Transient P2X 7 receptor activation triggers macrophage death independent of toll-like receptors 2 and 4, caspase-1, and pannexin-1 proteins

Hanley P., Kronlage M., Kirschning C., Del Rey A., Di Virgilio F., Leipziger J., Chessell I., Sargin S., Filippov M., Lindemann O., Mohr S., Königs V., Schillers H., Bähler M., Schwab A.

Abstract

The function of P2X 7 receptors (ATP-gated ion channels) in innate immune cells is unclear. In the setting of Toll-like receptor (TLR) stimulation, secondary activation of P2X 7 ion channels has been linked to pro-caspase-1 cleavage and cell death. Here we show that cell death is a surprisingly early triggered event. We show using live-cell imaging that transient (1-4 min) stimulation of mouse macrophages with high extracellular ATP ([ATP]e) triggers delayed (hours) cell death, indexed as DEV-Dase (caspase-3 and caspase-7) activity. Continuous or transient high [ATP]e did not induce cell death in P2X 7-deficient (P2X 7-/-) macrophages or neutrophils (in which P2X 7 could not be detected). Blocking sustained Ca 2+ influx, a signature of P2X 7 ligation, was highly protective, whereas no protection was conferred in macrophages lacking caspase-1 or TLR2 and TLR4. Furthermore, pannexin-1 (Panx1) deficiency had no effect on transient ATP-induced delayed cell death or ATP-induced Yo- Pro-1 uptake (an index of large pore pathway formation). Thus, "transient" P2X 7 receptor activation and Ca 2+ overload act as a death trigger for native mouse macrophages independent of Panx1 and pro-inflammatory caspase-1 and TLR signaling. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.