The role of peroxynitrite in cyclooxygenase-2 expression of rheumatoid synovium.

OBJECTIVE: Reactive oxygen intermediates play an important role in the inflammatory processes of rheumatoid arthritis. Cyclooxygenase-2 is an inducible form of an enzyme involved in prostanoid biosynthesis. This study linked peroxynitrite (ONOO-) to the signaling pathways that induce COX-2. RESULTS: Exposure of rheumatoid synovial cells to peroxynitrite resulted in COX-2 protein expression in a dose-dependent manner. RT-PCR analysis also demonstrated that COX-2 mRNA was induced in peroxynitrite-treated rheumatoid synovial cells. Dexamethasone markedly inhibited this peroxynitrite-mediated COX-2 expression at therapeutic concentrations. CONCLUSION: This study demonstrates that oxidant stress is an important inducer of COX-2 in rheumatoid synovium. This induction may contribute to the amplification of prostanoids in the rheumatoid inflammatory process.

Atypical mycobacteriosis in two patients with rheumatoid arthritis.

Abstract We report two cases of rheumatoid arthritis (RA) with atypical mycobacteriosis. Opportunistic infections are critical complications for rheumatic diseases. The use of steroids or immunosuppressants may increase the risk of opportunistic infections. However, these reports are rare in that they demonstrate atypical mycobacterial infections as complications of RA, even though no immunosuppressive agents were used. We discuss the characteristics of atypical mycobacterial infections of the lung in RA.

Regulation of rheumatoid synoviocyte proliferation by endogenous p53 induction.

The p53 tumour suppressor protein protects cells from tumorigenic alterations by inducing either cell growth arrest or apoptosis. In the present study, we investigated the role of endogenous p53 expressed in rheumatoid arthritis synovial fibroblasts which show transformed-appearing phenotypes. Type B synovial cells (fibroblast-like synovial cells) were exposed to a proteasome inhibitor, carbobenzoxyl-leucinyl-leucinyl-leucinal (MG-132). During this process, the expressions of p53 and p21 were examined by Western blot. Cell cycle analysis of the synovial cells was determined by DNA staining using propidium iodide (PI). Inhibition of proteasome resulted in the accumulation of p53 which was followed by an increase in the amount of a cyclin-dependent kinase (CDK)-inhibitor, p21. As a consequence, the retinoblastoma gene product, Rb, remained in the hypophosphorylated state, thus preventing PDGF-stimulated synovial cells from progressing into S-phase. This study shows that endogenous p53, which is inducible in rheumatoid synovial cells, is functionally active based on the findings that its expression blocks the G1/S transition by inhibiting the CDK-mediated phosphorylation of Rb via p21 induction. Thus the induction of p53 using proteasome inhibitor may provide a new approach in the treatment of RA.

Expression of membrane-type 1 matrix metalloproteinase in rheumatoid synovial cells.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is thought to be a putative regulator of pro-gelatinase A (MMP-2) in the rheumatoid synovium. In this study, we examined the effects of IL-1beta, one of the inflammatory cytokines, on the expression of MT1-MMP and the activation of pro-MMP-2 using rheumatoid synovial cells. We also studied the effects of KE-298 (2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid), a new disease-modifying anti-rheumatic drug (DMARD), on MT1-MMP expression of rheumatoid synovial cells. Type B synovial cells (fibroblast-like synovial cells) were cultured with KE-298 (25-100 microg/ml) in the presence of IL-1beta for 48 h. Activation of pro-MMP-2 secreted from synovial cells was analysed by gelatin zymography. Reverse transcription-polymerase chain reaction (RT-PCR) methods were used to detect MT1-MMP mRNA. MT1-MMP protein expression on synovial cells was examined by anti-MT1-MMP immunoblot. An active form of MMP-2 was demonstrated in the culture media conditioned by IL-1beta-stimulated synovial cells. In addition, MT1-MMP mRNA and protein expression of rheumatoid synovial cells were increased by IL-1beta treatment. KE-298 blocked this IL-1beta-induced pro-MMP-2 activation and MT1-MMP expression, but did not affect IL-1beta-induced tissue inhibitor of metalloproteinase-2 (TIMP-2) secretion from rheumatoid synovial cells. These findings indicate that activation of rheumatoid synovial cells by IL-1beta results in the induction of MT1-MMP expression. Given that MT1-MMP promotes matrix degradation by activating pro-MMP-2, these results suggest a novel mechanism whereby cytokine may contribute to articular destruction in rheumatoid arthritis (RA). KE-298 may prevent this process by down-regulating MT1-MMP expression.

