Purpose: To examine whether the vitronectin (VN) in serum is responsible for the serum stimulation of phagocytosis in the rod outer segment (ROS) by cultured retinal pigment epithelial (RPE) cells.
Methods: Vitronectin was removed from fetal bovine serum by heparin-agarose affinity chromatography. Concentrations in normal and depleted serum were determined by enzyme-linked immunosorbent assay, using a polyclonal antibody against bovine VN and commercially prepared human VN as a standard. A monoclonal antibody against human alpha v beta 5 was used in localization and in blocking experiments. Rod outer segment phagocytosis was measured using a flow cytometric assay.
Results: Affinity chromatography 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-free medium stimulated ROS phagocytosis in a dose-dependent manner. Pretreatment of RPE cells with an antibody against alpha v beta 5, an integrin receptor for VN, had no effect on phagocytosis in the absence of serum but completely blocked the serum stimulation of ROS phagocytosis. Antibody against alpha v beta 5 demonstrated a variable labeling pattern on the cultured RPE cell surface with morphologically distinct cell clusters exhibiting less labeling. Those cell clusters exhibiting less receptor labeling also showed less uptake of fluorescent-labeled ROS.
Conclusions: Vitronectin is the component responsible for serum stimulation of ROS uptake, and this uptake appears to be mediated by an alpha v beta 5 integrin. Although clearly important in vitro, a role for VN in ROS uptake by RPE cells in situ remains to be determined.