Leukocyte integrin Mac-1 (CD11b/CD18, αMβ2, CR3) acts as a functional receptor for platelet factor 4

Valeryi K Lishko,Valentin P Yakubenko,Tatiana P Ugarova,Nataly P Podolnikova

doi:10.1074/jbc.ra117.000515

Valeryi K Lishko, Valentin P Yakubenko + Show 2 more

Open Access

https://doi.org/10.1074/jbc.ra117.000515

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Abstract

Platelet factor 4 (PF4) is one of the most abundant cationic proteins secreted from α-granules of activated platelets. Based on its structure, PF4 was assigned to the CXC family of chemokines and has been shown to have numerous effects on myeloid leukocytes. However, the receptor for PF4 remains unknown. Here, we demonstrate that PF4 induces leukocyte responses through the integrin Mac-1 (αMβ2, CD11b/CD18). Human neutrophils, monocytes, U937 monocytic and HEK293 cells expressing Mac-1 strongly adhered to immobilized PF4 in a concentration-dependent manner. The cell adhesion was partially blocked by anti-Mac-1 mAb and inhibition was enhanced when anti-Mac-1 antibodies were combined with glycosaminoglycans, suggesting that cell-surface proteoglycans act cooperatively with Mac-1. PF4 also induced Mac-1-dependent migration of human neutrophils and murine WT, but not Mac-1-deficient macrophages. Coating of Escherichia coli bacteria or latex beads with PF4 enhanced their phagocytosis by macrophages by ∼4-fold, and this process was blocked by different Mac-1 antagonists. Furthermore, PF4 potentiated phagocytosis by WT, but not Mac-1-deficient macrophages. As determined by biolayer interferometry, PF4 directly bound the αMI-domain, the major ligand-binding region of Mac-1, and this interaction was governed by a Kd of 1.3 ± 0.2 μm Using the PF4-derived peptide library, synthetic peptides duplicating the αMI-domain recognition sequences and recombinant mutant PF4 fragments, the binding sites for αMI-domain were identified in the PF4 segments Cys12-Ser26 and Ala57-Ser70 These results identify PF4 as a ligand for the integrin Mac-1 and suggest that many immune-modulating effects previously ascribed to PF4 are mediated through its interaction with Mac-1.

Highlights

Platelet factor 4 (PF4) is one of the most abundant cationic proteins secreted from ␣-granules of activated platelets
To investigate whether integrin Mac-1 and HSPGs on macrophages are involved in promoting phagocytosis of PF4coated beads, we examined the effects of anti-Mac-1 reagents and heparin on phagocytosis of beads
PF4 has been assigned to the CXC chemokine subfamily based on its structure, its chemotactic activity for neutrophils and monocytes has remained controversial and it was concluded that PF4 does not behave as a classic chemokine [4, 6]

Summary

Results

We previously developed the computer program that allows the prediction of potential Mac-1 ligands by examining the presence of putative binding sites for the ␣MI-domain, a ligand recognition region of Mac-1 [12]. As expected, binding of active ␣LI-domain of the integrin ␣L␤2 (LFA-1), a receptor with a narrow ligand binding specificity [22] was low (Fig. 2D) These data demonstrate that soluble PF4 directly binds ␣MI-domain and this interaction requires the active form of ␣MI-domain. The finding that the ␣MI-domain directly interacts with PF4 suggests that PF4 may bind Mac-1 on the cell surface. To investigate this possibility, we performed adhesion with immobilized PF4 using various Mac-1-expressing cells. The role of Mac-1 in the interaction of Mac-1–HEK293 cells with PF4 was further determined using function-blocking mAb 44a directed to the human ␣M integrin subunit This mAb inhibited adhesion of Mac-1–HEK293 cells and U937 in a dose-dependent manner (Fig. S1) and at 10 ␮g/ml inhibited cell adhesion by 69 Ϯ 7 and 63 Ϯ 2% for Mac-1–HEK293 and U937 cells, respectively. Heparin at 10 ␮g/ml partially inhibited adhesion (ϳ30%)

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Discussion

Experimental procedures