Oxidant activity of rabbit synoviocytes (HIG-82) demonstrated by oxymetry and ethylene production.

Abstract

We are interested in a possible role of synoviocytes in the ROS production implicated in osteoarthritis, therefore we studied the response of a rabbit synoviocyte cell line (HIG-82) to variable oxygen tensions and the oxidant activity of these cells in response to stimuli. Synoviocytes were cultured at 5 and 21 % O2, their O2 consumption (cellular respiration, monitored with Clark electrode) was measured at 21% O2 and after anoxia, before and after stimulation with phorbol myristate acetate (PMA), and their oxidant response to PMA stimulation was quantified by measuring ethylene (gas chromatography) released when the substrate, alpha-keto-gamma-methylbutyric acid, is oxidised by the ROS produced by the cells. Cell growth was faster at 21 % O2 than at 5% O2, and microscopic observation revealed 2 cell populations: a few small round cells in suspension and many adherent cells. By oxymetry, we observed that a 106 synoviocytes suspension in 2 ml completely consumed O2 within 15 min, that anoxia (7 min) slightly slowed the respiration rate down and that PMA stimulation increased O2 consumption (150 % increase). The oxidant activity (ethylene production) of the cells was stimulated by PMA in a dose-dependent manner (10-9 to 10-7M) but the cell response was highly variable (from 150 to 1500 % increase) and was largely reduced by diphenyliodonium, an inhibitor of NADPH-oxidase and NO-synthase. The capacity to produce free radical species was confirmed for the small round cells by detection of an electron paramagnetic resonance (EPR) signal after stimulation. These results thus demonstrate a sensibility to O2 and an oxidant activity of synoviocytes at least related to ROS production by NADPH-oxidase activity