Masako Kita

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.

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‐1β or IFN‐γ, increasing amounts of SAA‐related derivatives were detected in culture supernatants. These findings suggest that activation of monocytes by IL‐1β or IFN‐γ 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.

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‐1β, 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 µg/ml) in the presence of IL‐1β 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‐1β‐stimulated synovial cells. In addition, MT1‐MMP mRNA and protein expression of rheumatoid synovial cells were increased by IL‐1β treatment. KE‐298 blocked this IL‐1β‐induced pro‐MMP‐2 activation and MT1‐MMP expression, but did not affect IL‐1β‐induced tissue inhibitor of metalloproteinase‐2 (TIMP‐2) secretion from rheumatoid synovial cells. These findings indicate that activation of rheumatoid synovial cells by IL‐1β 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.

Staphylococcal enterotoxin B‐specific adhesion of murine splenic T cells to a human endothelial cell line

The presence of a putative autoantigen of autoimmune disorder in a target organ may cause accumulation of specific T cells in the inflammatory region. One of the mechanisms of such accumulation involves the migration of specific‐circulating T cells through the endothelial cells into the target lesion. The presence of only a few specific T cells responsive to a putative autoantigen has hampered the investigation of specific migration of circulating T cells to the target organ. We used a superantigen to investigate specific T‐cell adhesion to endothelial cells, because it stimulates a large proportion of T cells with particular Vβ elements and adhesion of T cells to the endothelium is a vital step in the migration process. Adhesion of murine T cells to the human endothelial cell line, EA.hy926, was specifically increased in the presence of staphylococcal enterotoxin B (SEB). The increase was interferon‐γ (IFN‐γ)‐dependent, and consisted mainly of CD4+ T cells. Vβ8.1,2+ T cells preferentially adhered to endothelial cells in the presence of SEB compared with Vβ6+ T cells. Pretreatment of endothelial cells with SEB increased the adherence of Vβ8.1,2+ T cells, while anti‐human leucocyte antigen (HLA)‐DR and ‐DQ antibodies inhibited the increased adherence of Vβ8.1,2+ T cells. Our results demonstrate that increased T‐cell adhesion to endothelial cells is SEB specific, and that the specificity is dependent on major histocompatibility complex (MHC) class II molecules expressed on endothelial cells and on the recognition of the SEB–MHC class II complex by Vβ8.1,2+ T cells.