P2X7 receptor differentially couples to distinct release pathways for IL-1b in mouse macrophage

Abstract

The pro-inflammatory IL-1 cytokines, IL-1a, IL-1b and IL-18, are key mediators of the acute immune response to injury and infection. Mechanisms underlying their cellular release remain unclear. Activation of purinergic P2X7 receptors (P2X7R) by extracellular ATP is a key physiological inducer of rapid IL-1brelease from LPS-primed macrophage. We investigated patterns of ATP-mediated release of IL-1 cytokines from three macrophage types in attempts to provide direct evidence for or against distinct release mechanisms. We used peritoneal macrophage from P2X7R-/- mice and found that release of IL-1a, IL-18, as well as IL-1b, by ATP resulted exclusively from activation of P2X7R, that release of all these IL-1 cytokines involved pannexin-1 (panx1), and that there was both a panx1-dependent and independent component to IL-1b release. We compared IL-1 release patterns from LPS-primed peritoneal macrophage, RAW264.7 macrophage and J774A.1 macrophage. We found RAW264.7 macrophage readily release pro-IL- 1b independently of panx1 but do not release mature IL-1b because they do not express apoptotic speck-like protein with a caspase-activating recruiting domain (ASC) and so have no caspase-1 inflammasome activity. We delineated two distinct release pathways: the well-known caspase-1 cascade mediating release of processed IL-1b that was selectively blocked by inhibition of caspase-1 or panx1, and a calcium-independent, caspase-1/panx1-independent release of pro-IL-1b that was selectively blocked by glycine. None of these release responses were associated with cell damage or cytolytic effects. This provides the first direct demonstration of a distinct signaling mechanism responsible for ATP-induced release of pro-IL-1b.