A novel retinal pigment epithelial protein suppresses neutrophil superoxide generation. I. Characterization of the suppressive factor

Abstract

In acute intraocular inflammation, neutrophils release a variety of agents, that are potentially toxic to the surrounding tissues. The reactive oxygen metabolites, including superoxide are among these injurious agents. In the present study, retinal pigment epithelial (RPE) cells were found to inhibit by 70% to 80% the production of superoxide by neutrophils that were stimulated either by the receptor-coupled activator, N-formyl-methionyl-leucyl-phenylalanine, or by the non-receptor-coupled activator, phorbol myristate acetate. The inhibition is effective with a relatively small number of RPE cells (0.65 x 10(5)) mixed in a large pool of neutrophils (7.6 x 10(5)). The transduction of this effect does not require a neutrophil-RPE cell surface contact since the RPE culture supernatants also effectively inhibit the superoxide production. This protein is secreted specifically by cultured and noncultivated intact RPE cells, but not by fibroblasts, corneal epithelial cells or intact choroidal tissues. The protein is not cytotoxic to the neutrophils, and the inhibitory effect occurs in a dose-dependent manner up to the concentration tested. This factor is unrelated to either transforming growth factor-beta, or transferrin. There was no evidence of RPE scavenging of superoxide generated enzymatically by hypoxanthine-xanthine oxidase system, indicating that this factor does not have superoxide dismutase activity. When RPE cells were preincubated with 10 micrograms ml-1 cycloheximide, 60% of the activity was lost, suggesting that a de novo protein synthesis is required for the activity and that the protein is a significant steady-state product of RPE cells. Incubation of the released RPP with thermolysin (10 micrograms ml-1 15 min, 37 degrees C) also eliminated the activity. The degradation of activity by protease and inhibition of RPP activity by cycloheximide, therefore, confirmed the protein nature of the suppressive factor. This protein from RPE cells appears to act directly on the neutrophils, reducing the release of oxygen metabolites during activation, and thus limiting tissue injury during inflammation.