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The aim of this study was to investigate immunohistological changes in mitogen-activated protein kinases (MAPKs) in the synovium following treatment with golimumab, compared with methotrexate (MTX). We assessed synovial tissues for 13 different molecules to detect cytokine levels histologically from 10 methotrexate (MTX)-treated rheumatoid arthritis (RA) patients as controls and 10 golimumab plus MTX-treated RA patients. Synovium samples from both groups were assessed by hematoxylin and eosin (HE) staining and analyzed for expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP-3), CD4 (T cells), CD8 (T cells), CD20 (B cells), CD68 (macrophages), receptor activator of nuclear (kappa) B ligand (RANKL), bromodeoxyuridine (BrdU), CD29 (β-1 integrin), phospho-p38 MAPK (Tyr180/Tyr182), phospho-p44/42 MAPK (ERK1/ERK2), and phospho-c-Jun N-terminal kinase (JNK), by an immunohistological examination. HE staining showed that there was a significant decrease in cell proliferation in the synovium in RA patients who received golimumab compared with the controls. TNF-α, IL-6, MMP3, BrdU, p38, and ERK were not seen at significant levels in either group. On the other hand, CD4, CD8, CD20, CD29, CD68, RANKL, and JNK were significantly decreased in the golimumab group compared with the control. Based on a histological analysis of the synovium, it appears that the efficacy of the treatment with golimumab may involve the inhibition of cell proliferation, with decreases in T cells, B cells, macrophages, β-1 integrin, RANKL, and JNK in the synovium, compared with MTX treatment, in RA.

Resveratrol (trans-3,4'-trihydroxystilbene), a natural phytoalexin, possesses anti-inflammatory, anti-proliferative, and immunomodulatory properties and has the potential for treating inflammatory disorders. The present study was designed to investigate the effects of resveratrol on TNF-α-induced inflammatory cytokines production of IL-1β and MMP3 in Rheumatoid arthritis (RA) Fibroblast-like synoviocytes (FLS) and further to explore the role of PI3K/Akt signaling pathway by which resveratrol modulates those cytokines production. The levels of IL-1β, MMP-3 in cultural supernatants among groups were measured by enzyme-linked immunosorbent assay. Messenger RNA expression of IL-1β and MMP-3 in RA FLS was analyzed using a reverse transcription-polymerase chain reaction. Western blot analysis was used to detect proteins expression in RA FLS intervened by resveratrol. Resveratrol inhibited both mRNA and proteins expressions of IL-1β and MMP-3 on RA FLS in a dose-dependent manner. Resveratrol also decreased significantly the expression of phosphorylated Akt dose dependently. Activation of PI3K/Akt signaling pathway exists in TNF-α-induced production of IL-1β and MMP3 on RA FLS, which is hampered by PI3K inhibitor LY294002. Immunofluorescence staining showed that TNF-α alone increased the production of P-Akt, whereas LY294002 and 50 μM resveratrol suppressed the TNF-α-stimulated expression of P-Akt. Resveratrol attenuates TNF-α-induced production of IL-1β and MMP-3 via inhibition of PI3K-Akt signaling pathway in RA FLS, suggesting that resveratrol plays an anti-inflammatory role and might have beneficial effects in preventing and treating RA.

The aim of this study was to evaluate the morphological changes in the spleen, the thymus and the knee joints of rats with experimental adjuvant arthritis induced by Mycobacterium butyricum in the incomplete Freund's adjuvant and the effect of treatment with methotrexate (MTX). Particular attention was aimed on the redistribution of granulocytes in the tissues during the inflammatory process. Clinical parameters, e.g., joint edema, body weight and of gamma glutamyl transferase (GGT) activity as an inflammatory marker, have also been determined. Induction of adjuvant arthritis caused a significant decrease in granulocyte number in the spleen and vice versa a significant increase in the knee joints, but without significant changes in the thymus. Treatment with methotrexate reversed this phenomenon by increasing the granulocyte number in the spleen and decreasing it in knee joints. MTX decreased the joint edema as well as the activity of GGT in the spleen, modified the size of the white pulp of the spleen and increased the cortex/medulla ratio in the thymus. The observed changes support the anti-inflammatory and immunomodulatory properties of MTX supporting its use as the first-line medication in patients with rheumatoid arthritis.

