Dept of Medical Neurobiology, IMRIC, Hebrew University Hadassah Medical School, P.O.B 12272, Jerusalem 91120, Israel.
J Neuroinflammation. 2011 Mar 15;8:24. doi: 10.1186/1742-2094-8-24.
Traumatic injury to axons produces breakdown of axons and myelin at the site of the lesion and then further distal to this where Wallerian degeneration develops. The rapid removal of degenerated myelin by phagocytosis is advantageous for repair since molecules in myelin impede regeneration of severed axons. Thus, revealing mechanisms that regulate myelin phagocytosis by macrophages and microglia is important. We hypothesize that myelin regulates its own phagocytosis by simultaneous activation and down-regulation of microglial and macrophage responses. Activation follows myelin binding to receptors that mediate its phagocytosis (e.g. complement receptor-3), which has been previously studied. Down-regulation, which we test here, follows binding of myelin CD47 to the immune inhibitory receptor SIRPα (signal regulatory protein-α) on macrophages and microglia.
CD47 and SIRPα expression was studied by confocal immunofluorescence microscopy, and myelin phagocytosis by ELISA.
We first document that myelin, oligodendrocytes and Schwann cells express CD47 without SIRPα and further confirm that microglia and macrophages express both CD47 and SIRPα. Thus, CD47 on myelin can bind to and subsequently activate SIRPα on phagocytes, a prerequisite for CD47/SIRPα-dependent down-regulation of CD47+/+ myelin phagocytosis by itself. We then demonstrate that phagocytosis of CD47+/+ myelin is augmented when binding between myelin CD47 and SIRPα on phagocytes is blocked by mAbs against CD47 and SIRPα, indicating that down-regulation of phagocytosis indeed depends on CD47-SIRPα binding. Further, phagocytosis in serum-free medium of CD47+/+ myelin is augmented after knocking down SIRPα levels (SIRPα-KD) in phagocytes by lentiviral infection with SIRPα-shRNA, whereas phagocytosis of myelin that lacks CD47 (CD47-/-) is not. Thus, myelin CD47 produces SIRPα-dependent down-regulation of CD47+/+ myelin phagocytosis in phagocytes. Unexpectedly, phagocytosis of CD47-/- myelin by SIRPα-KD phagocytes, which is not altered from normal when tested in serum-free medium, is augmented when serum is present. Therefore, both myelin CD47 and serum may each promote SIRPα-dependent down-regulation of myelin phagocytosis irrespective of the other.
Myelin down-regulates its own phagocytosis through CD47-SIRPα interactions. It may further be argued that CD47 functions normally as a marker of "self" that helps protect intact myelin and myelin-forming oligodendrocytes and Schwann cells from activated microglia and macrophages. However, the very same mechanism that impedes phagocytosis may turn disadvantageous when rapid clearance of degenerated myelin is helpful.
轴突的创伤性损伤会导致病变部位以及病变部位远端的轴突和髓鞘的断裂，进而发生沃勒变性。吞噬细胞迅速清除变性髓鞘有利于修复，因为髓鞘中的分子会阻碍切断轴突的再生。因此，揭示调节巨噬细胞和小胶质细胞吞噬髓鞘的机制非常重要。我们假设髓鞘通过同时激活和下调小胶质细胞和巨噬细胞的反应来调节自身的吞噬作用。激活是在髓鞘与介导其吞噬作用的受体（例如补体受体-3）结合后发生的，这一点以前已经研究过。我们在这里测试的下调是在髓鞘 CD47 与巨噬细胞和小胶质细胞上的免疫抑制受体 SIRPα（信号调节蛋白-α）结合后发生的。
通过共聚焦免疫荧光显微镜研究 CD47 和 SIRPα 的表达，并通过 ELISA 研究髓鞘吞噬作用。
我们首先证明髓鞘、少突胶质细胞和施万细胞表达 CD47 而不表达 SIRPα，并进一步证实小胶质细胞和巨噬细胞表达 CD47 和 SIRPα。因此，髓鞘上的 CD47 可以与吞噬细胞上的 SIRPα 结合，并随后激活 SIRPα，这是 CD47/SIRPα 依赖性下调自身 CD47+/+ 髓鞘吞噬作用的前提。然后，我们证明当通过针对 CD47 和 SIRPα 的 mAbs 阻断吞噬细胞上髓鞘 CD47 和 SIRPα 之间的结合时，CD47+/+ 髓鞘的吞噬作用会增强，表明吞噬作用的下调确实取决于 CD47-SIRPα 结合。此外，在吞噬细胞中用 SIRPα-shRNA 慢病毒感染敲低 SIRPα 水平（SIRPα-KD）后，无 CD47（CD47-/-）髓鞘的吞噬作用在无血清培养基中增强，但 CD47+/+ 髓鞘的吞噬作用增强。因此，髓鞘 CD47 产生 SIRPα 依赖性下调吞噬细胞中的 CD47+/+ 髓鞘吞噬作用。出乎意料的是，当在存在血清的情况下测试时，SIRPα-KD 吞噬细胞对 CD47-/- 髓鞘的吞噬作用与正常情况相比没有改变，但增强了。因此，髓鞘 CD47 和血清都可以促进 SIRPα 依赖性髓鞘吞噬作用的下调，而无需考虑另一个。
髓鞘通过 CD47-SIRPα 相互作用下调自身吞噬作用。可以进一步认为，CD47 作为“自身”的标志物正常发挥作用，有助于保护完整的髓鞘和形成髓鞘的少突胶质细胞和施万细胞免受激活的小胶质细胞和巨噬细胞的侵害。然而，当迅速清除变性髓鞘有帮助时，阻碍吞噬作用的相同机制可能变得不利。
