Annexin 2 is Required for Phagocytosis of Rod Outer Segments by Retinal Pigment Epithelial Cells

Abstract

Abstract: : Purpose The aim of this study was to examine whether retinal pigment epithelial cell annexin 2 has a role in phagocytosis of rod photoreceptor outer segments (ROS). Methods We have used ARPE19 cells (a human retinal pigmented epithelial cell line) grown on lens capsule or tissue culture plates coated with extracellular matrix proteins to model the RPE monolayer in association with Bruch’s membrane. RPE cells expressing an annexin 2-green fluorescent protein fusion construct were cultured in the presence of Alexa red-labelled ROS, and imaged by confocal microscopy. ROS internalisation was quantified by analysis of digitized images, and comparisons were made between cells grown on different substrates in the presence or absence of small interfering RNAs against annexin 2. Results ARPE19 cells grown on polypropylene, fibronectin or vitronectin were poorly phagocytic but when the cells were grown on lens capsule, laminin or collagen IV their ability to phagocytose ROS improved significantly. The increase in phagocytic competence was paralleled by an increase in annexin 2 expression. siRNA-mediated depletion of annexin 2 in cells grown on these substrates attenuated the increase in phagocytic function, as did expression of a dominant negative mutant of annexin 2 in RPE cells grown on lens capsule. Using confocal microscopy we also observed enrichment of annexin 2 to the phagocytic cup during the preliminary stages of ROS adhesion, filopod extension and cup invagination. Conclusion We have shown that high levels of phagocytic competence in RPE cells correlate with high levels of annexin 2 expression. Interfering with annexin 2 function leads to signficantly impaired phagocytic function, indicating an essential role for annexin 2 in this process.

Keywords: retinal pigment epithelium • photoreceptors • retinal degenerations: cell biology