Influence of oxygen tension on nitric oxide and prostaglandin E2 synthesis by bovine chondrocytes.

OBJECTIVES: To determine the in vitro effects of oxygen tension on interleukin (IL)-1beta induced nitric oxide (*NO) and prostaglandin E(2) (PGE(2)) production by bovine chondrocytes. DESIGN: Enzymatically isolated bovine chondrocytes were cultured for different periods in suspension in 21 (atmospheric), 5 or 1% (low) oxygen tension and in the absence or in the presence of increased amounts (0.01 to 1nM) of IL-1beta. Nitrite and nitrate concentrations in the culture supernatants were determined by a spectrophotometric method based upon the Griess reaction. PGE(2) production was quantified by a specific radioimmunoassay (RIA). Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) mRNA steady state levels were also quantified by real-time polymerase chain reaction (PCR). RESULTS: In the absence of IL-1beta, ()NO production remained stable whatever the oxygen tension used. IL-1beta dose-dependently increased *NO production in both atmospheric and low oxygen conditions but the effect was more pronounced in low (1 and 5%) than in atmospheric (21%) oxygen tension (P<0.001). Under low and atmospheric oxygen tension, iNOS gene expression was increased by IL-1beta, but to a lesser extent in 21% than in 1 or 5% oxygen (P<0.01). In the basal condition, bovine chondrocytes spontaneously produced PGE(2) whatever the oxygen tension used. At 21% oxygen, IL-1beta dose-dependently increased PGE(2) production while no significant effect was observed at 1 or 5% oxygen. COX-2 gene expression was significantly upregulated by IL-1beta in both low and atmospheric oxygen tension. No significant difference between oxygen tension conditions was observed. CONCLUSIONS: This study demonstrates that a hypoxic environment fully blocks COX-2 activity but favours iNOS gene expression in chondrocytes culture. These findings indicate that O(2) tension modulates cellular behaviour in culture and supports the concept of chondrocyte culture in low oxygen tension to reproduce in vitro the life conditions of chondrocytes.

Reactive oxygen species downregulate the expression of pro-inflammatory genes by human chondrocytes.

OBJECTIVES: To determine the regulatory effects of reactive oxygen species (ROS) on the expression by human osteoarthritic chondrocytes of interleukin (IL)-1beta, -6 and -8, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene in response to interleukin (IL)-1beta or lipopolysaccharide (LPS). METHODS: Human chondrocytes in monolayer culture were incubated for 3 h with ROS generating molecules such as S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 100 microM), 3-morpholinosydnonimine (SIN-1, 100 microM), with chemically synthesised peroxynitrite (ONOO-, 10 microM) or hydrogen peroxide (H2O2, 100 microM). After treatment by ROS, chondrocytes were washed and then cultured for the next 24 h with or without lipopolysaccharide LPS (10 microg/ml) or IL-1beta (1.10(-11) M). IL-1beta, IL-6, IL-8, iNOS and COX-2 gene expression was analysed by real time and quantitative RT PCR. IL-6, IL-8 and prostaglandin (PG) E2 productions were assayed by specific immunoassays. Nitrite was measured in the culture supernatants by the Griess procedure. RESULTS: LPS and IL-1beta stimulated IL-1beta, IL-6, IL-8, iNOS and COX-2 gene expression. SNAP significantly downregulated LPS induced overall gene expressions, whereas SIN-1 had no effect. ONOO- inhibited iNOS and COX-2 gene expression but not that of the cytokine genes. When chondrocytes were incubated with IL-1beta, SIN-1 and ONOO dramatically decreased all gene expressions while SNAP was inefficient. H2O2 treatment inhibited both LPS and IL-1beta induced gene expressions. CONCLUSIONS: These data provide an evidence that ROS may have anti-inflammatory properties by depressing inflammatory gene expression. Further, we demonstrate that ROS effects are dependent on the nature of radical species and the signalling pathway that is activated. These findings should be taken into consideration for the management of antioxidant therapy in treatment of inflammatory joint diseases.

Evaluation of classical NSAIDs and COX-2 selective inhibitors on purified ovine enzymes and human whole blood.

Cyclooxygenase is the key enzyme in the biosynthesis of prostanoids, biologically active substances involved in several physiological processes and also in pathological conditions such as inflammation. It has been well known for 10 years that this enzyme exists under two forms: a constitutive (COX-1) and an inducible form (COX-2). Both enzymes are sensitive to inhibition by conventional non-steroidal anti-inflammatory drugs (NSAIDs). Observations were made that COX-1 was mainly involved in homeostatic processes, while the COX-2 expression was associated with pathological conditions leading to the development of COX-2 selective inhibitors. Several methods have been reported for the evaluation of the COX-1 and COX-2 inhibitory potency and selectivity of conventional or COX-2 selective NSAIDs. In this study, we evaluated the COXs inhibitory profile of both conventional NSAIDs and COX-2 selective inhibitors using two different in vitro methods: the first test was performed using purified enzymes while the second method consisted of a whole blood assay. The results obtained with reference drugs in these two assays will be discussed and compared in this article.