Superoxide inhibits proliferation and phagocytic internalization of photoreceptor outer segments by bovine retinal pigment epithelium in vitro

Abstract

Experiments were performed to investigate the effect of free radical damage on two aspects of retinal pigmented epithelium (RPE) metabolism, namely, proliferation and phagocytosis. Bovine RPE cells were maintained in monolayer cultures, either as passaged (for proliferation and lysosomal activity assays) or primary cultures (for phagocytosis measurements). Free radicals (superoxide anions) were generated by a xanthine oxidase (XO)-hypoxanthine (HX) reaction. Total phagocytosis (binding plus ingestion of rod outer segments (ROS)) was quantitated by radioimmunoassay using a specific anti-opsin antibody and iodinated secondary antibody. In some cases, agents with known or possible protective influences against oxidative damage, i.e., superoxide dismutase (SOD), vitamin E, and basic fibroblast growth factor (bFGF), were tested for their activity in this model system. RPE cell proliferation was inhibited in a HX-XO dose-dependent manner, in the absence of cell toxicity. Modifications of cell morphology were also noticed. Either simultaneous exposure of RPE cells to ROS membranes and HX-XO or pretreatment of ROS membranes with HX-XO prior to their addition to RPE monolayers led to a statistically significant 20-30% decrease in phagocytosis relative to control values. This decrease was essentially observed in the binding phase of phagocytosis, indicating damage to ROS surface molecules as the primary event. Addition of SOD or vitamin E prevented this loss of phagocytic activity, whereas bFGF had no effect. Superoxide radicals did not, however, affect phagocytosis when RPE cells were exposed to them alone, prior to incubation with ROS; nor did they alter a later stage in the phagocytic process, acid phosphatase activity. This tissue culture model represents a convenient system for analyzing free radical damage in different aspects of RPE-photoreceptor behavior and may be useful in studying this phenomenon in several retinal disorders.