Regulation by reactive oxygen species of interleukin-1β, nitric oxide and prostaglandin E2 production by human chondrocytes

Abstract

Objectives To determine the effects of two drugs, N-monomethyl-L-arginine (L-NMMA) and N-acetylcysteine (NAC), on interleukin-1β (IL-1β), nitric oxide (NO) and prostaglandin E2 (PGE2) production by human chondrocytes. The effect of aceclofenac (ACECLO), a non-steroidal antiinflammatory drug (NSAID), was also examined.Methods Human chondrocytes were enzymatically isolated from osteoarthritic knee cartilage and then maintained in culture in suspension for 48h in the absence or in the presence of lipopolysaccharide (LPS) (10μg/ml), L-NMMA (0.5mM), NAC (1mM) or ACECLO (6·10−6M). IL-1β and PGE2 productions were quantified by specific immunoassays. Nitrite was measured in the culture supernatants by a spectrophotometric method based upon the Griess reaction. Cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS) and IL-1β gene expressions were quantified by transcription of mRNA followed by real time and quantitative polymerase chain reaction. COX-2 protein expression was analysed by Western blot.Results LPS markedly increased the expression of IL-1β, iNOS and COX-2 genes. In parallel, NO2 and PGE2 amounts found in the culture supernatants were significantly enhanced whereas IL-1β was immunologically undetectable. The addition of L-NMMA (0.5mM) fully blocked LPS-induced NO production but greatly increased PGE2 production, suggesting a negative effect of NO on PGE2 synthesis. Inversely, NO production was stimulated by NAC while PGE2 production was not affected. Interestingly, NAC increased the IL-1β and iNOS mRNA levels but did not significantly modify COX-2 mRNA expression. L-NMMA did not significantly affect the expression of IL-1β, iNOS and COX-2. The amount of COX-2 protein did not change in the presence of the antioxidants. Finally, ACECLO fully blocked the production of PGE2 by chondrocytes without affecting the levels of COX-2 mRNA.Conclusions The stimulation of IL-1β, NO and PGE2 production by LPS is differentially controlled by reactive oxygen species (ROS). In fact, L-NMMA and NAC have different mechanisms of action on the regulation of NO and PGE2 productions. L-NMMA fully inhibits NO but increases PGE2 production whereas NAC up-regulates NO but does not modify PGE2 synthesis. The stimulating effect of L-NMMA on PGE2 production is not controlled at the transcriptional level. These findings suggest that antioxidant therapy could have different effects according to the oxygen radical species targeted. Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.