Characterization and application of an in vitro detection system for studying the binding and phagocytosis of rod outer segments by retinal pigment epithelial cells

Abstract

Direct and indirect radioactivity and fluorescent assays have been developed to study the interaction of rod outer segments (ROS) with retinal pigment epithelial (RPE) cells. In the direct assays ROS labelled with 125I or fluorescein isothiocyanate (FITC) have been used to measure total phagocytosis, i.e. surface binding and ingestion. In the indirect assays RPE cells were first treated with unlabelled ROS or biotinylated ROS and subsequently probed with [ 125I]Rho 4D2 antirhodopsin antibody or [ 125I]streptavidin for radioactivity measurements or with the Rho 4D2 antibody and FITC-goat anti-mouse Ig or FITC-streptavidin for fluorescent counting. In these indirect methods the number of surface bound ROS were distinguished from the number of ingested ROS by comparative labelling of non-permeabilized and permeabilized ROS-treated RPE cells. Using these assays, we have studied the binding and ingestion of bovine ROS with cultured bovine RPE cells. As in the case of newborn cultured rat RPE cells [Hall and Abrams 1987 Exp. Eye Res., 45, 907–922], binding and ingestion of bovine ROS by bovine RPE cells was saturable with respect to ROS concentration and time. At 37°C ROS binding reached a saturating concentration at 1 × 10 7 ROS per well; the number of bovine ROS ingested by bovine RPE cells, however, was less than the number of rat ROS ingested by rat RPE cells. When 1 × 10 7 ROS per well was used, maximal surface binding of bovine ROS to bovine RPE cells was obtained after 2–3 hr, whereas after an initial delay, ingestion rapidly increased to a maximum at 1–2 hr. In contrast to cultured newborn rat RPE cells, however, little, if any binding of ROS to bovine RPE cells was observed at 23°C or 17°C. Using direct binding assays and competitive inhibition assays, we have also shown that bovine RPE cells recognize rat ROS to the same degree as bovine ROS, and rat RPE cells bind bovine ROS in addition to rat ROS. This suggests that the ROS ligand and RPE receptor have conserved binding domains for these mammalian species. The assays described here should prove useful for future studies on the identification and characterization of components involved in ROS-RPE cell interaction during phagocytosis.