The root of Clematis chinensis Osbeck has been used widely in rheumatoid arthritis in Chinese traditional medicine, and AR-6 is a triterpene saponin isolated from it. In this present study, we investigated the in vivo effects of oral AR-6 in chronic rat with collagen-induced arthritis (CIA) and possible molecular mechanism. CIA was induced by immunizing 56 female Sprague-Dawley (SD) rats with chicken typeIIcollagen (CII). Following eighteen days, the immunization rats with CIA were treated with AR-6 (32, 16, 8 mg/kg), cyclophosphamide (7 mg/kg), and TGP (Total Glucosides of Paeonia) (180 mg/kg) for 7 days, and rats without CIA were given the same volume of purified water. TNF-α and IL-1β levels in peripheral blood will be measured by ELISA, and Western blot analysis will be used to detect the expression of NF-κB p65 subunits, TNF-α and COX-2, in synovial membrane. We found that therapeutic treatment with AR-6 markedly improves the paw swelling and histopathological changes. Moreover, the serum levels of pro-inflammatory cytokines TNF-α and IL-1β were markedly lowered, and the expression of NF-κB p65 subunits, TNF-α and COX-2, in the synovial membrane of CIA rats was significantly inhibited in the AR-6-treated groups. These results enable to prove that AR-6 has a potential anti-inflammatory effect in CIA rats, and its mechanism may relate to the inhibition of the expression of NF-κB p65 subunits, TNF-α and COX-2.

Gliostatin/thymidine phosphorylase (GLS/TP) is known to have angiogenic and arthritogenic activities. The purpose of this study was to determine the inhibitory effects of FK506 (tacrolimus) on GLS production in rheumatoid arthritis (RA). We investigated the modulation of serum GLS by FK506 therapy and the effect of FK506 on the production of GLS in fibroblast-like synoviocytes (FLSs). Serum samples were collected from 11 RA patients with active disease at baseline and after 12 weeks of FK506 treatment. Serum concentrations of GLS and matrix metalloproteinase (MMP)-3 were measured by ELISA and found to be down-regulated in responders evaluated with a disease activity score. Patient FLSs were cultured and stimulated by tumor necrosis factor (TNF)-α with or without FK506. The expression levels of GLS were determined using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme immunoassay and shown to be significantly increased. GLS levels in TNF-α-stimulated FLSs were reduced by FK506 treatment. Our data show a novel mechanism for the action of physiological concentrations of FK506 in RA that regulates the production of GLS in FLSs.

Unlabelled: To observe the level and the distribution of MMP-1 in cartilage and synovium over the progression of OA in rabbit ACLT model, and to explore the relationship between the amount of MMP-1 expression and the degree of OA cartilage degradation. OA was induced in 20 rabbits by anterior cruciate ligament transection (ACLT) and ten rabbits were killed at 4 and 8 weeks after the operation, respectively. A further ten rabbits received unilateral knee arthrotomy as controls. Cartilage degradation was observed under dissecting microscope, immunohistochemical, and morphometric analysis were adopted to record the tissue level and distribution of MMP-1 in cartilage and synovium. Cartilage degradation in both ACLT groups was more severe than that in control group, and the degradation in 8-week ACLT group was more severe than that in 4-week ACLT group. MMP-1 was expressed predominantly in the superficial and upper intermediate layers of cartilage and in the lining layer of synovium. Its expression was increased steadily over the progression of OA both in cartilage and in synovium, but there was a little difference in the increase pattern between them: increase of MMP-1 in synovium lagged behind that in cartilage in early stage of OA., Conclusion: MMP-1 was involved in OA development in rabbit ACLT model and the amount of its expression was related with the degree of cartilage degradation. The increase of MMP-1 expression in synovium lagged behind that in cartilage, suggesting OA pathology was originated from cartilage, but synovitis may also paticipate in cartilage degradation, especially in middle and late stage of OA.

Combined effects of hyaluronate and indomethacin in the treatment of rabbits with antigen-induced arthritis (AIA) were evaluated by assessing joint swelling, C-reactive protein (CRP) and prostaglandin E(2) (PGE(2)) levels with periodic intra-articular (ia) injections of hyaluronate alone (HA group) and with either a low or high concentration of indomethacin (LI-HA or HI-HA group). End-point analyses included matrix metalloproteinases-3 (MMP-3) activity and macroscopic and histological joint examinations. Results demonstrated that treatment in LI-HA and HI-HA groups resulted in statistically significant suppression of CRP, PGE(2, )and MMP-3 in comparison with those of HA group. Inhibition of serum CRP was only observed in LI-HA group. The order of serum MMP-3 inhibition was LI-HA>HI-HA>HA. Based on macroscopic and histological analyses of pannus formation, hyperplasia, inflammation, joint leakage and erosion, and loss of proteoglycan, the only statistically significant improvement was shown in LI-HA group compared to HA group and HI-HA group compared to control group.

