Recently, we showed that antibodies catalyze the generation of hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*) and water. Here, we show that this process can lead to efficient killing of bacteria, regardless of the antigen specificity of the antibody. H2O2 production by antibodies alone was found to be not sufficient for bacterial killing. Our studies suggested that the antibody-catalyzed water-oxidation pathway produced an additional molecular species with a chemical signature similar to that of ozone. This species is also generated during the oxidative burst of activated human neutrophils and during inflammation. These observations suggest that alternative pathways may exist for biological killing of bacteria that are mediated by potent oxidants previously unknown to biology.

T he antimicrobial activity of phagocytes depends in part on the cells' ability to reduce oxygen to reactive microbicidal oxidants by means of an NADPH oxidase. In resting phagocytes, the NADPH oxidase is in a dormant state, but exposure of the cell to any of a variety of stimuli can activate the enzyme, causing it to release large amounts of O 2 Ϫ by reducing oxygen at the expense of NADPH. The oxidase is activated by the phosphorylation of one of its cytosolic subunits, p47 PHOX , on particular serines (1). In whole cells, the stimulation of an appropriate receptor activates PKC and phosphatidylinositol 3-kinase (PI3-kinase) (2-4). Although NADPH oxidase activation by PKC has been well characterized (5, 6), the association between PI3-kinase and NADPH oxidase activation remains to be established. A possible connection is through the activation of Akt (7), whose role in oxidase activation is suggested by the finding that Akt is activated rapidly when neutrophils are treated with oxidase-activating agents (8) and by the finding that oxidase activation is inhibited by wortmannin (9). The experiments described here show that Akt is able to activate the oxidase by phosphorylating p47 PHOX on serines S304 and S328. Experimental ProceduresMaterials. Active Akt was obtained from Upstate Biotechnology (Lake Placid, NY) or from J.-H.L., whose recombinant material was 95% pure by SDS͞PAGE. [␥-32 P]ATP was purchased from New England Nuclear. Phosphorylated peptides were obtained from Sigma.Isolation and Fractionation of Neutrophils. Neutrophils were obtained from normal subjects by dextran sedimentation and Ficoll͞Hypaque fractionation of freshly drawn citrateanticoagulated blood (10). After treatment on ice for 10-20 min with 5 l of 0.54 M diisopropyl fluorophosphate, the neutrophils, suspended at 10 8 cells per ml in a modified relaxation buffer (0.1 M KCl͞3 mM NaCl͞3.5 mM MgCl 2 ͞10 mM Pipes buffer, pH 7.3), were subjected to nitrogen cavitation. Membranes and cytosol were separated by centrifugation through a Percoll gradient. Aliquots of membrane and cytosol were stored at Ϫ70°C until use.Recombinant Oxidase Components. Recombinant fusion proteins composed of an upstream GST linked to a downstream p47 PHOX , p67 PHOX , or Rac2 were isolated from Escherichia coli transformed with pGEX-6P3 plasmids containing cDNA inserts encoding the downstream proteins. The fusion proteins were isolated by the addition of a prewashed 50% slurry of glutathione-Sepharose 4B (Amersham Biosciences) in PBS (133 mM NaCl͞16 mM Na 2 HPO 4 ͞2.7 mM KCl͞1.5 mM KH 2 PO 4 , pH 7.3). The tube was rotated end-over-end for 1 h at 4°C and then microcentrifuged for 3 min at 200 ϫ g to sediment the glutathione-Sepharose beads. The beads were washed four times with 10 bead volumes of PBS, and the bound fusion proteins were eluted by incubating for 30 min at 4°C with three 1-ml washes of 50 mM Tris⅐HCl, pH 8.0͞20 mM glutathione͞0.2 M NaCl. Excess glutathione was removed from the purified recombinant protein by dialysis against relaxation buffer and conce...

