“Bundling” of SIRPα into plasma membrane microdomains is essential for effective monocyte and macrophage binding interactions with CD47 and signaling (P5117)

Abstract

Abstract SIRPα is an ITIMs-containing, cell surface signaling receptor expressed on myeloid leukocytes. Through binding interactions with CD47, a universally expressed counter-receptor, SIRPα regulates important immunological processes including neutrophil and monocyte transmigration to inflammatory loci and macrophage recognition of phagocytic targets. While their crucial roles have been demonstrated, the mechanisms underlying the dynamic interactions between SIRPα 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 SIRPα, 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 SIRPα binding. Labeling of SIRPα on the cell surface observed a diffuse distribution pattern on naïve monocytes, but highly punctate patterns on transmigrated monocytes and macrophages. Protein crosslinking and sucrose density co-sedimentation confirmed that SIRPα in latter cells form oligomerized complexes through which SIRPα achieves high avidity binding to CD47. Furthermore, such SIRPα complexes contain SHP-1 and are localized in cholesterol-rich lipid domains in the plasma membrane, and their dynamic formation involves Src-family tyrosine kinase-mediated phosphorylation.