VITRONECTIN IS RESPONSIBLE FOR SERUM-STIMULATED UPTAKE OF ROD OUTER SEGMENTS BY CULTURED RETINAL-PIGMENT EPITHELIAL-CELLS

Purpose. To examine whether the vitronectin (VN) in serum is responsib le for the serum stimulation of phagocytosis in the rod outer segment (ROS) by cultured retinal pigment epithelial (RPE) cells. Methods. Vit ronectin was removed from fetal bovine serum by heparin-agarose affini ty chromatography. Concentrations in normal and depleted serum were de termined by enzyme-linked immunosorbent assay, using a polyclonal anti body against bovine VN and commercially prepared human VN as a standar d. A monoclonal antibody against human alpha v beta 5 was used in loca lization and in blocking experiments. Rod outer segment phagocytosis w as measured using a flow cytometric assay. Results. Affinity chromatog raphy removed 95% of the VN from serum as determined by enzyme-linked immunosorbent assay. Vitronectin-depleted serum did not stimulate ROS phagocytosis by RPE cells. Commercially prepared VN added to serum-fre e medium stimulated ROS phagocytosis in a dose-dependent manner. Pretr eatment of RPE cells with an antibody against alpha v beta 5, an integ rin receptor for VN, had no effect on phagocytosis in the absence of s erum but completely blocked the serum stimulation of ROS phagocytosis, Antibody against alpha v beta 5 demonstrated a variable labeling patt ern on the cultured RPE cell surface with morphologically distinct cel l clusters exhibiting less labeling. Those cell clusters exhibiting le ss receptor labeling also showed less uptake of fluorescent-labeled RO S. Conclusions. Vitronectin is the component responsible fat serum sti mulation of ROS uptake, and this uptake appears to be mediated by an a lpha v beta 5 integrin. Although clearly important in vitro, a role fo r VN in ROS uptake by RPE cells in situ remains to be determined.