Gliostatin/thymidine phosphorylase (GLS/TP) is known to have angiogenic and arthritogenic activities. The purpose of this study was to elucidate whether GLS/TP is involved in the regulation of the angiogenic cytokine vascular endothelial growth factor (VEGF) in rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLSs) from patients with RA were cultured and stimulated with recombinant human GLS (rHuGLS) and interleukin (IL)-1beta. Immunohistochemistry showed that GLS/TP and VEGF were detectable in the synovial lining cells. In cultured FLSs, both VEGF mRNA and protein levels were markedly increased by rHuIL-1beta treatment. rHuGLS increased VEGF mRNA expression in a dose-dependent manner. We detected high concentrations of VEGF165 protein in culture supernatants from FLSs treated with rHuGLS (300 ng/ml), which were comparable to GLS levels found in synovial fluid of RA patients. These findings indicate that GLS/TP and VEGF have synergistic effects on angiogenesis in rheumatoid synovitis, and that GLS/TP has a role in regulating VEGF.

Interleukin-1 beta (IL-1beta) is an abundant cytokine, which, together with TNF-alpha, mediates inflammatory events in rheumatoid arthritis (RA). IL-1beta is known to induce the induction of inflammatory cytokines and metalloproteinases (MMPs) in rheumatoid synovial cells. Here, we assessed these inflammatory events by measuring IL-1beta levels in the human synovial cell line, MH7A. We observed that the activation of p38 MAP kinase by IL-1beta was involved in the induction of inflammatory cytokines, as well as several genes, including MMP-1 and MMP-3. SB203580, a specific p38 MAP kinase inhibitor, inhibited the production of IL-1beta-induced cytokines and MMPs, while the levels of the tissue inhibitor of metalloproteinase (TIMPs) were unchanged by treatment with SB203580. Moreover, the induction of suppressor of cytokine signaling 3 (SOCS3) and interferon regulatory factor 1 (IRF-1) were both found to be induced by the inhibition of p38 MAP kinase. Therefore, we suggested that the inhibition of p38 MAP kinase might enhance anti-inflammatory tendencies in the MH7A cells.

Cyclo-oxygenase (COX)-2 has been associated with inflammation in rheumatoid arthritis (RA), but its role in joint destruction remains unclear. In this study, we investigated the effect on cultured rheumatoid fibroblast-like synoviocytes (FLS) of the selective COX-2 inhibitor celecoxib on the expression of matrix metalloproteinases (MMPs), which play an important role in tissue degradation and angiogenesis in rheumatoid synovium. Treatment with nontoxic doses of celecoxib resulted in dose-dependent inhibition of MMP-1, -2, and -3 secretion from FLS when measured by enzyme-linked immunosorbent assay. Celecoxib suppressed proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-1beta) induced augmentation of the gelatinolytic activity on zymography. These results suggest that COX-2 inhibitors might influence matrix degradation or angiogenesis in RA by downregulating the expression of various MMPs in rheumatoid FLS.

Caspase-1 expression in synovial membrane-like interface tissue (SMLIT) around loosened hip prostheses and osteoarthritic synovial samples was studied. Caspase-1 mRNA was found in SMLIT and synovial tissue. There is no difference in the copy numbers of caspase-1 mRNA between these samples. Both precursor and active forms of caspase-1 proteins appeared in these samples, but the number of positive cells was higher in SMLIT than in synovial tissue. Double labeling revealed that most caspase-1-positive cells were macrophages and fibroblasts. In the lining-like layers and deep stroma of SMLIT, many cells were double positive for active caspase-1 and interleukin-1 beta (IL-1beta). In contrast, the number of active caspase-1/IL-18 double-positive cells was very low. We conclude that caspase-1 synthesis is increased in SMLIT. Caspase-1 can be involved in implant loosening by processing IL-1beta precursor into its mature form, which is a potent osteoclast-activating factor and a major proinflammatory mediator.

