Toshka A. Abrams

ROS ingestion by RPE cells is turned off by increased protein kinase C activity and by increased calcium

The activation of protein kinase C (PKC) by phorbol myristate acetate (PMA) rapidly inhibits the phagocytosis of rod outer segments (ROS) by cultured rat retinal pigment epithelial (RPE) cells. PMA, at a concentration between 3.3 and 10 nM, blocks ROS ingestion by 50%, but does not inhibit the binding of ROS. The Ca2+ ionophore, A23187, also inhibits ROS phagocytosis, with an IC50 of about 0.5-1.0 microM and interferes with the ability of RPE cells to bind ROS. The effects of both of these drugs are reversible after drug washout. When PMA and A23187 are applied to cells consecutively, the effects are additive. These results suggest either that PMA and A23187, act upon the same proteins in the pathway which controls ROS ingestion, or that A23187 affects phagocytosis at the ROS binding level, while PKC affects steps further along the ingestion path. The effect of this process is to shut down the ingestion of ROS, as is seen during the prolonged feeding of ROS to RPE cells in culture.

The phagocytosis of ROS by RPE cells is not inhibited by mannose-containing ligands

We have examined the ability of mannose and the mannose-rich ligands, mannan and mannosylated BSA, to inhibit the phagocytosis of rod outer segments (ROS) by cultured rat retinal pigment epithelial (RPE) cells. Mannose, at concentrations up to 0.25 M, had no effect on either the binding or the ingestion of ROS. At concentrations above 0.25 M, the cells were rounded and showed detachment from the substrate, and phagocytosis was markedly inhibited. Neither mannan (2 mg ml-1), nor mannosylated BSA(0.8 mg ml-1), affected the phagocytosis of ROS. These results suggest that the phagocytosis of ROS is probably not mediated by a mannose receptor on the surface of the RPE cells.

Integrin αvβ5 is not required for the phagocytosis of photoreceptor outer segments by cultured retinal pigment epithelial cells

Abstract The phagocytosis of photoreceptor outer segments (OS) by the retinal pigment epithelium (RPE) is a receptor mediated process. A key component of this process is the receptor tyrosine kinase, Mer. RPE cells from the RCS rat, which lacks a functional mer gene, and do not express Mer protein, are able to bind OS, but are unable to ingest them, suggesting that both a binding receptor and an ingestion receptor (Mer) are required for phagocytosis to occur. These rats become blind shortly after birth. To date the binding receptor has not been identified. Recent studies, using an SV40 transformed rat RPE cell line, RPE-J, or cultured human RPE cells, have suggested that the receptor for OS binding is the integrin αvβ5. However, the results presented here clearly show that this integrin plays at most a minor role in the phagocytosis of OS by primary cultures of rat RPE cells. OS phagocytosis by normal RPE cells is not affected by a function-blocking antibody to αvβ5 integrin, nor by the integrin-specific blocking peptide GRGDSP. Additionally, RPE-J cells do not express the Mer receptor protein, which has been shown to be obligatory for OS phagocytosis, or RPE65, a specific marker for RPE cells. We suggest that the RPE-J cell line is not a valid model with which to study the complex process of OS phagocytosis.

RPE cells from normal rats do not secrete a factor which enhances the phagocytosis of ROS by dystrophic rat RPE cells

Retinal pigment epithelial (RPE) cells from normal and dystrophic rats were grown separately and in mixed culture for 7 days, without a change of growth medium. Isolated rod outer segments (ROS) were suspended in the conditioned medium from these cells, and were fed to the mixed or pure RPE cell cultures. No increase or decrease in the phagocytosis of ROS by dystrophic or normal RPE cells, respectively, was observed. These results suggest that normal RPE cells do not secrete a diffusible factor(s) which enhances the phagocytosis of ROS by dystrophic RPE cells.

Further Studies on the Phagocytosis of Photoreceptor Outer Segments by Rat Retinal Pigment Epithelial Cells

The shedding of photoreceptor outer segments (OS) and their phagocytosis by the adjacent retinal pigment epithelium (RPE) is a fundamental process which occurs in all vertebrate species studied to date. Numerous studies have showed that OS shedding and phagocytosis is light entrained and follows a circadian rhythm (1, 2). When this process goes awry, such as in the RCS rat (3–5) and possibly in the vitiligo mouse (6, 7) degeneration of the OS soon follows. There is an accumulating body of evidence (8) that the phagocytosis of OS is a receptor mediated process, whereby a specific receptor on the RPE (the phagocytosis receptor) recognizes a ligand on the surface of the shed OS. After binding to the receptor, the shed OS is internalized by the RPE cell, where it undergoes digestion by lysosomal enzymes.