The P2X7 Receptor and Pannexin-1 Are Both Required for the Promotion of Multinucleated Macrophages by the Inflammatory Cytokine GM-CSF

Abstract

The P2X7 receptor (P2X7R), 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 P2X7R is critical for the induction of multinucleated MA by the inflammatory cytokine GM-CSF: 1) pharmacological inhibition of P2X7R 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 P2X7 low MA clone, selected for defective P2X7R function, was unresponsive; 3) MA from mice lacking P2X7R failed to respond to GM-CSF, in contrast to wild-type. GM-CSF also stimulated ATP-induced membrane permeabilization in J774 P2X7 high MA and rat alveolar MA, an effect absent in the P2X7 low MA clone and inhibited by the P2X7 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 P2X7 but not on Panx-1 expression and that its metabolism to adenosine mediates P2X7-dependent MA fusion. These data demonstrate that both P2X7 and Panx-1 are required for GM-CSF promotion of MA fusion but likely act independently through different signaling pathway(s).

Selective Impairment of P2Y Signaling by Prostaglandin E2 in Macrophages: Implications for Ca2+-Dependent Responses

“…It is well known that P2X7 activation by ATP participates in the regulation of the innate response in macrophages, including the killing of intracellular pathogens, the release and maturation of proinflammatory cytokines (i.e., IL-1b and IL-18) (51-55), and the increase in PGE 2 levels (19). The mechanisms involved in these actions implicate a rise in Ca 2+ influx and in the activation of the MAPK signaling pathways (56)(57)(58). However, less is known regarding the role of P2Y receptors in macrophage function.…”

Accelerated Tumor Progression in Mice Lacking the ATP Receptor P2X7

“…Supernatants were collected and assayed for IL-1b content. Peritoneal macrophages were isolated as described (20). Mice were killed by excess anesthesia, and 4.5 mL sterile PBS was injected into the peritoneal cavity.…”

P2X7 receptor activation leads to increased cell death in a radiosensitive human glioma cell line

“…M059J cells were pre-treated with the selective P2X7R antagonist A740003 (10 μM) [13][14][15][16][17][18] or with the two non-selective P2 receptor antagonists suramin (30 μM) or RB2 (100 μM), and after 20 min with ATP (5 mM) for 24 h. At the end of this period, the medium was removed, the cells were washed with calcium magnesium-free medium (CMF) and 100 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (MTT) (MTT 5 mg/ml in PBS in 90 % DMEM/10 % FBS) was added to the cells and incubated for 3 h. The formazan crystals were dissolved with 100 μl of dimethyl sulfoxide (DMSO). The absorbance was quantified in 96-well plates (Spectra Max M2e, Molecular Devices) at 595 nm.…”