EVALUATION OF OXIDATIVE PROCESSES IN HUMAN PIGMENT EPITHELIAL-CELLS ASSOCIATED WITH RETINAL OUTER SEGMENT PHAGOCYTOSIS

To investigate the nature of the oxidative event that occurs during ph agocytosis of retinal outer segments (ROS) by cultured human retinal p igment epithelial (RPE) cells, cells were incubated with isolated bovi ne ROS labeled with either the fluorescent probe carboxy-SNAFL-2 or th e nonfluorescent, oxidizable probe 2',7'-dichlorodihydrofluorescein (H 2DCF). The increase in fluorescence following phagocytosis was measure d by a flow cytometer. Other measurements included: oxygen consumption using a Clark-type oxygen electrode, extracellular superoxide release by superoxide dismutase inhibitable lucigenin chemiluminescence, intr acellular hydrogen peroxide (H2O2) production, and the effect of catal ase inhibition on cellular thiobarbituric acid-reactive substances (TB ARS) caused by phagocytosis. The activities of the enzymes NADPH oxida se and palmitoyl-CoA oxidase were also measured. H2DCF attached to bov ine ROS was oxidized during phagocytosis with a time course suggesting oxidation subsequent to ROS uptake. Measurements of oxygen consumptio n showed a time-dependent increase of 10%, 4 h after ROS feeding, attr ibutable to a doubling of the cyanide-resistant oxygen consumption. In tracellular H2O2 production also doubled 4 h after ROS phagocytosis. R OS uptake by RPE cells produced no significant extracellular superoxid e, while extracellular superoxide production was readily demonstrated in a control macrophage cell line. Enzyme activity measurements showed that incubation of RPE cells with ROS doubled catalase activity witho ut affecting superoxide dismutase or glutathione peroxidase activities . Inhibition of catalase during ROS uptake increased TBARS by 66%. Oth er enzyme activity measurements showed that human RPE cells possess bo th NADPH oxidase and palmitoyl-Coh oxidase activities. We conclude tha t ROS phagocytosis subjects RPE cells to an oxidative event on the sam e order of magnitude as measured in a macrophage. The event is not an extracellular macrophage-type respiratory burst and may be due to intr acellular H2O2 resulting from an NADPH oxidase in the phagosome or fro m beta-oxidation of ROS lipids in peroxisomes. Irrespective of case, t he enzyme catalase appears to be essential in protecting the RPE cell against reactive oxygen species produced during phagocytosis. (C) 1994 Academic Press, Inc.