Loss of cell surface CD47 ‘clustering’ formation and binding avidity to SIRPα facilitate apoptotic cell clearance by macrophage

CD47, a ‘self’ recognition marker expressed on tissue cells, interacts with immunoreceptor SIRPα expressed on the surface of macrophages to initiate inhibitory signaling that prevents macrophage phagocytosis of healthy host cells. Previous studies have suggested that cells may lose the surface CD47 during aging or apoptosis to enable phagocytic clearance. In the present study, we demonstrate that the level of cell surface CD47 is not decreased but the distribution pattern of CD47 is altered during apoptosis. On non-apoptotic cells, CD47 molecules are clustered in lipid rafts forming ‘punctates’ on the surface, whereas on apoptotic cells, CD47 molecules are diffused on the cell surface following the disassembly of lipid rafts. We show that clustering of CD47 in lipid rafts provides a high binding avidity for cell surface CD47 to ligate macrophage SIRPα, which also presents as clusters, and elicit SIRPα-mediated inhibitory signaling that prevents phagocytosis. In contrast, dispersed CD47 on the apoptotic cell surface is associated a significant reduction of the binding avidity to SIRPα and failure to trigger SIRPα signal transduction. Disruption of lipid rafts with methyl-β-cyclodextrin (MβCD) disrupted CD47 cluster formation on the cell surfaces, leading to decrease of the binding avidity to SIRPα and a concomitant increase of cells being engulfed by macrophages. Taken together, our study reveals that CD47 normally is clustered in lipid rafts on non-apoptotic cells but is diffused in the plasma membrane when apoptosis occurs, and this transformation of CD47 greatly reduces the strength of CD47-SIRPα engagement, resulting in the phagocytosis of apoptotic cells.