Bundling of SIRPalpha into plasma membrane lipid rafts is essential for effective monocyte and macrophage interaction with CD47.

摘要

SIRPalpha is a ITIMs-containing, cell surface signaling receptor expressed mainly on myeloid leu-kocytes. Through binding interactions with CD47, a universally expressed counter-receptor, SIRPalpha regulates important immunological processes including neutrophil and monocyte trans-migration to inflammatory loci and macrophage recognition of phagocytic targets. While their crucial roles have been demonstrated, the mechanisms underlying the dynamic interactions be-tween SIRPalpha and CD47 remain to be characterized. By cell adhesion assays, we found that freshly isolated peripheral monocytes and cultured monocytic cells (THP-1 and U937) express abundant SIRPalpha, but do not effectively bind to CD47. In contrast, monocytes that had transmigrated across endothelia, and macrophages differentiated from THP-1 or obtained from murine tissues, displayed a high avidity of SIRPalpha binding. Labeling of SIRPalpha on the cell surface observed a diffuse distribution patterns on peripheral monocytes, but highly punctate patterns on transmigrated monocytes and macrophages. Protein crosslinking and sucrose density co-sedimentation confirmed that SIRPalpha in latter cells form oligomerized complexes through which SIRPalpha achieves high avidity binding to CD47. Furthermore, such SIRPalpha complexes are localized in cholesterol-rich lipid domains in the plasma membrane, and their dynamic formation involves Src-family tyrosine kinase-mediated phosphorylation.