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Discordant cytokine production is characteristic of chronic inflammatory conditions like rheumatoid arthritis (RA), and anti-cytokine therapeutics are becoming routinely used to treat RA in the clinic. Fibroblasts from rheumatoid synovium have been shown to contribute to cytokine production in inflamed joints; likewise these cells also produce cytokines in response to inflammatory mediators signalling through Toll like receptors (TLRs). Tyrosine kinase activity is essential to LPS-induced cytokine production, and we have previously implicated a role for the Tec kinase, Bmx, in inflammatory cytokine production. Here we show that Bmx kinase activity in RASF is increased following LPS stimulation and that Bmx is involved in the regulation of LPS-induced IL-6 and VEGF production via mRNA stabilisation. This is an important insight into the regulation of VEGF, which is involved in a wide range of different pathologies, and may lead to more effective design of novel anti-inflammatory/angiogenic therapeutics for conditions such as RA.

Rheumatoid arthritis (RA), a chronic and systemic disease of unknown etiology, is characterized by hyperplasia of synovial cells, which ultimately lead to the destruction of cartilage and bone. To elucidate the molecular mechanisms that lead to RA, we analyzed synovial cells established from patients with RA by oligonucleotide microarrays. Gene expression profiles clearly suggested that oxidative stress is enhanced in RA synovial cells, which was confirmed by measuring cellular levels of reactive oxygen species. One of the highly up-regulated proteins in RA synovial cells was thioredoxin reductase 1 (TRXR1), a protein that plays an important role in antioxidant defense system. Subsequent analysis demonstrated that TRXR1 suppresses hydrogen peroxide and inhibits apoptosis of RA synovial cells. Thus, our results reveal a novel pathophysiologic function of RA synovial cells as a generator of oxidative stress, and a self-defense mechanism against self-generated oxidative stress.

Calpain is secreted by intra-articular synovial cells and degrades the main components of cartilage matrix proteins, proteoglycan, and collagen, causing cartilage destruction. Matrix metalloproteinase-3 (MMP-3) has also been detected in synovial fluid and serum, and is involved in the development and progression of rheumatoid arthritis by degradation of the extracellular matrix and cartilage destruction. To investigate the relationship between calpain and MMP-3 in rheumatic inflammation, we utilized the rheumatic synovial cell line, MH7A. Tumor necrosis factor (TNF-alpha) stimulation-induced increased expression of mu-calpain, m-calpain, and MMP-3 in these cells, as well as the release of calpain and MMP-3 into the culture medium. The calpain inhibitors, ALLN (calpain inhibitor I) and calpeptin, did not affect the intracellular expression of MMP-3, but reduced the secretion of MMP-3 in a concentration-dependent manner. Down-regulation of mu- but not m-calpain by small interfering RNAs abolished TNF-alpha-induced MMP-3 release from the synovial cells. These findings suggest that calpain, particularly mu-calpain, regulates MMP-3 release by rheumatic synovial cells, in addition to exerting its own degradative action on cartilage.

In the sera of rheumatoid arthritis (RA) patients, autoantibodies directed to citrullinated proteins are found with high specificity for RA. Peptidylarginine deiminases (PADIs) are enzymes responsible for protein citrullination. Among many isoforms of PADIs, only PADI4 has been identified as an RA-susceptibility gene. To understand the mechanisms of the initiation and progression of RA, we compared the properties of two PADIs, human PADI2 and human PADI4, which are present in the synovial tissues of RA patients. We confirmed their precise distribution in the RA synovium and compared the stability, Ca2+ dependency, optimal pH range, and substrate specificity. Small but significant differences were found in the above-mentioned properties between hPADI2 and hPADI4. Using LC/MS/MS analysis, we identified the sequences in human fibrinogen indicating that hPADI2 and hPADI4 citrullinate in different manners. Our results indicate that hPADI2 and hPADI4 have different roles under physiological and pathological conditions. Further studies are needed for the better understanding of the role of hPADIs in the initiation and progression of RA.

beta 2-Microglobulin (beta 2-m) is a major constituent of amyloid fibrils in hemodialysis-associated amyloidosis (HAA), a serious complication in patients on long-term hemodialysis. The most distinctive pathological feature of HAA is the deposition of amyloid fibrils with subsequent articular inflammation and destruction. However, the pathological role of beta 2-m is not well known at present. We investigated the effects of beta 2-m on the production of proteinases from synovial fibroblasts isolated from patients with rheumatoid arthritis. beta 2-m stimulated synovial fibroblasts to produce stromelysin, a neutral matrix metalloproteinase (MMP-3). The production of MMP-2 and of a tissue inhibitor of metalloproteinase-1 (TIMP-1) were not enhanced by beta 2-m-treated synovial fibroblasts. Stromelysin is capable of degrading several components of the extracellular matrix and believed to be the key enzyme causing articular destruction in inflammatory joint diseases. Our results suggest a novel role for beta 2-m in articular inflammation and destruction mediated by stromelysin in HAA., (Copyright 1997 Academic Press.)

