A distinct integrin-mediated phagocytic pathway for extracellular matrix remodeling by RPE cells

PURPOSE. To characterize the phagocytosis of extracellular matrix component s by retinal pigment epithelial cells and to determine which receptors and signal transduction pathways are involved. METHODS. Fluorescent latex beads were coated with fibronectin (FN), collage n type I or TV, or thrombospondin and incubated with human retinal pigment epithelial cells for 3 hours. Phagocytosis was quantified by flow cytometry . The effects of adhesion blocking antibodies to cell surface receptors (al pha 1, alpha 3, alpha 5, beta 1, alpha 5 beta 1, alpha nu beta 3, alpha nu beta 5 integrins and CD36) and inhibitors of specific intracellular signali ng pathways (tyrosine kinase phosphatidylinositol 3-kinase [PIS-kinase], pr otein kinase C [PKC], and mitogen-activated protein kinase) were determined using FN-coated beads. RESULTS. Phagocytosis of FN-coated beads was greate r than phagocytosis of beads coated with collagen type II collagen type IV, or thrombospondin or uncoated controls (P < 0.0005), Anti-alpha 5, -beta 1 and -alpha 5 beta 1 antibodies markedly inhibited FN phagocytosis (P < 0.0 005); the inhibitory effects of anti-alpha 5 antibody were stronger in the initial stages (binding) than in the later stages (internalization) of phag ocytosis. There was no significant effect on phagocytosis when anti-alpha 1 , -alpha 3, -alpha nu beta 5, -alpha nu beta 3 or -CD36 antibodies were use d. Fibronectin phagocytosis was decreased by inhibitors of tyrosine kinase (genistein, 100 mu g/ml, P < 0.005) and PI3-kinase (wortmannin, 5 mu M, P < 0.01), but these reagents did not affect the uncoated controls. The PKC in hibitor calphostin C (400 nM) nonspecifically increased the phagocytosis of FN-coated (P < 0.05) and uncoated beads (P < 0.01). CONCLUSIONS. Subconflu ent retinal pigment epithelial cells preferentially phagocytose FN over oth er extracellular matrix components. Phagocytosis of FN utilizes the alpha 5 beta 1 integrin, is mediated in part through tyrosine kinase and PIS-kinas e signaling pathways, and is modulated by PKC. Phagocytosis of extracellula r matrix by retinal pigment epithelial cells may represent a novel mechanis m for remodeling of thr provisional extracellular matrix during outer retin al wound healing.