MOLECULAR MECHANISM OF CELL DEATH AND ITS PHYSIOLOGICAL ROLE

在这个项目中，我们研究了凋亡DNA降解的分子机制，及其生理作用。也就是说，我们的特点是CAD（半胱天冬酶激活的DNA酶），以前在我们的实验室确定为在细胞凋亡过程中激活的DNA酶的酶性质。然后，我们通过建立CAD缺陷小鼠证明CAD是唯一负责凋亡细胞中的细胞自主DNA降解。对CAD缺陷小鼠凋亡细胞的分析表明，凋亡细胞的DNA可以在体内被巨噬细胞中存在的DNase II降解后，死亡细胞被吞噬细胞吞噬。然后建立了DNase II缺陷小鼠，对小鼠的分析表明，DNase不仅降解凋亡细胞的DNA，而且降解从红系前体细胞排出的DNA。DNase II敲除小鼠是胚胎致死的，这是由于先天免疫激活携带大量未消化DNA的巨噬细胞产生的干扰素-β。利用巨噬细胞吞噬凋亡细胞后能切割凋亡细胞DNA的知识，建立了一种可靠的凋亡细胞吞噬的检测方法。使用这个系统，我们确定了一个名为乳脂球-EGF 8（MFG-E8）的因子。MFG-E8通过与暴露于凋亡细胞的磷脂酰丝氨酸结合来识别凋亡细胞。MFG-E8还通过整联蛋白与巨噬细胞结合，表明其充当凋亡细胞和吞噬细胞之间的桥梁。发现MFG-E8在一组巨噬细胞中表达，包括脾的生发中心的带状体巨噬细胞和未成熟的树突状细胞。MFG-E8缺陷小鼠在脾脏的生发中心积累未吞噬的凋亡细胞，并发展成自身免疫性疾病，如肾炎，这表明如果凋亡细胞不能迅速从我们的身体中清除，它们将诱导自身免疫。

In this project, we have examined the molecular mechanism of apoptotic DNA degradation, and its physiological role. That is, we characterized the enzymatic properties of CAD (caspase-activated DNase) that was previously identified in our laboratory as a DNase activated during apoptosis. We then demonstrated by establishing the CAD-deficient mice that CAD is solely responsible for the cell-autonomous DNA degradation in apoptotic cells. Analysis of the apoptotic cells in the CAD-deficient mice showed that the DNA of apoptotic cells could be degraded in vivo by DNase II present in the macrophages after the dead cells were engulfed by phagocytes. The DNase II-deficient mice were then established, and the analysis of the mice showed that the DNase digests not only DNA of apoptotic cells, but also the DNA expelled from erythroid precursor cells. The DNase II-null mice were embryonic lethal, which was due to the interferon-β that was produced in macrophages carrying a large amount of undigested DNA by the activation of innate immunity. Using the knowledge that the DNA of apoptotic cells can be cleaved by macrophages after the dead cells are engulfed, we established a reliable assay method for engulfment of apoptotic cells. Using this system, we identified a factor called Milk Fat Globule-EGF8 (MFG-E8). MFG-E8 recognized apoptotic cells by binding to phosphatidylserine exposed to the apoptotic cells. MFG-E8 also bound to macrophages via integrin, indicating that it serves as a bridge between apoptotic cells and phagocytes. MFG-E8 was found to be expressed in a set of macrophages including tingible-body macrophages in the germinal centers of the spleen, and in immature dendritic cells. The MFG-E8-deficient mice accumulated unengulfed apoptotic cells in the germinal centers of the spleen, and developed autoimmune diseases such as nephritis, indicating that if apoptotic cells are not swiftly cleared from our bodies, they will induce the autoimmunity.