Nuclear receptors inhibit synthesis of collagenase-1 (matrix metalloproteinase-1; MMP-1), an enzyme that degrades interstitial collagens and contributes to joint pathology in rheumatoid arthritis. SMRT (Silencing Mediator for Retinoid and Thyroid hormone receptors) mediates the repressive effect of nuclear receptors at hormone responsive elements (HREs), prompting us to investigate whether this co-repressor could also regulate transcription of MMP-1, which lacks any known HREs. We find that primary synovial fibroblasts express SMRT. When over-expressed by transient transfection, SMRT inhibits MMP-1 promoter activity induced by interleukin-1 (IL-1), phorbol phorbol myristate acetate (PMA) or v-Src. SMRT apparently inhibits MMP-1 gene expression by interfering with one or more transcriptional elements clustered in a region between -321 and +63. We conclude that SMRT negatively regulates MMP-1 synthesis through a novel, HRE-independent mechanism that involves proximal regions of the MMP-1 promoter.

Superantigens activate a large number of T cells in a V beta-restricted manner after binding to MHC class II molecules on the antigen presenting cells (APC). Superantigens also activate APC directly by interacting with their ligands, MHC class II molecules. In the present study, we examined the effects of superantigens on matrix metalloproteinases (MMPs) secretion from rheumatoid synovial fibroblasts. We demonstrated that stimulation of interferon (IFN)-gamma-treated synovial fibroblasts with staphylococcal enterotoxin A (SEA) selectively induced the secretion of stromelysin, a neutral MMP, from synovial fibroblasts. Pretreatment of synovial fibroblasts with cycloheximide, an inhibitor of protein synthesis, prevented MMP-3 secretion from rheumatoid synovial cells suggesting that protein synthesis was required for SEA-induced MMP-3 secretion after SEA binding to MHC class II molecules. Our data suggest that in the synovium, bacterial superantigens are potent inducers of stromelysin which plays a critical role in articular destruction observed in inflammatory joint disease.

We recently reported that interleukin-4 (IL-4) inhibited prostanoid synthesis through inhibiting cyclooxygenase 2 biosynthesis. In the present study, we examined the effect of IL-4 on the expression of cytosolic phospholipase A2 (cPLA2). The amounts of protein and mRNA of cPLA2 were determined by western blotting and reverse transcription polymerase chain reaction (RT-PCR), respectively. Although interleukin-1alpha (IL-1alpha) and tumor necrosis factor alpha (TNFalpha) had little effect on the biosynthesis of cPLA2 in phorbol myristate acetate (PMA)-differentiated U937 cells, lipopolysaccharide (LPS) increased the protein level of cPLA2 in a dose-dependent manner. IL-4 inhibited the increased synthesis of cPLA2 at the mRNA level. In addition, IL-4 inhibited the biosynthesis of cPLA2 in untreated or LPS treated freshly prepared rheumatoid synovial cells at the mRNA level. These findings suggest that IL-4 inhibits prostanoid synthesis through inhibiting the expression of both cPLA2 and cyclooxygenase 2.

The histological features of rheumatoid arthritis (RA) consist of overgrowth of synovial cells. Several growth factors that cause synovial hyperplasia have been identified in RA synovium. The basic-fibroblast growth factor (b-FGF), representing one of these growth factors, may play an important role in the pathogenesis of RA. We examined the b-FGF-mediated intracellular signal pathway involved in synovial cell growth. b-FGF-induced synovial cell growth was inhibited by protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, but not by H7 that inhibits protein kinase C (PKC). Stimulation of synovial cells with b-FGF resulted in tyrosine phosphorylation of cellular proteins and MAP kinase activation. Our results also demonstrated that b-FGF-mediated activation of MAP kinase was inhibited by herbimycin A indicating that protein tyrosine kinase may be involved in the activation of MAP kinase in human synovial cells. However, inhibition of b-FGF-mediated MAP kinase activation by PKC downregulation did not occur.

