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P2X₇ Receptor Differentially Couples to Distinct Release Pathways for IL-1β in Mouse Macrophage¹

Pablo Pelegrin^*, Consuelo Barroso-Gutierrez and Annmarie Surprenant^2,*

^* Faculty of Life Science, University of Manchester, Manchester, United Kingdom; and Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom

The proinflammatory IL-1 cytokines IL-1, IL-1β, 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-1β release 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-1, IL-18, as well as IL-1β, by ATP resulted exclusively from activation of P2X₇R, release of all these IL-1 cytokines involved pannexin-1 (panx1), and that there was both a panx1-dependent and -independent component to IL-1β 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-1β independently of panx1 but do not release mature IL-1β because they do not express apoptotic speck-like protein with a caspase-activating recruiting domain 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-1β that was selectively blocked by inhibition of caspase-1 or panx1, and a calcium-independent, caspase-1/panx1-independent release of pro-IL-1β 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-1β.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by The Wellcome Trust and a postdoctoral fellowship (to P.P.) from AstraZeneca Charnwood, U.K.

² Address correspondence and reprint requests to Dr. Annmarie Surprenant, Faculty of Life Science, University of Manchester, Michael Smith Building D3308, Manchester M13 9PT, U.K. E-mail address: a.surprenant{at}manchester.ac.uk

³ Abbreviations used in this paper: ASC, apoptosis-associated speck-like protein containing a C-terminal caspase-activating recruiting domain; panx1, pannexin-1; AOM, 2,6-dimethylbenzoyloxymethyl ketone; LDH, lactate dehydrogenase; NIG, nigericin; MTX, maitotoxin.