRNA in synovial tissues from 8 patients with RA, 6 patients with OA, and 2 patients with noninflamed joints was evaluated using in situ hybridization with digoxigenin-labeled RNA probes. Results. Both RA and OA synovial tissue expressed cathepsins L and D as well as collagenase mRNA. The expression of the cathepsins was markedly higher in interstitial regions and, to some extent, in perivascular infiltrates of RA synovial tissue compared with OA specimens. Conclusion. Cathepsins L and D mRNA are expressed differently in RA and OA synovial tissues, supporting the concept that these enzymes may contribute to the influx of mononuclear cells into RA synovium. Moreover, the data reveal that the expression of collagenase and cathepsins in RA and OA synovial lining is otherwise largely similar, and suggest that the adhesion of synovial cells to cartilage mediates the invasive destructive process in RA.     

Beneficial effect of galectin 9 on rheumatoid arthritis by induction of apoptosis of synovial fibroblasts

OBJECTIVE: To compare the expression of galectin 9 (Gal-9) in synovial tissue (ST) from rheumatoid arthritis (RA) patients and osteoarthritis (OA) patients and to evaluate the effects of Gal-9 on fibroblast-like synoviocytes (FLS) in these patients. METHODS: The expression of Gal-9 in ST and FLS was compared using immunohistochemical techniques. Apoptotic cells in RA and OA ST samples were detected by TUNEL assay. Apoptosis of FLS was analyzed by the sub-G(1) method in vitro. The in vivo suppressive effects of Gal-9 on collagen-induced arthritis (CIA) in a mouse model were also elucidated. RESULTS: The percentage of Gal-9-positive cells in ST samples and the amount of Gal-9 in synovial fluid samples were significantly higher in patients with RA than in patients with OA, suggesting the involvement of Gal-9 in the development of RA. Compared with the 2 wild-type Gal-9 forms, stable Gal-9, a mutant protein resistant to proteolysis, significantly induced apoptosis of FLS from RA patients. In contrast, other galectins, such as Gal-1, Gal-3, and Gal-8, did not induce apoptosis or suppress the proliferation of human RA FLS. Stable Gal-9 preferentially induced apoptosis and suppressed the proliferation of RA FLS in vitro. It also induced apoptosis of cells in RA ST implanted into SCID mice in vivo. In a mouse model of CIA, apoptotic cells were detected in the joints of stable Gal-9-treated mice, but not phosphate buffered saline-treated mice, and suppressed CIA characterized by pannus formation with inflammatory cell infiltration and bone/cartilage destruction. CONCLUSION: Gal-9-induced apoptosis of hyperproliferative RA FLS may play a critical role in the suppression of RA.     

Fibroblast-like synoviocytes derived from patients with rheumatoid arthritis show the imprint of synovial tissue heterogeneity: evidence of a link between an increased myofibroblast-like phenotype and high-inflammation synovitis

OBJECTIVE: Given the heterogeneity of gene expression patterns and cellular distribution between rheumatoid arthritis (RA) synovial tissues, we sought to determine whether this variability was also reflected at the level of the fibroblast-like synoviocyte (FLS) cultured from RA synovial tissues. METHODS: Gene expression profiles in FLS cultured from synovial tissues obtained from 19 RA patients were analyzed using complementary DNA microarrays and hierarchical cluster analysis. To validate the subclassification, we performed prediction analysis and principal components analysis. Genes that differed significantly in their expression between FLS cultures were selected using Statistical Analysis of Microarrays software. Real-time quantitative polymerase chain reaction was performed to validate the microarray data. Immunocytochemistry was applied to study the expression of the genes of interest in FLS and synovial tissues. RESULTS: Hierarchical clustering identified 2 main groups of FLS characterized by distinctive gene expression profiles. FLS from high-inflammation synovial tissues revealed increased expression of a transforming growth factor beta/activin A-inducible gene profile that is characteristic of myofibroblasts, a cell type considered to be involved in wound healing, whereas increased production of growth factor (insulin-like growth factor 2/insulin-like growth factor binding protein 5) appeared to constitute a characteristic feature of FLS derived from low-inflammation synovial tissues. The molecular feature that defines the myofibroblast-like phenotype was reflected as an increased proportion of myofibroblast-like cells in the heterogeneous FLS population. Myofibroblast-like cells were also found upon immunohistochemical analysis of synovial tissue. CONCLUSION: Our findings support the notion that heterogeneity between synovial tissues is reflected in FLS as a stable trait, and provide evidence of a possible link between the behavior of FLS and the inflammation status of RA synovium.     

