Synovial Tissue Of RA Patients Research Articles

AB0012 Microrna-223 in synovial tissue of rheumatoid arthritis and osteoarthritis patients

BackgroundRheumatoid Arthritis (RA) is a systemic autoimmune disorder characterized by chronic synovitis and progressive joint destruction. MicroRNAs (miRNAs) are small non-coding RNA molecules able to modulate gene expression at post-transcriptional...

The periodontium of periodontitis patients contains citrullinated proteins which may play a role in ACPA (anti‐citrullinated protein antibody) formation

To determine the presence and location (stroma versus epithelium) of citrullinated proteins in periodontitis tissue as compared to non-periodontitis tissue and synovial tissue of RA patients. Periodontitis, healthy periodontal and RA-affected synovial tissue samples were collected in addition to buccal swabs. These samples were stained for the presence of citrullinated proteins using polyclonal (Ab5612) and monoclonal (F95) antibodies. Furthermore, Western blotting with F95 was performed on lysates prepared from periodontal and synovial tissues. In periodontitis stroma, increased citrullinated protein presence (80%) was observed compared with control stroma (33%), the latter was associated with inflammation of non-periodontitis origin. Periodontal epithelium always stained positive for Ab5612. Noteworthy, only periodontitis-affected epithelium stained positive for F95. All buccal mucosal swabs and 3 of 4 synovial tissue samples stained positive for both Ab5612 and F95. Western blotting with F95 showed presence of similar citrullinated proteins in both periodontitis and RA-affected synovial tissue. Within the periodontal stroma, citrullination is an inflammation-depended process. In periodontal epithelium, citrullination is a physiological process. Additional citrullinated proteins are formed in periodontitis, apparently similar to those formed in RA-affected synovial tissue. Periodontitis induced citrullination may play a role in the aetiology of rheumatoid arthritis.

VEGF induces RANKL in Rheumatoid Arthritis synovial fibroblasts

BACKGROUDVEGF is the most endothelial cell‐specific angiogenic factor characterised to date. Several recent reports have demonstrated that VEGF is also implicated in the pathogenesis of RA, and serum levels of VEGF correlate well with RA disease activity, particularly with swollen joint counts.OBJECTIVEThis study aims to determine the effect and mechanism of VEGF on bony destructive process in RA through RANKL regulation from synovial fibroblasts.METHODSynovial fibroblasts were isolated from synovial tissues of RA patients. After RA synovial fibroblasts were treated with rhVEGF, the expression of RANKL mRNA was determined using real‐time PCR. After inhibition of pro‐inflammatory cytokines or intracellular signal molecules, the change of the VEGF‐induced RANKL expression was determined using real‐time PCR.RESULTSThe expression of RANKL mRNA in RA synovial fibroblasts was increased after VEGF stimulation in a dose dependent manner. VEGF‐induced RANKL expression was also diminished after inhibition of Src, PKC and ERK.CONCLUSIONOur results show that VEGF upregulates RANKL expression in RA synovial fibroblasts. These effects are mediated by the Src/PKC and ERK pathways.This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF‐2010‐R1A4A007‐0024198)

Stromal cell‐derived factor(SDF)‐1 induces RANKL in Rheumatoid Arthritis synovial fibroblasts and CD4+ T cells

BACKGROUDSDF‐1 is a chemokine promoting the migration of inflammatory cells into the inflamed synovium in rheumatoid arthritis.OBJECTIVEThis study aims to determine the effect and mechanism of SDF‐1 on bony destructive process in RA through RANKL regulation from synovial fibroblasts and CD4+ T cells.METHODSynovial fibroblasts were isolated from synovial tissues of RA patients and CD4+ T cells were isolated from PBMC of RA patients. After RA synovial fibroblasts and CD4+ T cells were treated with rhSDF‐1, the expression of RANKL mRNA was determined using real‐time PCR.RESULTSThe expression of RANKL mRNA in RA synovial fibroblasts and CD4+ T cells was increased after SDF‐1 stimulation in a dose dependent manner. After RA synovial fibroblasts were culture with neutralizing antibodies of TNF‐α and IL‐6, the expression of RANKL mRNA was decreased in RA synovial fibroblasts. SDF‐1‐induced RANKL expression was also diminished after inhibition of PI3 kinase, p38 MAP kinase, and JNK.CONCLUSIONThis result suggests that SDF‐1 could induce RANKL from RA synovial fibroblasts mediated by TNF‐α and IL‐ 6. In RA CD4+ T cells, SDF‐1‐induced RANKL expression is mediated by IL‐1β as well as TNF‐α and IL‐6.This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF‐2010‐ NRF‐ 2010‐R1A4A002‐0008205)

