Cellular and molecular studies of retinal pigment epithelium phagocytosis

Retinal pigment epithelium (RPE) cells lose epithelial phenotype and are transformed into a fibroblast-like morphology after exposure to vitreous. The current studies are designed to investigate RPE phagocytic ability after vitreous transformation and the involvement of two phagocytic candidate receptors: the mannose receptor and the alphavbeta5 integrin. A phagocytic assay indicates that phagocytosis of photoreceptor outer segments (ROS) is significantly decreased in the transformed RPE cells. The molecular basis of this phenomenon has been studied and the mannose receptor has been examined first. A peptide antibody against porcine mannose receptor has been generated and characterized which specifically recognizes macrophage mannose receptor from porcine lung in immunoblots. However, this antibody does not recognize a protein around 175 kDa from cultured porcine RPE cells, which is the molecular weight of the mannose receptor. An antiserum against human mannose receptor shows the same results, but does recognize the 175 kDa protein associated with RPE apical membrane proteins freshly isolated from the porcine retina. Studies using RT-PCR show a 248 bp mannose receptor PCR product from both normal and transformed RPE. However, the mRNA level is too low to be accurately quantitated. A ligand-binding assay with mannose-BSA indicates that a mannose-dependent recognition system exists in the cultured RPE and that some of the mannose binding proteins on the transformed cells are decreased. The other phagocytic receptor, alphavbeta5 integrin, has been examined by immunoprecipitation coupled with immunoblotting and immunofluorescent localization and found to be decreased and redistributed in the transformed cells. Furthermore, a functional blocking antibody to alphavbeta5 significantly inhibits ROS phagocytosis in both normal and transformed RPE with a residual phagocytic activity remaining at saturating antibody concentrations. These data indicate that the decrease in ROS phagocytosis observed in the transformed RPE cultures is due, at least in part, to a corresponding down regulation of alphavbeta5 integrin. The fact that the functional blocking antibody cannot completely inhibit phagocytic activity suggests that other phagocytic receptor pathways are involved in ROS uptake by RPE cells. The mannose receptor is not likely to mediate porcine RPE phagocytosis in vitro, however, it may play a role in retinal phagocytosis in vivo.