Nitric oxide protects cultured rheumatoid synovial cells from Fas-induced apoptosis by inhibiting caspase-3.

Nitric oxide (NO) is elevated in the synovial fluids and sera of patients with rheumatoid arthritis (RA) and is thought to be an important proinflammatory mediator in the rheumatoid synovium. To test the hypothesis that NO might modulate the apoptosis-inducing signal pathway, we investigated the effects of NO on rheumatoid synovial-cell apoptosis induced by Fas ligation with anti-Fas antibody. Pretreatment of synovial cells with the NO donor S-nitro-N-acetylpenicillamine (SNAP) prevented the Fas-mediated induction of apoptosis. The activation of caspase-3 was required to mediate Fas-induced synovial cell apoptosis. The NO donor SNAP inhibited Fas-induced caspase-3 activation in rheumatoid synovial cells. However, NO did not interrupt Fas-induced caspase-8 cleavage or subsequent cytochrome c release into the cytosol in rheumatoid synovial cells. These data indicate that NO prevents apoptosis in rheumatoid synovial cells by directly inhibiting caspase-3 activation. Thus, we propose that NO interferes with cell death signal transduction and may contribute to rheumatoid synovial cell proliferation by inhibiting induction of apoptosis.

A novel role for interleukin-18 in human natural killer cell death: high serum levels and low natural killer cell numbers in patients with systemic autoimmune diseases.

OBJECTIVE: Patients with systemic autoimmune diseases have been reported to have reduced numbers of peripheral blood natural killer (NK) cells compared with healthy subjects. The ability of selected cytokines to trigger NK cell death prompted us to compare the levels of peripheral blood cytokines with the numbers of NK cells in patients with various systemic autoimmune diseases. METHODS: We used enzyme-linked immunosorbent assays to measure the concentration of selected cytokines (interleukin-18 [IL-18], IL-15, IL-12, IL-2, interferon-gamma [IFNgamma], and tumor necrosis factor alpha [TNFalpha]) in sera from 58 patients with systemic autoimmune diseases and 33 healthy controls. The absolute number of T cells and NK cells in the peripheral blood was measured in parallel using flow cytometry. The ability of selected cytokines to induce NK cell death was then measured using 3,3'-dihexyloxacarbocyanine iodide dye, propidium iodide staining, and caspase 3 activity. RESULTS: Levels of IL-18, IL-15, IFNgamma, and TNFalpha were elevated in sera from patients with systemic autoimmune diseases compared with normal controls. The percentage of NK cells and natural killer T cells were significantly decreased in the peripheral blood of patients with systemic autoimmune diseases compared with normal controls. Serum concentrations of IL-18, IL-15, and TNFalpha were inversely related to the number of NK cells in both patients and healthy controls. The combination of IL-18 and IL-15 or IL-18 and IL-12 induced NK cell death in vitro. The combination of IL-18 and IL-15 or IL-18 and IL-12 enhanced IFNgamma and TNFalpha production by NK cells in vitro. Cytokine-induced NK cell death is caspase-dependent and is partially blocked by neutralizing antibodies against TNFalpha. CONCLUSION: High levels of IL-18 and IL-15 are associated with the decreased number of NK cells that is observed in patients with systemic autoimmune diseases.

