The Journal of Immunology The P2X 7 Receptor and Pannexin-1 Are Both Required for the Promotion of Multinucleated Macrophages by the Inflammatory Cytokine GM-CSF Irma Lemaire,* Simonetta Falzoni, †,‡ Bin Zhang,* Patrizia Pellegatti, †,‡ and Francesco Di Virgilio † The P2X 7 receptor (P2X 7 R), an ATP-gated ion channel, has been implicated in the process of cell-to-cell fusion into multinucleated macrophages (MA), but its contribution to MA fusion driven by physiological/pathological stimuli is not clearly established. Based on several lines of evidence, we demonstrate that P2X 7 R is critical for the induction of multinucleated MA by the inflammatory cytokine GM-CSF: 1) pharmacological inhibition of P2X 7 R with oxidized ATP (oATP), KN-62, and the selective antagonist A740003 abrogated GM-CSF action on rat alveolar MA and murine peritoneal MA; 2) a murine J774 P2X 7 low MA clone, selected for defective P2X 7 R function, was unresponsive; 3) MA from mice lacking P2X 7 R failed to respond to GM-CSF, in contrast to wild-type. GM-CSF also stimulated ATP-induced membrane permeabilization in J774 P2X 7 high MA and rat alveolar MA, an effect absent in the P2X 7 low MA clone and inhibited by the P2X 7 blockers oATP and KN-62. Notably, the stimulatory effects of GM-CSF on pore formation and MA fusion were both inhibited by blocking functional Pannexin-1 (Panx-1), and GM- CSF failed to stimulate MA fusion in cells from Panx-1 knockout mice. We provide further evidence that extracellular ATP release from peritoneal MA is dependent on P2X 7 but not on Panx-1 expression and that its metabolism to adenosine mediates P2X 7 - dependent MA fusion. These data demonstrate that both P2X 7 and Panx-1 are required for GM-CSF promotion of MA fusion but likely act independently through different signaling pathway(s). The Journal of Immunology , 2011, 187: 3878–3887. M acrophages (MA) are present in all tissues, and cell-to- cell communication between MA themselves and with other cells is crucial in a wide range of biological processes such as tissue repair, immune reactions, and metastasis (1, 2). Homotypic fusion of MA into multinucleated giant cells has long been recognized both in homeostasis and disease. This specialized form of MA differentiation is critical for the normal development of osteoclasts, the cells responsible for bone re- sorption (3, 4). Such process is also an important component of host responses in various pathological conditions. For instance, the presence of multinucleated MA is a hallmark of chronic inflammatory reactions that take place in infective granulomas, foreign body reactions, and sterile inflammation (4, 5). Notably, multinucleated MA have been observed in tuberculosis (5), yeast infection (6), HIV infection (7), failure of orthopedic implants (8), and sarcoidosis (5, 9). Accordingly, a number of cytokines pro- duced by immune cells including GM-CSF, M-CSF, IL-4, IL-13, and IL-6, alone or in combination, have been reported to stimulate the appearance of multinucleated MA in various cell culture sys- tems (4, 10–14). Despite the widespread observation of MA fusion in vitro and in vivo, the mechanisms underlying this process remain poorly understood. It is thought that MA–MA fusion is mediated by receptor–ligand interaction, and several cell membrane molecules such as b-integrins receptors (4), CD44 (15), CD47 (16), CD98 (17), the transmembrane glycoprotein ADAM 9 (18), and the dendritic cell-specific transmembrane protein (DC-STAMP) (12) have been identified as mediators of the fusion process. In partic- ular, DC-STAMP was found to be essential for cell–cell fusion in osteoclasts and MA, as mice lacking the DC-STAMP gene failed to form multinucleated MA and osteoclasts (12). Significant evidence also supports the implication of the puri- nergic P2X 7 membrane receptor in osteoclast formation (19, 20) and in the fusion of various MA preparations (21, 22) including those from patients with sarcoidosis (9, 23). The P2X 7 receptor (P2X 7 R), a member of the P2X family of nucleotide receptors, displays several attributes that make it an attractive candidate as a potential modulator of intercellular communication and MA fu- sion in inflammation. The P2X 7 R is expressed by MA (21, 22) and osteoclasts (19) and is activated by extracellular ATP, a danger signal known to be present at sites of injury and inflammation (24). Quite notably, activation of P2X 7 R by ATP could trigger further release of ATP (25), an extracellular messenger that can propagate cell-to-cell signaling (26). Moreover, because of its ability to destabilize lipid bilayer and form a membrane pore (27), P2X 7 is a relevant membrane protein as a mediator of MA cell– cell interaction. In this respect, the permeabilization pore coupled to P2X 7 R has been ascribed to the hemichannel protein Pannexin- 1 (Panx-1) (28, 29). Panx-1 has been shown to be a component of P2X 7 R signaling pathways and has been implicated in the release *Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada; † Section of General Pathology, De- partment of Experimental and Diagnostic Medicine, University of Ferrara, 44100 Ferrara, Italy; and ‡ Interdisciplinary Center for the Study of Inflammation, University of Ferrara, 44100 Ferrara, Italy Received for publication August 16, 2010. Accepted for publication July 26, 2011. This work was supported by the Natural Sciences and Engineering Research Council of Canada, the Canadian Institute of Health Research, the Italian Association for Cancer Research (Grant IG 5354), Telethon of Italy (Grant GGP06070), the Italian Space Agency, the Commission of European Communities (7th Framework Program HEALTH-F2-2007-202231), the Regione Emilia Romagna (research programs “In- novative approaches to the diagnosis of inflammatory diseases” and “Moniter”), and by institutional funds from the University of Ferrara. Address correspondence and reprint requests to Dr. Irma Lemaire, Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada. E-mail address: ilemaire@uottawa.ca Abbreviations used in this article: CX-43, connexin-43; DC-STAMP, dendritic cell- specific transmembrane protein; EtBr, ethidium bromide; f.i., fusion index; MA, macrophage; MGC, multinucleated giant cell; oATP, oxidized ATP; Panx-1, Pan- nexin-1; PMB, polymyxin B; P2X 7 R, P2X 7 receptor. Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002780