Fig. 5.

MFG-E8 enhances phagocytosis of neutrophils with PLB-985 macrophage-induced PS externalization. (A) HMDM were pretreated with MFG-E8 (WT) or D89E MFG-E8 mutant protein (1 μg/ml) for 30 min, and the recombinant proteins were added again at the time of cocultivation. Neutrophils were cocultured with HMDM for 1 h, and the percentages of phagocytosis-positive macrophages were determined. Neutrophils undergoing spontaneous apoptosis and neutrophils maintained in culture for 2 h in the absence of PLB-985 macrophages were included for comparison. Data are derived from four independent experiments and are reported as mean values ± sem. (B) Cocultivation of neutrophils and HMDM was performed as above, and the percentages of macrophages that carried one or greater than or equal to two target cells in the presence or absence of MFG-E8 (1 μg/ml) were determined. Data shown are mean values ± sem (n=3). (C) Representative images showing HMDM engulfment of TAMRA-labeled target neutrophils (red) in the presence or absence of MFG-E8 (1 μg/ml). Hoechst 33342 (blue) was used for visualization of cell nuclei. Arrows indicate macrophages that have ingested more than one target cell. Original magnification, ×40.