Histochem Cell Biol (2008) 130:281–297 DOI 10.1007/s00418-008-0461-4 R E V I E W Reactive oxygen species in phagocytic leukocytes John M. Robinson Accepted: 5 June 2008 / Published online: 3 July 2008 Springer-Verlag 2008 Abstract Phagocytic leukocytes consume oxygen and generate reactive oxygen species in response to appropriate stimuli. The phagocyte NADPH oxidase, a multiprotein complex, existing in the dissociated state in resting cells becomes assembled into the functional oxidase complex upon stimulation and then generates superoxide anions. Biochemical aspects of the NADPH oxidase are brieﬂy discussed in this review; however, the major focus relates to the contributions of various modes of microscopy to our understanding of the NADPH oxidase and the cell biology of phagocytic leukocytes. Keywords NADPH oxidase Respiratory burst Phagocytosis Neutrophils Cytochemistry Introduction Phagocytic leukocytes, when appropriately stimulated, consume oxygen and produce superoxide (O2-) in a process often referred to as the respiratory burst. Much of the work that has led to our current understanding of the regulatory events and the molecular organization underlying the respiratory burst is derived from studies of neutrophils, the This paper is dedicated to the memory of late John A. Badwey, an insightful and creative scientist, with whom the author enjoyed a fruitful collaboration studying neutrophil biology that spanned over 20 years. J. M. Robinson (&) Department of Physiology and Cell Biology, Ohio State University, Columbus, OH 43210, USA e-mail: robinson.21@osu.edu most abundant of the phagocytic cells in blood. Therefore, this review will likewise focus on the neutrophil. The respiratory burst is mediated by the NADPH-oxi- dase complex, a multicomponent system that is rapidly assembled following activation of neutrophils. As we have discussed previously, the neutrophil NADPH oxidase pro- duces O2- and hydrogen peroxide (H2O2) following activation (Robinson et al. 2004). The NADPH oxidase catalyzes the production of O2-: NADPH þ 2O2 ! NADPþ þ 2O2 + Hþ A dismutation reaction occurs with O2- to produce peroxide: O2 þ O2 þ 2Hþ ! H2O2 þ O2 Both H2O2 and O2- are components of the oxygen-depen- dent, antimicrobial system of phagocytic leukocytes (e.g., Robinson and Badwey 1995). In part, the cytotoxic effects of O2- and H2O2 relate to their ability to react with products of other microbicidal systems in these cells to generate additional reactive oxygen species (ROS) [e.g., hydroxyl radical (OH ), singlet oxygen 1 ( O2), ozone (O3)]. Superoxide can react with either hypo- chlorous acid (HOCl) or nitric oxide (NO ) to produce OH (Beckman et al. 1990; Ramos et al. 1992). O2 þ HOCl ! OH þ O2 þ Cl O2 þ NO þ Hþ ! OH þ NO2 Hypochlorous acid and nitric oxide are produced in phagocytic leukocytes by myeloperoxidase and nitric oxide synthase, respectively (Harrison and Schultz 1976; Xie et al. 1992). Singlet oxygen can form by nonenzymatic dismutation of superoxide (Corey et al. 1987). Additionally, hydrogen peroxide and the 123