Inhibition of neutrophil-mediated chondrocyte cytotoxicity by nitric oxide (NO) generated by articular chondrocytes.

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Nitric oxide (NO) was identified as an active product of the oxidative cleavage of L-arginine. NO is generated by endothelial cells, macrophages and neutrophils and the higher concentration of nitrite, which is the stable endproduct of NO,is detected in the rheumatoid synovial fluid and serum. It is recently reported that NO inhibits neutrophil superoxide anion production and, in this way, associated with tissue degradation in inflammatory process. We have reported that articular chondrocytes were damaged by hydrogen peroxide generated by activated neutrophils. We have studied, in this investigation, the effect of NO on neutrophil-mediated chondrocyte cytotoxicity. Primary monolayr culture of chondrocytes was obtained from bovine articular cartilage by collagenase digestion and neutrophils were separated from healthy human doners. Chondrocyte cytotoxicity was measured by chromium-51 (^<51>Cr) release from the prelabeled chondrocytes in the presence of neutrophils. The concentration of NO was measured as nitrite concentration by Grisse reaction. Articular chondrocytes synthesized NO by the stimulation of interleukin (IL-1alpha) and TNF-alpha for 24h in a dose-dependent fashion. In the presence of 100 muM of L-arginine, 200 U/ml of IL-1alpha yielded the significant generation (P<0.01) of NO (2muM nitrite equivalent) from chondrocytes. Neutrophils increased ^<51>Cr-release from articular chondrocytes, and this chondrocyte cytotoxicity was significantly (p<0.05) reversed by addition of 100 muM of L-arginine in the neutrophil-chondrocyte co-cultures In contrast, D-arginine failed to inhibit the neutrophil mediated chondrocyte cytotoxicity. These result suggested that NO generated from articular chondrocytes have a protective effect against the oxidative stress in inflammatory cartilage.