Native type V collagen molecules resist mammalian collagenase but are cleaved by certain gelatinases. We report a prominent site of cleavage within the collagen type V molecules by 92 kDa gelatinase (MMP-9). The enzyme was purified from conditioned medium of a rabbit synovial cell line (HIG-82). It cleaved native type V collagen from bovine bone in solution at two molecular sites, one near the amino-terminus, the other producing a 3/5 C-terminal fragment. Amino-terminal sequence analysis of the individual alpha chains from this latter fragment showed that MMP-9 had cleaved between residues Gly439-Val in both alpha 1(V) and alpha (XI) and between residues Gly445-Leu in the alpha 2(V) chain. These sites are close to the previously reported trypsin-cleavage site. The findings imply that gelatinases may be necessary for initiating or completing degradation of type I/type V copolymeric fibrils for growth and remodeling of extracellular collagen.

Phospholipase A2 (PLA2) activities in rheumatoid synovial fibroblasts (RSF) stimulated with interleukin-1 beta (IL-1 beta) were investigated. RSF incubated in the presence of IL-1 beta (120 pg/ml) for 18 h secreted 35 fold more PGE2 than did those incubated without IL-1 beta. IL-1 beta treatment did not increase the level of secretory PLA2 (sPLA2) activity or sPLA2 protein in the conditioned medium or subcellular fractions of lysed RSF. In contrast, the cell-associated PLA2 activity increased 3 to 4 fold in IL-1 beta stimulated RSF when compared with the control. The IL-1 beta stimulated, cell-associated PLA2 required submicromolar concentrations of calcium for activity, a characteristic consistent with the calcium sensitivity of cytosolic PLA2 (cPLA2) activity reported in other cell types, such as U937 cells. These findings demonstrate that an elevation in a cytosolic PLA2, rather than a sPLA2, is associated with increased PGE2 production in IL-1 beta stimulated RSF.

Cytokine regulation of synovial cell function has been considered to be involved in the pathogenesis of rheumatoid arthritis. Synoviocyte urokinase-type plasminogen activator (u-PA) expression may be relevant to the tissue remodelling, as well as to the cell migration and transformation occurring in rheumatoid joints. We report here that purified recombinant human oncostatin M (greater than or equal to approximately 0.2 U/ml = 1 pM) stimulated within six hr the u-PA activity of non-rheumatoid synovial fibroblast-like cells and raised their u-PA mRNA levels. Oncostatin M could augment PGE2 production and DNA synthesis in these cells; however, the increase in PGE2 was small compared with that caused by IL-1. Since oncostatin M is produced by immune cells, it may have a role in immune and inflammatory reactions by interacting with fibroblast populations, such as synoviocytes, in the manner described.

Human synoviocytes, rabbit articular chondrocytes and human skin fibroblasts in culture were examined for their ability to express elastase activity. Latent enzyme activity degrading insoluble elastin was detected in the culture media of the three cell types and was completely abolished by metal chelating agents. Triton X-100 cell extracts were found to degrade a synthetic elastase substrate, N Succinyl-(Ala)3p-nitroanilide (SANA). The SANA-degrading activity of cell extracts could be attributed to a metalloprotease for fibroblasts and synoviocytes (100%) and to a metalloprotease associated with a cysteine protease for chondrocytes (70 and 30% respectively). This SANA-degrading activity was partly due to the combined action of an endo and an exopeptidase. Tumor Necrosis Factor-alpha (TNF-alpha) and Interferon-gamma (IFN-gamma) significantly enhanced the elastin degrading activity present in the culture media of both synoviocytes and chondrocytes. Interleukin-1 beta significantly increased the secretion of elastase by chondrocytes. By contrast, Transforming Growth Factor-beta (TGF-beta) reduced by 80 per cent the secretion of elastinolytic activity by chondrocytes but had not effect on other cell types.

Plasma membranes were isolated from lapine synovial cells grown in monolayer culture using discontinuous sucrose gradient centrifugation techniques. 5'nucleotidase was detected in great abundance while glucose-6 phosphate dehydrogenase and cytochrome oxidase were present at low to undetectable levels. Plasma membranes incubated at 37 degrees C for 60 min with [3H]-arachidonyl-phosphatidylinositol/phosphatidylserine synthesized [3H]-diacylglycerides. Little if any [3H]-diacylglyceride synthesis was measured when [3H]-arachidonyl-phosphatidylcholine or [3H]-arachidonyl-phosphatidylethanolamine were used as substrates. These results are consistent with a plasma membrane-associated phosphatidylinositol-specific phospholipase C from lapine synovial cells in culture.