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，我们建立了一种检测吞噬凋亡细胞的方法。我们已经表明，表达ICAD突变体的凋亡细胞仅在被巨噬细胞吞噬后才发生DNA片段化，我们利用这些发现进行了测定。即，将巨噬细胞与表达ICAD突变体的凋亡胸腺细胞一起培养，并用抗Mac 1抗体和TUNEL染色。凋亡细胞的巨噬细胞呈Mac-1和TUNEL双阳性。使用该测定，我们可以定量吞噬凋亡细胞的巨噬细胞的百分比。我们接下来用该测定筛选了针对小鼠腹腔巨噬细胞产生的一系列单克隆抗体，并发现抗体（命名为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2422 Ab识别的分子，并对蛋白质进行质谱分析，鉴定它们为分泌蛋白小鼠乳脂球-EGF-因子8（MFG-E8）。MFG-E8通过识别暴露在外细胞膜上的磷脂酰丝氨酸特异性结合凋亡细胞。MFG-E8通过其RGD基序与结合至细胞，特别是结合至表达avb 3整联蛋白的细胞的磷脂接合。MFG-E8的加入使NIH 3 T3细胞吞噬凋亡细胞。MFG-E8在RGB基序中携带点突变，表现为显性负性形式，并在体外和体内抑制腹腔巨噬细胞对凋亡细胞的吞噬作用。这些结果表明，从活化的巨噬细胞分泌的MFG-E8与凋亡细胞结合，并将它们带到吞噬细胞中进行吞噬。

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.