To investigate the mechanism of synovial hyperplasia by human T-lymphotropic virus type I (HTLV-I) infection, the enzymatic activity of telomerase and expression of telomerase-related factors in HTLV-I infected synoviocytes were examined. Cultured synoviocytes obtained from four patients with osteoarthritis (OA) and four with traumatic joint disease (TJD) were infected by HTLV-I. Telomerase activity was detected by telomeric repeat amplification protocol (TRAP) assay. Expression of telomerase-related mRNAs such as telomerase reverse transcriptase (hTERT), telomerase RNA component (hTERC), and telomeric repeat binding factor 2 (TRF2) were also examined. Telomerase activity was detected in all HTLV-I-infected synoviocytes but not in uninfected synoviocytes. A remarkable induction of hTERT mRNA was observed in four of eight HTLV-I-infected synoviocytes, whereas expressions of hTERC, TRF2, and TEP-1 mRNAs were not changed. Our results clearly demonstrate that HTLV-I upregulates telomerase activity in synoviocytes probably via upregulation of hTERT activity. These findings suggest that telomerase activation in synoviocytes has an important role in upregulated proliferative activity of HAAP synoviocytes.

To investigate the mechanism of persistent proliferation of rheumatoid arthritis (RA) synoviocytes in situ, we examined the activity of telomerase enzyme and the expression of telomerase related factors in cultured synoviocytes. Cultured synoviocytes obtained from patients with rheumatoid arthritis (n = 29), osteoarthritis (OA, n = 18), and traumatic joint disease (TJD, n = 4) were examined. Telomerase activity was detected by TRAP (telomeric repeat amplification protocol) assay, and 12 out of 29 samples of synoviocytes (41%) from RA patients showed a positive telomerase activity, whereas none of the samples from OA and TJD patients showed this activity. Results were confirmed by PCR-ELISA. The telomerase activity was enhanced by basic fibroblast growth factor (bFGF). The mRNA expression of telomerase related factors, such as hTERC, TRF2, and TEP-1, showed no difference between RA and OA synoviocytes. Our results suggest that telomerase is activated in rheumatoid synoviocytes, and that bFGF upregulates the activity of this enzyme in RA synoviocytes.

Mast cells are believed to play a novel part in the development of destructive synovial pannus in rheumatoid arthritis (RA). This study was undertaken to investigate the localization of vascular endothelial growth factor (VEGF) in the synovial membrane using a unique immunostaining technique. Synovial specimens of RA patients were examined immunohistochemically and were compared with specimens from non-RA controls. Multi-labelling subtraction immunostaining, a modification of double- and triple-labelling immunostaining, revealed that the VEGF-positive cells were identical to tryptase-positive cells (mast cells). No other cell types were found to be positive for VEGF. The synovium of RA patients showed a larger number of VEGF-positive mast cells than that of non-RA controls (P<0.001). The study suggests that mast cell-derived VEGF may contribute to the development of synovial pannus in RA.

Matrix metalloproteinases such as collagenase and stromelysin are recognised as important cartilage-degrading enzymes in the pathophysiology of rheumatoid arthritis. Synovial fibroblasts and macrophages are the major cellular components of rheumatoid synovium, but the regulation and relative expression of collagenase and stromelysin by these two cell types remains uncertain. Using in vitro cultures of adherent rheumatoid synovial cells we have examined the coordinate or separate expression of collagenase and stromelysin-1 by dual immunolocalisation and Western blotting techniques. Synovial fibroblasts, when activated by macrophage-derived products in primary culture or by interleukin-1/phorbol myristate acetate in subcultures, released significant quantities of collagenase and stromelysin in their inactive, precursor forms. The ratio of released procollagenase: prostromelysin varied between different synovial cell preparations. Dual immunolocalisation studies demonstrated both coordinate and separate expression of the two enzymes by single cells. Approximately 80% of the activated fibroblasts, especially those with stellate morphology, showed co-expression of both enzymes. By contrast synovial macrophages had a modest or negligible capacity to elaborate either enzyme under the same in vitro conditions. In many fibroblastic cells both collagenase and stromelysin were co-localised to the perinuclear Golgi region and the same cytoplasmic compartments. Vesicular structures appear to provide intracellular transport for both enzymes to sites of secretion. Both enzymes showed preferential pericellular binding to a collagenous substratum rather than any association with the plasma membrane/cell surface.

In view of the important role of interstitial collagenase in the pathogenesis of rheumatoid arthritis (RA), we studied the expression of fibroblast-type collagenase in rheumatoid synovium and searched for its potential transcription factors, namely the oncoprotein c-fos and the early-growth-response gene-1 (egr-1), an inducible zinc-finger encoding gene. Elevated levels of RNA sequences complimentary to c-fos and egr-1 cDNA probes could be detected in cytoplasmic extracts of collagenase-expressing synovial fibroblast-like cells when compared to equivalent RNA amounts isolated from control fibroblasts. Utilizing immunocytochemistry, immunoreactivity for c-fos oncoprotein was found in 13 of 19 joint specimens obtained from patients with active RA. These oncoprotein data were positively correlated to the collagenase expression in the same specimens. Moreover, immunohistochemical analysis confirmed the localization of both oncoprotein c-fos and fibroblast-type collagenase within synovial fibroblast-like cells attached to bone erosions.

