Molecular mechanism for phagocytosis of apoptotic cells

点击翻译按钮获取中文摘要

To elucidate the molecular mechanism for phagocytosis of apoptotic cells by macrophages, we have established an assay to detect phagocytosis of apoptotic cells. We have shown that the apoptotic cells expressing ICAD mutant undergo DNA fragmentation only after they are phagocytosed by macrophages, and we took advantage of the findings for the assay. That is, macrophages were cultured with apoptotic thymocytes expressing the ICAD mutant, and were stained with anti-Mac 1 antibody and TUNEL. The macrophages with apoptotic cells showed Mac-1 and TUNEL double positive. Using the assay, we could quantitate the percentage of macrophages that phagocytose apoptotic cells. We next screened the series of monoclonal antibodies raised against mouse peritoneal macrophages with the assay, and found that an antibody (designated 2422 Ab) increased the percentage of macrophages that engulfed the apoptotic cells. A molecule recognized by 2422 Ab was purified by affinity chromatography, and a mass spectrometry analysis of the protein identified them as mouse milk fat globule-EGF-factor 8 (MFG-E8), a secreted protein. MFG-E8 specifically bound to apoptotic cells by recognizing phosphatidylserine exposed on outer cell membrane. MFG-E8 engaged by phospholipid bound to cells, in particular to avb3-integrin-expressing cells via its RGD motif. The addition of MFG-E8 rendered NIH3T3 cells to engulf apoptotic cells. MFG-E8 carrying a point mutation in the RGB motif behaved as a dominant negative form, and inhibited the phagocytosis of apoptotic cells by peritoneal macrophages in vitro and in vivo. These results indicated that MFG-E8 secreted from activated macrophages binds to apoptotic cells, and brings them to phagocytes for engulfment.