Inhibitory Effects of Chloride on the Activation of Caspase-1, IL-1β Secretion, and Cytolysis by the P2X7 Receptor

Abstract

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.