Mertk Is Directly Involved In Ingestion Of Photoreceptor Outer Segments During RPE Phagocytosis

Abstract

Abstract: : Purpose:To investigate whether Mertk, which complements the phagocytic defect of cultured RCS retinal pigment epithelial (RPE) cells, is directly involved in ingestion of photoreceptor outer segments (OS). Methods:Primary rat RPE cultures were incubated with purified OS for 1, 2 or 3 hrs and the unbound OS were removed. Localization of the remaining OS and Mertk was examined by double immunolabeling with an anti-rhodopsin monoclonal antibody and an anti-Mertk polyclonal antiserum. Mertk phosphorylation during RPE phagocytosis was also studied by immunoprecipitation and subsequent immunoblotting. A chimeric protein composed of the ectodomain of Mertk fused to the Fc segment of IgG (Mer-Fc) was tested for its ability to inhibit phagocytosis of OS. Results:Mertk progressively co-localized with OS during RPE phagocytosis; a small amount of co-localization was observed after 1 hr, with nearly complete co-localization after 3 hrs. Preliminary studies indicated that phosphorylation of Mertk increased after 2 hrs, as compared to a control without OS. Mer-Fc did not affect OS binding, but diminished OS ingestion by 70%. Conclusion:The time course of Mertk phosphorylation and co-localization with OS suggests that accumulation of Mertk around OS is necessary for ingestion. Inhibition of OS ingestion by Mer-Fc indicates that interaction of the Mertk ectodomain with a factor on the outside of the RPE cell triggers OS ingestion. Support: grants from the NIH, the Foundation Fighting Blindness, the Ruth and Milton Steinbach Fund, the Karl Kirchgessner Foundation, That Man May See, Inc., the Macula Vision Research Foundation and Research to Prevent Blindness.

Keywords: 513 phagocytosis and killing • 517 photoreceptors • 567 retinal pigment epithelium