Figure 5.

ROS binding is inhibited by αvβ5 antibody and by an RGD containing peptide but not by vitronectin. Internalization of surface-bound ROS is independent of αvβ5 integrin. (A) Cells were challenged with FITC-ROS for 2 h after 30 min preincubation in the presence of the indicated reagents. Inhibition of phagocytosis by αvβ5 antibody was concentration dependent, other antibodies did not alter ROS binding. Species-specific antibodies F11 (rat) and LM609 (human) were used solely on RPE-J or ARPE-19 cells, respectively. GRGDSP, but not a control peptide or vitronectin, competed with ROS binding. Phagocytosis of untreated samples represents 100%. Values are mean ± SD, n = 8, using RPE-J cells or ARPE-19 cells. Inhibition values were identical for both cell lines. (B) To determine the effect of αvβ5 inhibition on ROS uptake, RPE cells were challenged with FITC-ROS in the absence (1, 2) or presence (3, 4) of αvβ5 blocking antibody for 2 h; at this time, unbound ROS were washed out. Uptake of prebound ROS during the following three hours was in the presence (2, 4, ░⃞) or absence (1, 3, ▪) of freshly added αvβ5 blocking antibody. The flowchart outlines the treatment of different samples. For each sample, total fluorescence was measured after 5 h, then extracellular fluorescence quenched with trypan blue and the sample rescanned to measure the internalized fraction. The internalized fluorescence is given as percent of the total fluorescence of the sample at 5 h. Note that the total uptake at 2 h for samples 3 and 4 is 16% of the uptake by samples 1 and 2. Values represent mean values of two experimental sets; n = 4 for each sample in each experiment.