Recent studies have suggested that antibodies can catalyze the generation of previously unknown oxidants including dihydrogen trioxide (H 2O3) and ozone (O3) from singlet oxygen ( 1 O 2 * ) and water. Given that neutrophils have the potential both to produce 1 O 2 * and to bind antibodies, we considered that these cells could be a biological source of O 3. We report here further analytical evidence that antibody-coated neutrophils, after activation, produce an oxidant with the chemical signature of O 3. This process is independent of surface antibody concentration down to 50% of the resting concentration, suggesting that surface IgG is highly efficient at intercepting the neutrophil-generated 1 O 2 * . Vinylbenzoic acid, an orthogonal probe for ozone detection, is oxidized by activated neutrophils to 4-carboxybenzaldehyde in a manner analogous to that obtained for its oxidation by ozone in solution. This discovery of the production of such a powerful oxidant in a biological context raises questions about not only the capacity of O 3 to kill invading microorganisms but also its role in amplification of the inflammatory response by signaling and gene activation. N eutrophils (PMNs) are the most abundant leukocytes in the bloodstream. Their function is the killing of bacteria and fungi, in part by the triggering of an oxidative burst that is composed of a set of enzymatic and chemical reactions ultimately leading to the formation of hypohalous acid, 1 O 2 * , and hydroxyl radical (HO • ) (1, 2). The first step in this cascade, the reduction of dioxygen, is initiated by the enzyme NAD(P)H oxidase. This oxidase is a complex enzyme composed of five components: gp91 phox (with phox being phagocyte oxidase), a heavily glycosylated 56-kDa protein that contains the electroncarrying components of the oxidase; p67 phox , p47 phox , and p22 phox , which are proteins named according to their approximate molecular weights; and rac2, a low molecular weight GTPase. In the resting cell, p47 phox and p67 phox form a complex in the cytosol (which also contains p40 phox , a protein whose effect on oxidase activity is unclear), whereas gp91 phox and p22 phox are in the membrane. When the PMN is activated by antibody-coated bacteria, p47 phox is phosphorylated on particular serines and moves to the membrane to assemble the active oxidase, carrying with it its cargo of p67 phox and the enigmatic p40 phox . Rac 2, also necessary for oxidase activity, picks up a GTP and moves into the oxidase assembly. The NAD(P)H oxidase then produces superoxide anion (O 2•Ϫ ) (Eq.

The leukocyte NADPH oxidase catalyzes the production of O(2)(-) from oxygen at the expense of NADPH. Activation of the enzyme requires interaction of the cytosolic factors p47(PHOX), p67(PHOX), and Rac2 with the membrane-associated cytochrome b(558). Activation of the oxidase in a semirecombinant cell-free system in the absence of an amphiphilic activator can be achieved by phosphorylation of the cytosolic factor p47(PHOX) by protein kinase C. Another cytosolic factor, p40(PHOX), was recently shown to be phosphorylated on serine and threonine residues upon activation of NADPH oxidase, but both stimulatory and inhibitory roles were reported. In the present study, we demonstrate that the addition of phosphorylated p40(PHOX) to the cell-free system inhibits NADPH oxidase activated by protein kinase C-phosphorylated p47(PHOX), an effect not observed with the unphosphorylated p40(PHOX). Moreover phosphorylated p40(PHOX) inhibits the oxidase if added before or after full activation of the enzyme. Direct mutagenesis of protein kinase C consensus sites enables us to conclude that phosphorylation of threonine 154 is required for the inhibitory effect of p40(PHOX) to occur. Although the phosphorylated mutants and nonphosphorylated mutants are still able to interact with both p47(PHOX) and p67(PHOX) in pull-down assays, their proteolysis pattern upon thrombin treatment suggests a difference in conformation between the phosphorylated and nonphosphorylated mutants. We postulate that phosphorylation of p40(PHOX) on threonine 154 leads to an inhibitory conformation that shifts the balance toward an inhibitory role and blocks oxidase activation.