Lysozyme-producing cells were analysed by enzyme histochemistry in paraffin sections of synovial tissue of 60 patients with rheumatoid arthritis (RA) and 20 patients with osteoarthritis (OA). For lysozyme detection three enzyme histochemical systems - peroxidase-anti-peroxidase, alkaline phosphatase and biotin-avidin - were used in parallel experiments. Lysozyme was found to be produced by polymorphonuclear cells, mononuclear phagocytes and part of synovial lining cells. All types of lysozyme-producing cells were increased in RA compared with OA. Subgrouping of RA synovitis according to histomorphological criteria allowed the demonstration of an inverse relationship between the number of lysozyme-producing cells and the grade of proliferation of fibroblasts, called mesenchymoid transformation by Fassbender [19]. The different methods of lysozyme detection differed in specificity and sensitivity. The immunoenzymatic staining of lysozyme allows specific and quantitative evaluation of phagocytizing cells in RA and OA.

Fibroblast-like synovial cells isolated from intact joints of non-arthritic human donors released up to nine-times higher activity of the lysosomal acid hydrolase N-acetyl-beta-glucosaminidase (NAG) than controls when incubated in conditioned medium from homologous peripheral blood mononuclear cells (MCCM). This increase occurred without decrease in cell numbers or other evidence of cytotoxicity. An increase in cell-associated NAG activity was also suggested, but this was not statistically significant. Indomethacin present during production of MCCM or added with MCCM to fibroblast cultures did not alter the response to MCCM, indicating that the effect of MCCM was not due to the presence of products from the cyclo-oxygenase pathway. At a concentration known to block protein synthesis in most cells (10(-5) M), cycloheximide markedly suppressed the NAG releasing response to MCCM. The secretion of NAG due to MCCM was not affected by all-trans retinoic acid (10(-6) M) but was suppressed by the corticosteroid, dexamethasone.

During 5 days of culture, explants of normal rabbit synovium produced no active collagenase, negligible latent collagenase, but significant levels of free collagenase inhibitor. Synovium from joints exhibiting a proliferative arthritis produced greatly elevated levels of collagenase; the appearance of active enzyme in the medium during the second day of culture was associated with the disappearance of free inhibitor. Enzyme levels in the media correlated well with the arthritic status of joints, when explants were prepared up to 10 weeks after the induction of the model arthritis. Synovium from the contralateral joints of rabbits with unilaterally induced arthritis produced no active collagenase, but approximately one-third as much latent collagenase as found with arthritic joints. Enzymatic activities against gelatin and cartilage proteoglycan substrates were demonstrated in synovial culture media in addition to collagenolytic activity. Gel filtration showed that these activities were not due to a single enzyme, and further characterisation confirmed that the enzymes were metalloproteinases. The results are considered in the light of published data, and the involvement of metalloproteinases and their specific inhibitor in the development of arthritic lesions is discussed.

Human articular cartilage of different ages was cultured for 8 days and proteoglycan (PG) release into the medium was measured. Retinol and synovial co-culture increased the PG release of cartilage of all ages. The effect of retinol was dose-dependent. Synovium increased also the PG release of dead cartilage, whereas retinol did not. The increased PG release by synovial co-culture is therefore mainly the result of synovial enzymes acting directly on the matrix rather than of a factor inducing chondrocyte-mediated breakdown.

Using a rabbit model arthritis we have investigated the ability of dexamethasone to alter the production of collagenase and the specific metallo-proteinase inhibitor TIMP by explants of synovium and cartilage in vitro. The patterns of collagenase and TIMP production by untreated explants from arthritic joint tissues in culture were similar to those described previously [1, 2]. Dexamethasone dramatically altered the patterns of production of collagenase and TIMP. At a dose of 10 nM, or above, the patterns of production by treated synovium resembled those of normal rabbit synovium: collagenase production was suppressed and TIMP increased compared with untreated arthritic synovium. The levels of latent collagenase in cartilage also fell with increasing doses of dexamethasone and TIMP levels were higher, although normal levels were not reached. These experiments have been conducted as a prelude to testing the effects of various anti-rheumatic drugs in vivo, and attempting to correlate changes in clinical parameters with the subsequent production of collagenase and TIMP in vitro. The data are discussed in relation to the therapeutic use of corticosteroids and to their mode of action on joint tissues.