Mechanisms and clinical relevance of TRAIL-triggered responses in synovial fibroblasts of rheumatoid arthritis patients

ObjectiveWe have shown that TRAIL induces apoptosis only in a portion of RA fibroblast-like synoviocytes (RAFLS) and that in the surviving cells, TRAIL induced proliferation. In the present study, we...

Apoptosis in the rheumatoid arthritis synovial membrane: modulation by disease-modifying anti-rheumatic drug treatment

RA is characterized at the synovial tissue level by synovial lining hyperplasia, angiogenesis and mononuclear cell infiltrates. A failure of apoptotic pathways may explain these pathological changes in RA synovial tissue. This study aims to demonstrate the presence of initiators and inhibitors of apoptosis in RA synovial tissue and the effect of treatment with DMARDs on apoptotic pathways in RA. Synovial biopsy specimens were obtained at arthroscopy from 16 RA patients before and at 3- or 6-month intervals after commencing treatment with a DMARD. Apoptosis (by the terminal deoxynucleotidyl transferase mediated dUTP nick end labelling method and polyADP-ribose polymerase staining), proteins regulating apoptosis [Fas, FADD-like IL1b converting enzyme inhibitory protein (FLIP), Bcl-2, Survivin and X-linked inhibitor of apoptosis protein (XIAP)] and the presence of activated caspases (caspases 3 and 8) were detected by immunohistochemistry and quantified using image analysis and semiquantitative techniques. Fifteen patients responded to treatment, with an ACR response of > or =20%, 13 achieving an ACR response of > or =50% and 3 achieving an ACR remission. There was a significant reduction in SM macrophages and memory T cells, with an increase in fibroblast-like synovial lining cells following DMARD treatment. Apoptosis was not detected in the inflamed synovial tissue of RA patients before starting treatment, despite evidence of caspase activation, but was detectable after successful treatment with DMARDs. Inhibitors of activated caspases (FLIP, Survivin and XIAP) were detected in RA synovial tissue and were down-modulated with successful DMARD treatment. Apoptotic pathways are defective in RA synovial tissue from patients with active disease, despite the presence of activated caspases, possibly due to the abundant expression of inhibitors of the caspase pathway in RA synovial tissue. DMARD treatment can modulate apoptosis in the RA SM, which may lead to restoration of the SM architecture towards that of normal synovial tissue.

Macrophage migration inhibitory factor upregulates expression of receptor activator of NF-kappa B ligand(RANKL) in rheumatoid arthritis synovial fibroblasts (98.16)

Abstract Rheumatoid arthritis(RA) is characterized by chronic progressive inflammatory destruction of bone and cartilage, which is mediated by inflammatory cytokines. Macrophage migration inhibitory factor (MIF) is abundantly expressed in the sera, synovial fluids and synovial tissues of RA patients, and its level correlates with disease activity. The aim of this study is to explore the role of MIF in regulating RANKL expression and osteoclastogenic activity in RA synovial fibroblasts (FLS). FLS were separated from the synovium of RA patients and stimulated with rMIF. RANKL expression was determined by real time PCR, western blot and immunostaining. Osteoclast precursor cells were cocultured with MIF-stimulated RA FLS and subsequently stained for tartrate-resistant acid phosphatase (TRAP) activity. MIF upregulated the expression of RANKL in RA-FLS. The increased expression of RANKL mRNA and protein was completely blocked by AP-1 inhibitor (curcurmin), p38 MAPK inhibitor (SB203580) or JAK II-STAT2(AG490). Increased number of TRAP positive cells were observed when osteoclast precursor cells were incubated with MIF-pretreated RA FLS. These results suggest that MIF could be involved in the inflammatory bone destruction by regulating RANKL expression in RA FLS. The interaction between MIF and RANKL could be a potential gateway leading to new therapeutic approach in treating bone destruction in RA.

