Investigating antibody-catalyzed ozone generation by human neutrophils

Abstract

Recent studies have suggested that antibodies can catalyze the generation of previously unknown oxidants including dihydrogen trioxide (H(2)O(3)) and ozone (O(3)) 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.

Figure 1

Oxygen-dependent microbicidal action of PMNs with the new antibody-catalyzed water-oxidation pathway shown. MPO, myeloperoxidase.

Figure 2

Fluorescence-activated cell sorter analysis of human PMNs. (A) Human PMNs ± secondary goat anti-human FITC-labeled antibody (FITC-labeled antibody, mean fluorescence 31.75 arbitrary units). (B) Human PMNs after acid treatment (pH 4.0) + secondary anti-human FITC-labeled antibody (mean fluorescence 14.61 arbitrary units). (C) Human PMNs after heating to 37°C + secondary anti-human FITC-labeled antibody (mean fluorescence 16.45 arbitrary units).

Figure 3

Oxidation of indigo carmine 1 to isatin sulfonic acid 2.

Figure 4

Oxidation of indigo carmine by activated human PMNs. (A) Effect of surface IgG concentration. ♦, unactivated PMNs; ▴, PMNs activated with PMA after acid treatment (pH 4.0); □, PMA-activated PMNs. (B) Effect of catalase on the time course of indigo carmine bleaching. ♦, unactivated PMNs; ■, PMNs activated with PMA and no catalase; ▴, PMNs activated with PMA and 100 units/ml catalase. (C) Effect of catalase on isatin sulfonic acid 2 (ISA) formation. ♦, PMA-activated PMNs and 100 units/ml catalase; □, PMA-activated PMNs and no catalase.