282 deprotonated form of hypochlorous acid (OCl-) can react to form 1O2 (Steinbeck et al. 1992). H2O2 þ 2OCl !1 O2 þ H2O2 þ 2Cl More recently it was shown that 1O2 and H2O2 react in an antibody catalyzed reaction to form ozone and that ozone and H2O2 can react to form hydroxyl radical and superoxide (Wentworth et al. 2002). 21O2 þ H2O ! H2O2 þ O3 O3 þ H2O2 ! OH þ O2 þ O2 þ Hþ The NADPH oxidase Physiological and biochemical changes associated with leukocyte phagocytosis were noted some time ago (Bald- ridge and Gerard 1933; Sbarra and Karnovsky 1959; Iyer et al. 1961). It was subsequently shown that superoxide is a product of the respiratory burst and that superoxide pro- duction is defective in neutrophils from patients with the genetic disease chronic granulomatous disease (CGD) (Babior et al. 1973; Curnutte et al. 1974). However, a fuller understanding of the underlying mechanisms associated with the respiratory burst had to await a number of metho- dological and technological advances. Neutrophils from CGD patients are unable to generate normal amounts of O2- owing to the loss of components of the NADPH-oxidase complex (reviewed in Segal et al. 2000; Heyworth et al. 2003; Malech and Hickstein 2007). Regulation of the activation of the NADPH oxidase in neutrophils is exquisite since the oxidase is dormant in resting cells but is rapidly activated following appropriate stimulation of these cells. Due to the toxicity of its products, it is not surprising that the NADPH oxidase is highly regu- lated and limited in its sites of action. Physiological stimuli capable of activating the neutrophil oxidase include phago- cytizable particles such as bacteria and yeast, certain molecules that induce chemotaxis in these cells, certain bioactive lipids, and antibodies. The chemotatic peptides, such as f-Met-Leu-Phe, have served as models for the study of receptor-ligand mediated activation of the NADPH oxi- dase. Other ‘‘non-physiological’’ agents can also activate the oxidase and have been important in understanding the regu- lation of the phagocyte NADPH oxidase. This latter group of compounds includes certain phorbol esters, fatty acids, and retinoids as well as sodium ﬂuoride. The time course of activation of the oxidase and the amounts of reactive oxygen species produced varies depending on the agent used to stimulate cells and activate the oxidase. The rapid activation of the oxidase and the response of neutrophils to agonists can be appreciated by comparing the kinetics of the production 123 Histochem Cell Biol (2008) 130:281–297 Fig. 1 The levels of O-2 produced and the rate at which it is produced can vary depending upon the stimulus. Real-time kinetics of O2- production by thioglycolate-elicited guinea pig neutrophils stimulated in three different ways is shown; the cytochrome c reduction spectrophotometric assay was employed. Representative curves of cells stimulated with either (A) NaF (12.5 mM) plus AlCl3 (100 lM), (B) the chemotatic peptide f-Met-Leu-Phe (10-7 M), or (C) a combination of f-Met-Leu-Phe and NaF plus AlCl3 at the same concentrations used in curves A and B. Cells (1 9 106) were added and allowed to equilibrate at 37 C for 3 min before addition of the stimulus; during that time the resting cells did not generate O2-. Following the equilibration period, a stimulus was added (arrow). With NaF plus AlCl3 there was a long lag period prior to the release of modest amounts of O2- (curve A). This lag period is likely due to the slow entry of the stimulus into the cells. The lag period was eliminated when cells were permeabilized by electroporation in the presence of NaF plus AlCl3 (Hartﬁeld and Robinson 1990). When cells were stimulated with the chemotatic peptide, production of O2- was essentially instantaneous and was largely shut down by 5 min post stimulation (curve B). When cells were stimulated by a combination of f-Met-Leu-Phe and NaF plus AlCl3, at the same concentrations used in curves A and B, the initial rate of O2- production was essentially the same as that for the chemotatic peptide alone; however, the shut off of the f-Met- Leu-Phe stimulated O2- was abrogated and the cells continued to produce O2- at a high rate throughout the course of the incubation period. This experiment shows that: (1) resting neutrophils in suspension do not generate O2-; (2) activation of the NADPH oxidase and the production of O2- can be very rapid in intact neutrophils; and (3) different stimulatory conditions can lead to different levels of O2- production and the kinetics of O2- production vary with different stimulatory conditions. The continuous spectrophotometric assay for O2- production was as we have described previously (Robinson et al. 1987)