Elsevier

Free Radical Biology and Medicine

Volume 49, Issue 12, 15 December 2010, Pages 1834-1845
Free Radical Biology and Medicine

Review Article
Intracellular generation of superoxide by the phagocyte NADPH oxidase: How, where, and what for?

https://doi.org/10.1016/j.freeradbiomed.2010.09.016Get rights and content

Abstract

Professional phagocytes increase their consumption of molecular oxygen during the phagocytosis of microbes or when encountering a variety of nonparticulate stimuli. In these circumstances, oxygen is reduced by the phagocyte NADPH oxidase, and reactive oxygen species (ROS), which are important for the microbicidal activity of the cells, are generated. The structure and function of the NADPH oxidase have been resolved in part by studying cells from patients with chronic granulomatous disease (CGD), a condition characterized by the inability of phagocytes to assemble a functional NADPH oxidase and thus to produce ROS. As a result, patients with CGD have a predisposition to infections as well as a variety of inflammatory symptoms. A long-standing paradigm has been that NADPH oxidase assembly occurs exclusively in the plasma membrane or invaginations thereof (phagosomes). A growing body of evidence points to the possibility that phagocytes are capable of NADPH oxidase assembly in nonphagosomal intracellular membranes, resulting in ROS generation within intracellular organelles also in the absence of phagocytosis. The exact nature of these ROS-producing organelles is yet to be determined, but granules are prime suspects. Recent clinical findings indicate that the generation of intracellular ROS by NADPH oxidase activation is important for limiting inflammatory reactions and that intracellular and extracellular ROS production are regulated differently. Here we discuss the accumulating knowledge of intracellular ROS production in phagocytes and speculate on the precise role of these oxidants in regulating the inflammatory process.

Introduction

Neutrophils and monocytes are professional phagocytes that are central to innate host defense and are indispensable for the rapid eradication of pathogenic microbes. These phagocytes are equipped with a number of antimicrobial systems, including the NOX2-containing NADPH oxidase complex, which reduces molecular oxygen to microbicidal reactive oxygen species (ROS) [1], [2], [3]. The entire complex is sometimes incorrectly called NOX2, even though this name refers to only one of the subunits (gp91phox) that make up the complex. The enzyme complex is also commonly referred to as the phagocyte oxidase and is known to generate substantially higher levels of ROS than other cellular oxidases. Although other ROS-generating systems are present in phagocytes (e.g., NADPH oxidases based on NOX members other than NOX2 [4], nitric oxide synthases [5], as well as mitochondria [6]), this review mainly discusses ROS derived from the NOX2-containing NADPH oxidase.

Section snippets

Structure of the NADPH oxidase

The phagocyte oxidase is a multicomponent, electron-transfer complex (Fig. 1). Two of the subunits, p22phox (phox for phagocyte oxidase) and gp91phox (the subunit also known as NOX2), form a membrane-bound, heterodimeric flavohemoprotein referred to as cytochrome b (cytochrome b558). Cytochrome b constitutes the catalytic, electron-transferring part of the NADPH-oxidase. In the absence of cellular activation, the cytosolic components of the NADPH oxidase, namely p40phox, p47phox, and p67phox,

NADPH oxidase deficiencies

Mutations or deletions in the genes that encode the components of the NADPH oxidase result in a rare immunodeficiency known as chronic granulomatous disease (CGD), originally called “fatal granulomatous disease of childhood” [14]. In the 1960s, CGD was shown to be associated with decreased phagocytic oxygen consumption [15] as well as defective microbial killing [16], thus explaining the predisposition of CGD patients to infection. CGD patients also suffer from a variety of inflammatory

Measuring icROS

Over the years, a number of techniques have been developed to monitor phagocyte ROS production, all of which have their advantages and limitations. Because there are a number of recent reviews on the subject [23], [24], [25], [26], [27], [28] we do not include a detailed description of all these techniques here. However, in the context of icROS production, some aspects of commonly used techniques (Table 1) are important to mention.

Our definition of icROS for the purpose of this discussion is

Phagolysosomal ROS production

Neutrophil defense against infecting microorganisms largely depends on the phagocytic process, initiated by invagination of the plasma membrane and resulting in a membrane-enclosed phagosome. The phagosome is further processed through heterotypic fusion of granules (gelatinase, specific, and azurophil granules) forming the mature phagolysosome [43] (Fig. 2A). Matrix components stored in the granules are delivered into the phagolysosome concomitant with a mixing of the membranes. Plasma

Particulate stimuli and their receptors

There are a number of physiological and nonphysiological stimuli that, through receptor ligation (or other mechanisms), induce icROS production detected as luminol-amplified CL (in the presence of extracellular scavengers) or DHR-123 activity (Fig. 3). As previously mentioned, the most obvious stimulant of icROS production is the uptake of phagocytic particles into a phagosome (Fig. 2A). Receptors that trigger phagocytic uptake in neutrophils include Fcγ receptors that recognize opsonizing

Signals for NADPH oxidase activation

To decipher the intracellular signaling network that regulates NADPH oxidase activity, reductionist systems have been set up to define the contribution of individual molecules. Several established signaling pathways have been shown to take part, in parallel and in conjunction with one another, ultimately leading to the translocation of the cytosolic NADPH oxidase components to cytochrome b and activation of the complete NADPH oxidase.

The most extensively studied signal transduction pathways in

Specific signals for icROS

Although signaling to the NADPH oxidase is complex and employs a number of pathways, the selective role for p40phox in icROS production [22] indicates that there are differences in the signaling routes and/or in the docking domain composition between the different cytochrome b-containing membranes (plasma membrane and granule membranes). This is in line with in vitro studies on the role of p40phox in icROS production induced by phagocytosis (phROS) and PMA (nphROS) [88], [89], [90], [91].

We

Biological consequences of altered phagocyte ROS production

To our knowledge, no human disorder has been characterized in which phagocytes display higher than normal ROS production due to aberrant function/assembly of the NADPH oxidase. Instead, the functions of NADPH-oxidase-derived ROS have to a large extent been elucidated from the study of patients with phagocytes that lack a functional NADPH oxidase and produce no or subnormal levels of ROS. As mentioned, the most important disease for characterization of the NADPH oxidase is CGD, a primary

ROS and inflammatory signaling

The recent reports describing exuberant, aseptic inflammatory manifestations accompanied by a selective loss of phagocyte nphROS in SAPHO [133] and p40phox CGD [22] have opened up the possibility that these nonphagosomal, intracellular radicals are key suppressors of inflammation. Exactly how nphROS could dampen inflammatory reactions is still unknown, but cellular oxidants are known to influence a variety of cell signaling pathways, some of which are highly involved in inflammation, by

Concluding remarks

The research community is gradually opening up to the existence of nphROS, i.e., retained, intravesicular ROS that are produced independent of phagosome formation. We foresee expansion and new directions in this field of research in the close future, not least thanks to recent clinical findings that indicate the biological and clinical importance of nphROS. The findings further implicate the importance of knowing the limitations of existing techniques for ROS measurements, both experimentally

Acknowledgments

This work was supported by the Swedish Medical Research Council (J.B., C.D., A.K.), the King Gustaf V Memorial Foundation (J.B., C.D., A.K.), the European Community's Seventh Framework Programme (FP7/2007–2013) under Grant Agreement 221094 (J.B./K.L.B.), the Ingabritt and Arne Lundberg Research Foundation, and the Swedish state under the LUA-ALF agreement. We thank My Erwander for expert assistance in preparing the figures.

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