Lucigenin-dependent chemiluminescence in articular chondrocytes

Abstract

We were recently able to measure intracellular levels of hydrogen peroxide within normal articular chondrocytes using the trapped indicator 2′,7′-dichlorofluorescein diacetate. Further studies have shown that stimulated chondrocytes produce luminol-dependent chemiluminescence, suggesting that these cells produce hydrogen peroxide and singletoxygen. In the present study, we have investigated the lucigenin-dependent chemiluminescence response in normal articular chondrocytes. Chondrocytes either in suspension or adhered to cover slips showed lucigenin-dependent chemiluminescence. There was a dose-dependent increase in chemiluminescence response when chondrocytes were incubated with soluble stimuli like phorbolmyristate-acetate, concanavalin A, and f-met-leu-phe. Catalase and the metabolic inhibitor, sodium azide, which inhibits the enzyme myeloperoxidase, had no inhibitory effect on lucigenin-dependent chemiluminescence production. Only the antioxidant, superoxide dismutase, prevented lucigenin-dependent chemiluminescence, indicating that this assay measures the production of superoxide anions by chondrocytes. We confirmed that chondrocytes release superoxide radicals using the biochemical assay of ferricytochrome c reduction. Since cartilage tissue is semitransparent, we were able to measure chemiluminescence response in live cartilage tissue, showing that chondrocytes which are embedded within the matrix can also generate superoxide anion radicals. Reactive oxygen intermediates have been shown to play a significant role in the degradation of matrix in arthritis. Our previous and present studies suggest that oxygen radiccals produced by chondrocytes may be an important mechanism by which chondrocytes induce cartilage matrix degradation.