CD36 Specific Effects of Oxidized Lipids on OS Phagocytosis by the Retinal Pigment Epithelium

Abstract

Abstract: : Purpose: To examine the effect of oxidized phospholipids that serve as specific ligands for CD36 on phagocytosis of photoreceptor outer segment fragments (OS) by the retinal pigment epithelium (RPE). Methods: RPE cell lines and primary RPE prepared from 12 to 16 day old CD36 knockout mice and of wild-type mice of the same genetic background were grown in culture. Specific oxidized phosphatidylcholine species recently identified as high affinity ligands for CD36 (J. Biol. Chem., 277:38517-23 (2002)) were synthesized. Oxidized low densitiy lipoprotein (oxLDL) was prepared exposing LDL to copper in ambient air. RPE cells received isolated OS in the presence of oxidized lipids (or non-oxidized controls). Binding and internalization kinetics were determined using fluorescence scanning. Results: Incubation of RPE with oxLDL, a multivalent ligand for scavenger receptors, increased the rate of OS internalization (but not of OS binding) by mouse RPE as previously shown for rat and human RPE. Soluble oxidized phospholipids that are specific CD36 ligands markedly inhibited OS internalization and completely reversed the increasing effect of oxLDL. This suggested that these lipids acted as monovalent ligands for CD36 inhibiting CD36 activation. Importantly, oxidized derivatives of phosphatidylcholine species containing sn-2 esterified docosahexaenoic acid (DHA), which structurally resembled the established CD36 ligands, also reduced RPE phagocytosis. Compared to wild-type RPE, CD36 null RPE phagocytosed OS faster at early time points but did not differ in overall OS binding or internalization capacity. Neither oxLDL nor CD36 specific lipid ligands affected OS uptake by CD36 null RPE. Conclusions: The activating effect of oxLDL on OS uptake by RPE is exclusively mediated by CD36 although RPE cells likely express related receptors for oxLDL (e.g. SRBI/II). Since CD36 null RPE retains its phagocytic capacity but loses its sensitivity to CD36 specific lipids and oxLDL, their phagocytic mechanism does not employ other oxLDL receptors to compensate for the loss of CD36. Interestingly, oxidation of phospholipid-bound DHA, which is most abundant in rod outer segments, generates CD36 ligands that modulate OS phagocytosis by RPE. We therefore propose that oxidation of outer segment lipids may promote timely phagocytosis of shed OS via CD36 receptors of RPE in the eye.

Keywords: retinal pigment epithelium • phagocytosis and killing • oxidation/oxidative or free radical damage