Kallikreins, kininogens and kinin receptors on circulating and synovial fluid neutrophils: role in kinin generation in rheumatoid arthritis

Neutrophils traffic into and have the capacity to generate kinins in SF of RA patients. The aim of this study was to assess the expression of kallikreins, kininogens and kinin receptors in circulating and SF neutrophils, as well as synovial tissue of RA patients, and to assess kinin generation in SF. Neutrophils were isolated from blood and SF of RA patients and blood of healthy volunteers. Expression of kallikreins, kininogens and kinin receptors in neutrophils and synovial tissue was assessed by immunocytochemistry using specific antibodies, with visualization by brightfield and confocal microscopy. Levels of basal and generated kinins in SF of RA patients were measured by ELISA. Kinin labelling was significantly reduced, indicating the loss of the kinin moiety from kininogen on circulating (P < 0.001) and SF neutrophils (P < 0.05) of RA patients. Immunolabelling of tissue kallikrein was also decreased, whereas kinin B(1) and B(2) receptor expression was increased in circulating and SF neutrophils of RA patients. Immunolabelling of kallikreins and kinin receptor proteins was similar in RA and normal synovial tissues. The basal kinin level in SF of RA patients was 5.7 +/- 6.1 ng/ml and the mean concentration of kinins generated in vitro was 80.6 +/- 56.3 ng/ml. The capacity for kinin generation was positively correlated with measures of disease activity. Kallikrein-kinin proteins on neutrophils play an important role in kinin generation and the pathophysiology of RA. Specific kallikrein and kinin receptor antagonists may be useful as IA therapies for inflamed joints.

Functional expression of the chemokine receptor CCR7 on fibroblast-like synoviocytes

We have characterized the expression and the function of the chemokine receptor CCR7 on fibroblast-like synoviocytes (FLS) of patients with RA and OA and on dermal fibroblasts. FLS were obtained after enzymatic digestion of synovial tissue (ST) of patients with RA and OA undergoing knee replacement surgery and taken into culture for chemokine receptor analysis by RT-PCR, flow cytometry and functional tests. Immunofluorescence for CCR7, fibroblast and T-cell markers was performed on ST of RA and OA patients. To study the response of FLS to CCR7 ligands and other chemokines, migration assays were performed in modified Boyden chambers. After stimulation of FLS with CCR7 ligands, the secretion of VEGF was evaluated by ELISA and Luminex. CCR7 is expressed on FLS of patients with RA and OA, but not on dermal fibroblasts. FLS migrated in response to the CCR7 ligands, CCL19 and CCL21. Stimulation of FLS with CCL19 resulted in a significantly increased secretion of VEGF of RA- and OA-FLS. Apart from the migration of FLS in response to CCL19 and CCL21, it was shown that activation of the CCR7 receptor on FLS results in an enhanced VEGF secretion. A considerable expression of CCR7 ligands in proximity to perivascular infiltrates has previously been described in inflamed synovial tissue of RA patients. Stimulation of FLS via CCR7 could thereby contribute to angiogenesis in the synovial tissue.

Synoviolin and Rheumatoid Arthritis

DEVELOPMENT Rheumatoid arthritis (RA) afflicts nearly 1% of the worldwide adult population. Cytokines released from immune cells cause chronic inflammation and stimulate the proliferation of synovial cells that destroy bone and cartilage of joints. But nearly 25% of RA patients do not respond to anti-cytokine therapy. This may be because synovial cell growth is also controlled by an enzyme called synoviolin. Amano et al. detected elevated synoviolin expression in the synovial tissue of RA patients. Mice overexpressing this molecule exhibited spontaneous arthropathy and a progressive synovial cell hyperplasia characteristic of RA patients. Reduced expression of synoviolin in mice correlated with protection from arthritis. This resistance was not due to an impaired cytokine response or reduced inflammatory cell infiltration, but to an increase in synovial cell apoptosis. Knockdown of synoviolin expression in rheumatoid synovial cells by RNA interference suppressed growth responses to cytokines. Synoviolin-null mouse fibroblasts also showed enhanced apoptosis induced by endoplasmic reticulum-related stress, suggesting a possible anti-apoptotic mechanism, and marking synoviolin as a possible drug target to treat RA. Synoviolin is an E3 ubiquitin ligase, but the role of its enzymatic activity in the pathogenesis of RA is not yet known. — LDC Genes Dev . 10.1101/gad.1096603 (2003).