Impaired degradation of serum amyloid A (SAA) protein by cytokine-stimulated monocytes.

Secondary amyloidosis (AA amyloidosis) is a systemic disease characterized by the extracellular tissue deposition of insoluble amyloid A (AA) protein. Aberrant metabolism of serum amyloid A (SAA) by macrophages is only one of many putative mechanisms which may be important in AA amyloidogenesis. In this study, we investigated the effects of cytokines on human monocyte-mediated SAA proteolysis. Human peripheral blood mononuclear cells (PBMC) or CD14(+) monocytes were cultured with SAA, and the culture supernatants were then subjected to anti-SAA immunoblot. CD14(+) monocytes degraded SAA completely. Whereas, when CD14(+) monocytes were pretreated with IL-1 beta or IFN-gamma, increasing amounts of SAA-related derivatives were detected in culture supernatants. These findings suggest that activation of monocytes by IL-1 beta or IFN-gamma hampers the proteolysis of a precursor protein and leads to a partial degradation of SAA. This down-regulated proteolysis of SAA protein by cytokine-stimulated monocytes may play a role in the mechanism of AA amyloid formation as well as its removal.

Effects of nitric oxide on matrix metalloproteinase-2 production by rheumatoid synovial cells.

Nitric oxide (NO) is a multifunctional messenger molecule generated from L-arginine by a family of enzymes, including nitric oxide synthase (NOS). This study was performed to examine whether NO modulates the production of matrix metalloproteinases (MMPs), which degrade all components of extracellular matrix (ECM), in rheumatoid synovial cells. We investigated the effects of exogenously generated NO by a NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the MMPs production by rheumatoid synovial cells. Culture media conditioned by SNAP-treated synovial cells were examined by gelatin zymography and immunoblot analysis. Incubation of synovial cells with SNAP resulted in gelatinase A production in a dose-dependent fashion. Furthermore, RT-PCR analysis demonstrated that MMP-2 mRNA expression was induced in SNAP-treated synovial cells. In contrast, SNAP did not influence the production of TIMP-1 and TIMP-2, which preferentially inhibit MMP-2, by rheumatoid synovial cells. Our data indicate that NO could modulate MMP production by rheumatoid synovial cells and therefore contribute to ECM degradation of articular components in RA.

Successful plasmapheresis in alveolar hemorrhage associated with systemic lupus erythematosus.

Abstract The case of an 18-year-old Japanese man with systemic lupus erythematosus (SLE) complicated by alveolar hemorrhage is described. The patient presented with fever, butterfly rash, and polyarthralgia, and was diagnosed with SLE. He suddenly developed alveolar hemorrhage during steroid pulse therapy. Treatment with plasmapheresis was initiated, with prompt clearing of the chest radiograph. This experience suggests that the prompt initiation of plasmapheresis should be considered for SLE patients with life-threating alveolar hemorrhage resistant to conventional immunosuppressive therapies.

Studies on cardiac ingredients of plants. XIII: Chemical modification of gitoxin to cardiotonic compounds without vascular effect.

Nitrated gitoxins (4) and bufotoxin homologues with various lengths of alkyl chain at C-3 of the steroid nucleus (10) were prepared from gitoxin (1). The pharmacological activities of the resulting compounds (4 and 10) were evaluated by measurement of inhibitory effect on NA+, K(+)-adenosine triphosphatase (ATPase) prepared from dog kidney, positive inotropic effect (PIE) on isolated guinea-pig papillary muscle preparations, and the effect on smooth muscle using the mesenteric artery from spontaneously hypertensive rats. Most of the compounds showed a smaller contractile effect on the arterial muscle. Among these compounds, gitoxin 3"-nitrate (4g) exhibited the most desirable biological activities, such as PIE comparable to that of 1, 1.25 times wider concentration-dependent range than 1, and lack of contractile activity on vascular muscle.