Tetracyclines inhibit nitrosothiol production by cytokine-stimulated osteoarthritic synovial cells

Objective and design: To evaluate the capacity of doxycycline and minocycline to inhibit NO production and N-nitrosation reactions in vitro. Methods: Synovial cells obtained from 6 patients with osteoarthritic joint disease were incubated for 24 hours with (i) or without (ii) IL-1 beta (1ng/ml), TNF-alpha (500 pg/ml), IFN-gamma (10(4) U/ml) plus minocycline or doxycycline (10(-4) to 10(-6) M), diclofenac (10(-5) M), or cortisol (10(-5) M). Nitrosothiols were determined by fluorimetry, nitrite by the Griess reaction, nitrate by a spectrophotometric assay using oxidation by nitrate reductase and iNOS by immunoblotting. Results: After 24 hours of stimulation, the level of NO production was much higher than that in untreated cells: about 5.5 times higher for nitrosothiols, 5.2 times higher for nitrate and about 3.5 times higher for nitrite, Doxycycline and minocycline induced a dose-dependent decrease in the production of nitrosothiols, nitrate and nitrite, and inhibited the synthesis of the iNOS protein. Doxycycline and minocycline inhibited the N-nitrosation reaction of DAN effectively, with IC50 values close to 100 muM. Diclofenac and cortisol had no effect. Conclusion: This study provides new information on the mechanism by which tetracyclines exert anti-inflammatory effects, via inhibiting nitrosothiols.