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P2X7 Receptor-Dependent Blebbing and the Activation of Rho-Effector Kinases, Caspases, and IL-1β Release1

In response to ATP binding, the P2X7R facilitates cation channel activation, nonspecific pore formation, rapid changes in plasma membrane morphology, and secretion of IL-1β from LPS-primed macrophages. To investigate the relationship between the P2X7R-dependent changes in plasma membrane organization and the release of IL-1β, we generated time-lapse movies of ATP-stimulated BAC1 murine macrophages in conjunction with biochemical analyses of IL-1β release. Similar image analyses in human embryonic kidney 293 cells expressing recombinant P2X7R (HEK-P2X7) permitted comparison of P2X7R-dependent effects in macrophage vs nonmacrophage backgrounds. Whereas HEK-P2X7 cells exhibit zeiotic blebbing within 5 min of ATP treatment, BAC1 macrophages initiated a distinct “tethered” blebbing 10 min after ATP addition. This blebbing was comparably induced by the P2X7R-selective agonist BzATP and was blocked by P2X7R inhibitors KN-62 and oxidized ATP. Blebbing was initiated at ATP concentrations ≥3 mM, but optimal IL-1β release occurred at 1 mM ATP. P2X7R-dependent blebbing was abrogated in the presence of Rho-effector kinase inhibitors Fasudil and Y-27632, but ATP-induced IL-1β release was unaffected. ATP-induced activation of RhoA could be detected in both HEK-P2X7 cells and BAC1 murine macrophages. Thus, P2X7R activation signals distinct, novel membrane blebbing events (dependent on RhoA activation and Rho-effector kinase activity) and simultaneously initiates release of IL-1β. Our observations that blebbing and IL-1β release are dissociable suggest these events occur via parallel rather than convergent signaling pathways.