The Cis Interaction of CD47 with Integrin Mac‐1 (α M β 2 , CD11b/CD18) Regulates Macrophage Responses

Abstract

CD47 is a widely expressed cell surface receptor that has originally been identified as an integrin-associated protein. In immune cells, CD47 has been shown to associate in cis with several integrins of the β1 and β3 subfamilies and the one member of the β2 subfamily, αLβ2. Surprisingly, the association of CD47 with integrin Mac-1 (αMβ2, CD11b/CD18), the major adhesion receptor on the surface of myeloid cells has not been documented. Furthermore, while previous studies focused on the mechanism by which CD47 on various host cells prevents phagocytosis by macrophages, the question as to how CD47 on macrophages may influence the responses of these cells has not been addressed. Here, we demonstrated that CD47 modulates Mac-1-dependent macrophage functions. In particular, adhesion of macrophages isolated from CD47-/- mice to fibrinogen and ICAM-1, the established physiological ligands of Mac-1 was significantly decreased compared to wild-type counterparts. Also, spreading of CD47-deficient macrophages was decreased by four- and two-fold on fibrinogen and ICAM-1, respectively. Compared to wild-type macrophages, migration of CD47-deficient macrophages to the Mac-1 ligand, the cathelicidin peptide LL-37 was significantly reduced. The lack of CD47 on the surface of macrophages impaired their ability to fuse in the presence of IL-4. Finally, the deficiency of CD47 also reduced phagocytosis of opsonized latex beads, a process fully dependent on Mac-1. The functional link between CD47 and Mac-1 was confirmed by their physical association in cis on the surface of macrophages and Mac-1-expressing HEK293 cells using co-immunoprecipitation and the proximity ligation assay. Studies with HEK293 ceIls expressing individual αM or β2 integrin subunits of Mac-1 showed that both subunits can interact with CD47. The activation of cells with PMA, Mn2+, and mAb MEM48 increased the amount of CD47 recovered in immune complexes with Mac-1, suggesting that CD47 has increased affinity for integrin in the extended conformation. The binding site in CD47 for Mac-1 was identified in its constituent IgV domain as recombinant IgV blocked the Mac-1-CD47 complex formation and inhibited adhesion and phagocytosis. Collectively, these results indicate that Mac-1 forms a lateral complex with CD47, which regulates important macrophage functions.