Agonistic anti‐human Fas monoclonal antibody induces fibroblast‐like synoviocyte apoptosis in haemophilic arthropathy: potential therapeutic implications

Haemophilic arthropathy (HA) is characterized by chronic proliferative synovitis leading to cartilage destruction and shares some pathological features with rheumatoid arthritis (RA). Apoptosis has been implicated in RA pathogenesis, and an agonistic anti‐Fas monoclonal antibody (mAb) was found to induce RA fibroblast‐like synoviocyte (FLS) apoptosis and suppress synovial hyperplasia in animal models of RA. The aim of this study was to evaluate the effect of anti‐Fas mAb on HA‐FLS. FLS were isolated from knee synovial biopsies from six HA patients, six RA patients and six healthy subjects. The expression of Fas in synovial biopsies was investigated by immunohistochemistry. FLS were stimulated with anti‐Fas mAb at different concentrations, alone or in combination with tumour necrosis factor‐α (TNF‐α) and basic fibroblast growth factor (bFGF). Fas expression in FLS was assessed by Western blot. Cell viability was studied with the WST‐1 assay. Active caspase‐3 levels were measured using ELISA and Western blot. A strong Fas‐immunoreactivity was observed in different cells of HA synovium, including FLS, inflammatory cells and endothelial cells. Fas antigen was constitutively overexpressed in cultured HA‐FLS. Anti‐Fas mAb had a significant cytotoxicity on HA‐FLS in a dose‐dependent manner, either alone or in combination with TNF‐α and bFGF. These cytotoxic effects were due to the ability of anti‐Fas to induce HA‐FLS apoptosis, as shown by the increased active caspase‐3 levels. Anti‐Fas mAb exhibited a more pronounced pro‐apoptotic effect on HA‐FLS than RA‐FLS. Fas antigen is highly expressed on HA‐FLS and its stimulation by anti‐Fas mAb may be an effective strategy to induce HA‐FLS apoptosis.     

Citrullination of fibronectin in rheumatoid arthritis synovial tissue

OBJECTIVES: Citrullination, catalysed by peptidylarginine deiminase (PAD), is the post-translational modification of peptidylarginine to citrulline, which is intimately involved in the pathogenesis of rheumatoid arthritis (RA). Fibronectin (Fn), a large glycoprotein, is expressed at high levels in arthritic joints and it mediates various physiological processes through interactions with cell-surface integrin receptors and growth factors. We investigated the citrullination of Fn and its potential contribution to the pathogenesis of RA. METHODS: We localized Fn expression and citrullination in RA synovial tissue by immunohistochemistry, immunoprecipitation and western blotting. We also determined levels of citrullinated Fn in plasma from RA patients using sandwich enzyme-linked immunosorbent assay (ELISA). After incubating Fn with rabbit skeletal muscle PAD, we examined the binding ability of citrullinated Fn to vascular endothelial growth factor (VEGF) and integrin beta1 using a solid-phase receptor binding assay as well as the effect of the citrullinated Fn on apoptosis using cultured HL-60 cells. RESULTS: Immunohistochemistry and western blotting analysis indicated that Fn formed extracellular aggregates that were specifically citrullinated in RA synovial tissue. No Fn deposits were observed in synovial tissues of osteoarthritis (OA). Sandwich ELISA detected higher levels of citrullinated Fn in plasma from patients with RA than from healthy controls or those with systemic lupus erythematosus. Following citrullination in vitro, the affinity of Fn for VEGF increased, but binding activity to integrin beta1 decreased and Fn no longer stimulated the apoptosis of monocytes induced from cultured HL-60 cells. CONCLUSIONS: Our results suggest that the citrullination of Fn is a specific event for RA synovium, although others have detected citrullinated total proteins in inflamed synovial tissue of RA and non-RA patients. Citrullination of Fn could alter interactions between Fn and its receptors and growth factors, consequently contributing to mechanisms of RA pathogenesis such as perturbed angiogenesis and apoptosis.