The proinflammatory cytokines IL-1β and IL-18 are inactive until cleaved by the enzyme caspase-1. Stimulation of the P2X7 receptor (P2X7R), an ATP-gated ion channel, triggers rapid activation of caspase-1. In this study we demonstrate that pretreatment of primary and Bac1 murine macrophages with TLR agonists is required for caspase-1 activation by P2X7R but it is not required for activation of the receptor itself. Caspase-1 activation by nigericin, a K+/H+ ionophore, similarly requires LPS priming. This priming by LPS is dependent on protein synthesis, given that cyclohexamide blocks the ability of LPS to prime macrophages for activation of caspase-1 by the P2X7R. This protein synthesis is likely mediated by NF-κB, as pretreatment of cells with the proteasome inhibitor MG132, or the IκB kinase inhibitor Bay 11-7085 before LPS stimulation blocks the ability of LPS to potentiate the activation of caspase-1 by the P2X7R. Thus, caspase-1 regulation in macrophages requires inflammatory stimuli that signal through the TLRs to up-regulate gene products required for activation of the caspase-1 processing machinery in response to K+-releasing stimuli such as ATP.

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The P2X7 receptor (P2X7R) is an ATP-gated cation channel that activates caspase-1 leading to the maturation and secretion of IL-1β. Because previous studies indicated that extracellular Cl− exerts a negative allosteric effect on ATP-gating of P2X7R channels, we tested whether Cl− attenuates the P2X7R→caspase-1→IL-1β signaling cascade in murine and human macrophages. In Bac1 murine macrophages, substitution of extracellular Cl− with gluconate produced a 10-fold increase in the rate and extent of ATP-induced IL-1β processing and secretion, while reducing the EC50 for ATP by 5-fold. Replacement of Cl− with gluconate also increased the potency of ATP as an inducer of mature IL-1β secretion in primary mouse bone marrow-derived macrophages and in THP-1 human monocytes/macrophages. Our observations were consistent with actions of Cl− at three levels: 1) a negative allosteric effect of Cl−, which limits the ability of ATP to gate the P2X7R-mediated cation fluxes that trigger caspase-1 activation; 2) an intracellular accumulation of Cl− via nonselective pores induced by P2X7R with consequential repression of caspase-1-mediated processing of IL-1β; and 3) a facilitative effect of Cl− substitution on the cytolytic release of unprocessed pro-IL-1β that occurs with sustained activation of P2X7R. This cytolysis was repressed by the cytoprotectant glycine, permitting dissociation of P2X7R-regulated secretion of mature IL-1β from the lytic release of pro-IL-1β. These results suggest that under physiological conditions P2X7R are maintained in a conformationally restrained state that limits channel gating and coupling of the receptor to signaling pathways that regulate caspase-1.

1 Periodate-oxidized ATP (oATP), which covalently modifies nucleotide-binding proteins, can significantly attenuate proinflammatory signaling. Although the P2X 7 nucleotide receptor (P2X 7 R) is irreversibly antagonized by oATP, it is unclear whether anti-inflammatory actions of oATP are predominantly mediated via its actions on P2X 7 R. Here, we describe inhibitory effects of oATP on proinflammatory responses in three human cell types that lack expression of P2X 7 R: human umbilical vein endothelial cells (HUVEC), HEK293 cells, and 1321N1 astrocytes. 2 oATP decreased by 40 -70% the secretion of interleukin (IL)-8 stimulated by tumor necrosis factor-a (TNF-a) in all three cell types, by IL-1b in HUVEC and 1321N1 cells, and by endotoxin in HUVEC. Attenuation of TNF-a-stimulated IL-8 secretion by oATP was similar in wild-type HEK cells or HEK cells stably expressing recombinant P2X 7 R. 3 oATP also attenuated cytokine-stimulated expression of nuclear factor-kB-luciferase reporter genes expressed in HEK or 1321N1 cells, but did not affect the rapid downregulation of IkB. 4 oATP had no effect on uridine triphosphate-induced activation of native P2Y 2 receptors in HEK cells, but reduced the potency and efficacy of ADP as an agonist of native P2Y 1 receptors. However, inhibition of P2Y 1 receptors with the specific antagonist MRS2216 did not mimic the effects of oATP on TNF-a-stimulated IL-8 secretion. 5 Although 1321N1 astrocytes lack expression of any known P2 receptor subtypes, oATP markedly inhibited ecto-ATPase activity in these cells, resulting in a significant accumulation of extracellular ATP. 6 In summary, oATP can attenuate proinflammatory signaling by mechanisms independent of the expression or activation of known P2 receptor subtypes.