No abstract

Macrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for initiating this process is unknown. Here, we used live cell imaging to show that actin-based protrusions at the leading edge initiate macrophage fusion. Phase-contrast video microscopy demonstrated that in the majority of events, short protrusions (∼3 µm) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (∼12 µm). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent protrusions formed at sites enriched in podosomes. Inducing fusion in mixed populations of GFP- and mRFP-LifeAct macrophages showed rapid spatial overlap between GFP and RFP signal at the site of fusion. Cytochalasin B strongly reduced fusion and when rare fusion events occurred, protrusions were not observed. Fusion of macrophages deficient in Wiskott-Aldrich syndrome protein and Cdc42, key molecules involved in the formation of actin-based protrusions and podosomes, was also impaired both in vitro and in vivo. Finally, inhibiting the activity of the Arp2/3 complex decreased fusion and podosome formation. Together these data suggest that an actin-based protrusion formed at the leading edge initiates macrophage fusion.

Pleiotrophin (PTN) is a multifunctional, cationic, glycosaminoglycan-binding cytokine and growth factor involved in numerous physiological and pathological processes, including tissue repair and inflammation-related diseases. PTN has been shown to promote leukocyte responses by inducing their migration and expression of inflammatory cytokines. However, the mechanisms through which PTN mediates these responses remain unclear. Here, we identified the integrin Mac-1 (αMβ2, CD11b/CD18) as the receptor mediating macrophage adhesion and migration to PTN. We also found that expression of Mac-1 on the surface of human embryonic kidney (HEK) 293 cells induced their adhesion and migration to PTN. Accordingly, PTN promoted Mac-1-dependent cell spreading and initiated intracellular signaling manifested in phosphorylation of Erk1/2. While binding to PTN, Mac-1 on Mac-1-expressing HEK293 cells appears to cooperate with cell-surface proteoglycans because both anti-Mac-1 function-blocking mAb and heparin were required to block adhesion. Moreover, biolayer interferometry and NMR indicated a direct interaction between the αI domain, the major ligand-binding region of Mac-1, and PTN. Using peptide libraries, we found that in PTN the αI domain bound sequences enriched in basic and hydrophobic residues, indicating that PTN conforms to the general principle of ligand-recognition specificity of the αI domain toward cationic proteins/peptides. Finally, using recombinant PTN-derived fragments, we show that PTN contains two distinct Mac-1-binding sites in each of its constitutive domains. Collectively, these results identify PTN as a ligand for the integrin Mac-1 on the surface of leukocytes and suggest that this interaction may play a role in inflammatory responses.

Macrophage fusion leading to the formation of multinucleated giant cells is a hallmark of chronic inflammation. Several membrane proteins have been implicated in mediating cell-cell attachment during fusion, but their binding partners remain unknown. Recently, we demonstrated that interleukin-4 (IL-4)induced fusion of mouse macrophages depends on the integrin macrophage antigen 1 (Mac-1). Surprisingly, the genetic deficiency of intercellular adhesion molecule 1 (ICAM-1), an established ligand of Mac-1, did not impair macrophage fusion, suggesting the involvement of other counter-receptors. Here, using various approaches, including signal regulatory protein ␣ (SIRP␣) knockdown, recombinant proteins, adhesion and fusion assays, biolayer interferometry, and peptide libraries, we show that SIRP␣, which, similar to ICAM-1, belongs to the Ig superfamily and has previously been implicated in cell fusion, interacts with Mac-1. The following results support the conclusion that SIRP␣ is a ligand of Mac-1: (a) recombinant ectodomain of SIRP␣ supports adhesion of Mac-1-expressing cells; (b) Mac-1-SIRP␣ interaction is mediated through the ligand-binding ␣ M I-domain of Mac-1; (c) recognition of SIRP␣ by the ␣ M Idomain conforms to general principles governing binding of Mac-1 to many of its ligands; (d) SIRP␣ reportedly binds CD47; however, anti-CD47 function-blocking mAb produced only a limited inhibition of macrophage adhesion to SIRP␣; and (e) co-culturing of SIRP␣-and Mac-1-expressing HEK293 cells resulted in the formation of multinucleated cells. Taken together, these results identify SIRP␣ as a counter-receptor for Mac-1 and suggest that the Mac-1-SIRP␣ interaction may be involved in macrophage fusion. Cell-cell fusion is a fundamental biological process that is required for development and homeostasis (1-3). Cellular cro ARTICLE

Macrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for initiating macrophage fusion is unknown. Here, we used live cell imaging to show that actin-based protrusions at the leading edge initiate macrophage fusion. Phase contrast video microscopy demonstrated that in the majority of events, short protrusions (3 ± 1 µm) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (16 ± 7 µm). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent actinbased protrusions formed at sites enriched in podosomes. Inducing fusion in mixed populations of GFP-and mRFP-LifeAct macrophages showed rapid spatial overlap between GFP and RFP signal at the site of fusion. Cytochalasin B strongly reduced fusion and when rare fusion events occurred, protrusions were not observed. Fusion of macrophages deficient in Wiskott-Aldrich syndrome protein and Cdc42, key molecules involved in the formation of actin-based protrusions and podosomes, was also impaired both in vitro and in vivo. Finally, inhibiting the activity of the Arp2/3 complex decreased fusion and podosome formation. Together these data indicate that an actin-based protrusion formed at the leading edge initiates macrophage fusion.