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Cell-Free NADPH Oxidase Activation Assays: “In Vitro Veritas”

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Neutrophil Methods and Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1124))

Abstract

The superoxide (O2 ∙−)-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22phox) and four cytosolic regulatory proteins, p47phox, p67phox, p40phox, and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 ∙− generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron “flow” along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 ∙−. Methodological difficulties in the dissection of this complex mechanism led to the design “cell-free” systems (also known as “broken cells” or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of “NADPH oxidase assembly,” thus on the formation of the complex between cytochrome b 558 and the cytosolic components and the resulting O2 ∙− generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purified recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantified by measuring the primary product of the reaction, O2 ∙−, trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O2 ∙− production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount role in the identification and characterization of the components of the NADPH oxidase complex, the deciphering of the mechanisms of assembly, the search for inhibitory drugs, and the diagnosis of various forms of chronic granulomatous disease (CGD).

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Acknowledgments

The research described in this report was supported by the Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, the Ela Kodesz Institute of Host Defense against Infectious Diseases, Israel Science Foundation Grants 428/01, 19/05, and 49/09, the Roberts-Guthman Chair in Immunopharmacology, the Walter J. Levy Benevolent Trust, the Roberts Fund, the Milken—Lowell Fund, the Wallis Foundation, the Rubanenko Fund, and the Joseph and Shulamit Salomon Fund. It is important to point out that the cell-free system was discovered almost simultaneously by several investigators. R.A. Heyneman and R.E. Vercauteren, in Belgium, and Linda McPhail and John Curnutte, in the USA, each independently, contributed greatly to the birth of the “cell-free” paradigm. Edgar Pick would like to thank his fellow scientists, too many to name, who provided materials and invaluable advice, for making this work possible. There is no greater satisfaction than the realization of the fact that, on so many occasions, what started as collaboration (called “networking” these days) or competition, evolved into long-lasting friendship.

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  1. The Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research and the Ela Kodesz Institute of Host Defense against Infectious Diseases, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

    Edgar Pick

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  1. Edgar Pick
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  1. Montana State University Dept. Veterinary Molecular Biology, Bozeman, Montana, USA

    Mark T. Quinn

  2. Lab. Human Bacterial Pathogenesis, National Institutes of Health Nat. Inst. Allergy & Infectious Diseases, Hamilton, Montana, USA

    Frank R. DeLeo

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Pick, E. (2014). Cell-Free NADPH Oxidase Activation Assays: “In Vitro Veritas”. In: Quinn, M., DeLeo, F. (eds) Neutrophil Methods and Protocols. Methods in Molecular Biology, vol 1124. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-845-4_22

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  • DOI: https://doi.org/10.1007/978-1-62703-845-4_22

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