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NADPH Oxidase NOX2 Mediates Rapid Cellular Oxidation following ATP Stimulation of Endotoxin-Primed Macrophages¹

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

The phagocytic NADPH oxidase (NOX2) plays a fundamental role in host defense and innate immunity. Here we demonstrate that external ATP triggers rapid cellular oxidation inhibited by diphenyleneiodonium in endotoxin-primed J774 macrophages and primary murine bone marrow-derived macrophages. To identify the source of reactive oxygen species (ROS), we compared responses between wild-type and NOX2-deficient macrophages. ATP-mediated ROS production was strongly attenuated in NOX2-deficient macrophages where responses were comparable to inhibition with diphenyleneiodonium. Notably, spatial differences in superoxide anion formation were observed where ROS formation was partially antagonized by extracellular superoxide dismutase in primary bone marrow-derived macrophages but unaffected in J774 macrophages. Loss of NOX2 was not observed to affect ATP-induced cell death. However, ATP-evoked cell death was found to be partially dependent on caspase-1 and cathepsin B activation. In conclusion, NOX2 plays a fundamental role in conferring macrophages with the ability to respond to extracellular ATP stimulation with robust changes in cellular oxidation.

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 the University of Bath and Heart Research UK.

² Address correspondence and reprint requests to Dr. Amanda B. MacKenzie, Department of Pharmacy and Pharmacology, 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: ROS, reactive oxygen species; ASC, apoptosis-associated speck-like protein containing a caspase-recruitment domain; BMDM, bone marrow-derived macrophage; DPI, diphenyleneiodonium; EtBr, ethidium bromide; H₂DCFDA, 2',7'-dichlorodihydrofluorescein diacetate; LDH, lactate dehydrogenase; NAC, N-acetylcysteine; P2X7R, P2X7 receptor; SOD, superoxide dismutase; Z-YVAD-FMK, Tyr-Val-Ala-Asp-fluoromethyl ketone.

⁴ The online version of this article contains supplemental material.