Membrane PR3 expression decreased human macrophage phagocytosis of RBL/PR3 and RBL/PR3S203A independently of its serine-proteinase activity. (A) Analysis of membrane PS and PR3 externalizations by flow cytometry after etoposide-induced apoptosis (bold line) compared with untreated cells (thin line). This representative experiment shows PS externalization on RBL/CT, RBL/PR3, and RBL/PR3S203A after etoposide-induced apoptosis; PR3 was expressed on apoptotic RBL/PR3 and RBL/PR3S203A but not apoptotic RBL/CT. Double labeling of membrane PR3 and annexin-V showed their close relationship. No relationship was observed under basal conditions because no annexin-V labeling was observed (data not shown). (B) Confocal fluorescence microscopy quantification of human macrophage phagocytosis of apoptotic RBL-2H3. TAMRA-labeled apoptotic RBL (red) were incubated with FITC-conjugated anti-CD14–labeled macrophages derived from human monocytes (green). Phagocytosis yielded a red-labeled apoptotic RBL engulfed by a green-labeled macrophage. Images were obtained as described in Figure 2. (C) Quantification of annexin-V–positive RBL cells after etoposide-induced apoptosis. All apoptotic RBL transfectants showed significantly enhanced PS externalization (**P < .01, ANOVA). However, there was no significant difference in PS externalization between all apoptotic transfectants. (D) Quantification of macrophage phagocytosis. The phagocytosing macrophages were identified by confocal microscopy to ensure that the RBL cells had been engulfed and were not just sitting on the macrophage surface. Phagocytosis rates were calculated as the ratio between the number of phagocytosing macrophages on total macrophages. As expected, phagocytosis rates of apoptotic RBL/CT, RBL/PR3, or RBL/PR3S203A were higher than viable RBL lines (P < .001, for all transfectants using ANOVA; not indicated in the figure). The rates of apoptotic RBL/PR3 and RBL/PR3S203A phagocytosis were significantly lower than that of apoptotic RBL/CT (**P < .01, ANOVA) but RBL/PR3 and RBL/PR3S203A rates were comparable (not significant [NS], ANOVA). Data are means (± SEM) from 8 independent experiments.