Nucleotides are emerging as an ubiquitous family of extracellular signaling molecules. It has been known for many years that adenosine diphosphate is a potent platelet aggregating factor, but it is now clear that virtually every circulating cell is responsive to nucleotides. Effects as different as proliferation or differentiation, chemotaxis, release of cytokines or lysosomal constituents, and generation of reactive oxygen or nitrogen species are elicited upon stimulation of blood cells with extracellular adenosine triphosphate (ATP). These effects are mediated through a specific class of plasma membrane receptors called purinergic P2 receptors that, according to the molecular structure, are further subdivided into 2 subfamilies: P2Y and P2X. ATP and possibly other nucleotides are released from damaged cells or secreted via nonlytic mechanisms. Thus, during inflammation or vascular damage, nucleotides may provide an important mechanism involved in the activation of leukocytes and platelets. However, the cell physiology of these receptors is still at its dawn, and the precise function of the multiple P2X and P2Y receptor subtypes remains to be understood.

Extracellular ATP is a mediator of intercellular communication and a danger signal. Release of this and other nucleotides modulates microglia responses via P2Y and P2X receptors, among which the P2X₇ subtype stands out for its proinflammatory activity and for up-regulation in a transgenic model of Alzheimer disease and in brains from Alzheimer disease patients. Here we show that amyloid β (Aβ) triggered increases in intracellular Ca²⁺ ([Ca²⁺]_i), ATP release, IL-1β secretion, and plasma membrane permeabilization in microglia from wild-type but not from P2X₇-deleted mice. Likewise, intra-hippocampal injection of Aβ caused a large accumulation of IL-1β in wild-type but not in P2X₇^−/− mice. These observations suggest that Aβ activates a purinergic autocrine/paracrine stimulatory loop of which the P2X₇ receptor is an obligate component. Identification of the P2X₇ receptor as a non-dispensable factor of Aβ-mediated microglia stimulation may open new avenues for the treatment of Alzheimer disease.

Endotoxin-dependent release of IL-1β from mouse microglial cells is a very inefficient process, as it is slow and leads to accumulation of a modest amount of extracellular cytokine. Furthermore, secreted IL-1β is mostly in the procytokine unprocessed form. Addition of extracellular ATP to LPS-primed microglia caused a burst of release of a large amount of processed IL-1β. ATP had no effect on the accumulation of intracellular pro-IL-1β in the absence of LPS. In LPS-treated cells, ATP slightly increased the synthesis of pro-IL-1β. Optimal ATP concentration for IL-1β secretion was between 3 and 5 mM, but significant release could be observed at concentrations as low as 1 mM. At all ATP concentrations IL-1β release could be inhibited by increasing the extracellular K+ concentration. ATP-dependent IL-1β release was also inhibited by 90 and 60% by the caspase inhibitors YVAD and DEVD, respectively. Accordingly, in ATP-stimulated microglia, the p20 proteolytic fragment derived from activation of the IL-1-β-converting enzyme could be detected by immunoblot analysis. These experiments show that in mouse microglial cells extracellular ATP triggers fast maturation and release of intracellularly accumulated IL-β by activating the IL-1-β-converting enzyme/caspase 1.

The P2X7 receptor (P2X7R) is a known and powerful activator of the NOD‐like receptor (NLR)P3 inflammasome; however, the underlying pathways are poorly understood. Thus, we investigated the molecular mechanisms involved. The effect of P2X7R expression and activation on NLRP3 expression and recruitment was investigated by Western blot, RT‐PCR, coimmunoprecipitation, and confocal microscopy in microglial mouse cell lines selected for reduced P2X7R expression and in primary cells from P2X7R ^‐/‐ C57BL/6 mice. We show here that P2X7R activation by ATP (EC ₅₀ = 1 mM) or benzoyl‐ATP (EC ₅₀ = 300 μM) and P2X7R down‐modulation caused a 2‐ to 8‐fold up‐regulation of NLRP3 mRNA in mouse N13 microglial cells. Moreover, NLRP3 mRNA was also up‐regulated in primary microglial and macrophage cells from P2X7R ^‐/‐ mice. Confocal microscopy and immunoprecipitation assays showed that P2X7R and NLRP3 closely interacted at discrete subplasmalemmal sites. Finally, P2X7R stimulation caused a transient (3‐4 min) cytoplasmic Ca ²⁺ increase localized to small (2‐3 μm wide) discrete subplasmalemmal regions. The Ca ²⁺ increase drove P2X7R recruitment and a 4‐fold increase in P2X7R/NLRP3 association within 1‐2 min. These data show a close P2X7R and NLRP3 interaction and highlight the role of P2X7R in the localized cytoplasmic ion changes responsible for both NLRP3 recruitment and activation.— Franceschini, A., Capece, M., Chiozzi, P., Falzoni, S., Sanz, J. M., Sarti, A. C., Bonora, M., Pinton, P., Di Virgilio, F. The P2X7 receptor directly interacts with the NLRP3 inflammasome scaffold protein. FASEB J. 29, 2450‐2461 (2015). www.fasebj.org

Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X₇. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X₇ receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207– 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X₇ receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X₇ blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X₇ receptor is involved in macrophage fusion.