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A Key Role for Redox Signaling in Rapid P2X₇ Receptor-Induced IL-1β Processing in Human Monocytes¹

Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom

P2X₇ receptors (P2X₇Rs) are ATP-gated ion channels that trigger caspase-1 activation in the presence of TLR ligands. Inflammatory caspase-1 is responsible for the proteolytic activation of IL-1β. However, the signaling events that couple P2X₇Rs to caspase-1 activation remain undefined. In this study we demonstrate that ATP-induced cellular oxidation is critical for caspase-1 activation and subsequent IL-1β processing. Purinergic receptor stimulation, including P2X₇Rs, of endotoxin-primed human monocytes augments NADPH oxidase activity whereas concurrent purinergic receptor stimulation triggers protein denitroyslation, leading to the formation of peroxynitrite. IL-1β cleavage is blocked under conditions where superoxide anion formation is blocked or monocytes are treated with antioxidants or a peroxynitrite scavenger. Nigericin, a K⁺/H⁺ antiporter, also increases NADPH oxidase activity, leading to IL-1β and caspase-1 processing that is blocked by a peroxynitrite scavenger or inhibition of NADPH oxidase. These data demonstrate that signaling via NADPH oxidase activity is fundamental for the processing of mature IL-1β induced by P2X₇R stimulation.

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 grants from British Heart Foundation and Heart Research U.K.

² Address correspondence and reprint requests to Dr. Amanda B. MacKenzie, University of Bath, Claverton Down, Bath BA2 7AY, U.K. E-mail address: a.mackenzie{at}bath.ac.uk

³ Abbreviations used in this paper: P2X₇R, P2X₇ receptor; DCF, 2',7'-dichlorofluorescein; DCFH, 2',7'-dichlorodihydrofluorescein; DCFH-DA, DCFH diacetate; DPI, diphenyleneiodonium; EtBr, ethidium bromide; FeTPPS, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) chloride; KN-04, N-[1-[N-methyl-p-(5 isoquinoline-sulfonyl)benzyl]]-2-(4-phenylpiperazine)ethyl]-5-isoquinoline-sulfonamide; NAC, N-acetyl cysteine; NOS, NO synthase; RONS, reactive oxygen and nitrogen species; SOD, superoxide dismutase; z-YVAD-fmk, Tyr-Val-Ala-Asp-fluoromethyl ketone.