Molecular mechanism for phagocytosis of apoptotic cells

为了阐明巨噬细胞吞噬凋亡细胞的分子机制，我们建立了巨噬细胞吞噬凋亡细胞的检测方法。我们已经证明表达ICAD突变体的凋亡细胞只有在被巨噬细胞吞噬后才会发生DNA断裂，我们利用这一发现进行了实验。即用表达ICAD突变体的凋亡胸腺细胞培养巨噬细胞，用抗mac 1抗体和TUNEL染色。凋亡细胞的巨噬细胞显示Mac-1和TUNEL双阳性。利用该方法，我们可以定量测定巨噬细胞吞噬凋亡细胞的百分比。接下来，我们用该实验筛选了一系列针对小鼠腹腔巨噬细胞的单克隆抗体，发现一种抗体（指定为2422 Ab）增加了吞噬凋亡细胞的巨噬细胞的百分比。通过亲和层析纯化了一个被2422 Ab识别的分子，并对该蛋白进行质谱分析，鉴定其为小鼠乳脂球- egf -因子8 (MFG-E8)，一种分泌蛋白。MFG-E8通过识别暴露在外细胞膜上的磷脂酰丝氨酸特异性地与凋亡细胞结合。MFG-E8通过磷脂与细胞结合，特别是通过RGD基序与表达avb3整合素的细胞结合。MFG-E8的加入使NIH3T3细胞能够吞噬凋亡细胞。携带RGB基序点突变的MFG-E8表现为显性阴性形式，在体外和体内均能抑制腹腔巨噬细胞对凋亡细胞的吞噬。这些结果表明，活化巨噬细胞分泌的MFG-E8与凋亡细胞结合，并将其带到吞噬细胞中吞噬。

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.