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.

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.

A panel of monoclonal antibodies to the 69 kDa glycosyl phosphatidylinositol anchored lymphocyte differentiation antigen ecto-5'-nucleotidase (ecto-5'-NT, CD73) was produced using highly purified human placental 5'-NT as immunogen. Antibodies 1E9.28.1 and 7G2.2.11 inhibit soluble placental 5'-NT activity and recognize lymphocyte CD73 in indirect immunofluorescence and immunoprecipitation assays. In addition, 1E9.28.1 induces vigorous T cell proliferation in the presence of submitogenic doses of phorbol myristate and F(ab')2 goat anti-mouse Ig. Both antibodies can be used to purify the three major forms of placental 5'-NT by affinity chromatography. By two-color immunofluorescence, CD73 was found to be expressed on 19 +/- 5% of CD3+, 11 +/- 4% of CD4+, 51 +/- 14% of CD8+, 25 +/- 8% of CD28+, 15 +/- 5% of CD29+, 27 +/- 7% of CD45RA+, and 70 +/- 6% of CD19+ lymphocytes. Within T cells, CD73 expression is restricted to the CD28+ subset. Thus, CD73 is found on subsets of both T and B lymphocytes, with the highest expression on B cells and CD8+ T cells. In sections of hyperplastic tonsil, CD73 expression is restricted to the small lymphocytes of the follicular mantle zone, a small subset of extrafollicular lymphocytes situated within the epithelium of the tonsillar crypt, and to follicular dendritic cells within the lower part of the "light-zone." CD73 is also detected on subsets of endothelial cells of capillaries and venules and the basal layer of non-keratinizing squamous epithelium and transitional cell type mucosa of many tissues. Given the tissue distribution of CD73, along with its glycosyl phosphatidylinositol membrane anchoring and the observation that some CD73 antibodies are mitogenic, we propose that this interesting antigen may play a role in cell activation, lymphocyte homing, and/or cell adhesion.

Resting and phorbol-activated human neutrophils were separated by treatment with Triton X-100 into detergent-extractable and cytoskeleton fractions. Respiratory burst oxidase activity was restricted entirely to the cytoskeleton. The cytoskeleton also contained 15% of the neutrophil cytochrome b5!, an oxidase-associated heme protein, as well as most of the oxidase-related cytosolic polypeptide p67x. In contrast, the components of the oxidase-associated phosphoprotein family p47'" were found almost exclusively in the detergent extract, suggesting that p47' is needed for oxidase activation but not for 02 production by the activated oxidase. Activation of the oxidase had no apparent effect on the distribution of any of these species between the cytoskeleton and the detergent extract. Our results support earlier studies implying that the cytoskeleton participates in an important way in regulating the activity of the 02-forming respiratory burst oxidase of neutrophils.