H2O2 Release from Human Granulocytes during Phagocytosis

Abstract

A B S T R A C T Normal and cytochalasin B-treated human granulocytes have been studied to determine some of the interrelationships between phagocytosisinduced respiration and superoxide and hydrogen peroxide formation and release into the extracellular medium by intact cells. By using the scopoletin fluorescent assay to continuously monitor extracellular hydrogen peroxide concentrations during contact of cells with opsonized staphylococci, it was demonstrated that the superoxide scavengers ferricytochrome c and nitroblue tetrazolium significantly reduced the amount of H202 released with time from normal cells but did not abolish it. This inhibitory effect was reversed by the simultaneous addition of superoxide dismutase (SOD), whereas the addition of SOD alone increased the amount of detectable H202 in the medium. The addition of sodium azide markedly inhibited myeloperoxidase-H202-dependent protein iodination and more than doubled H202 release, including the residual amount remaining after exposure of the cells to ferricytochrome c, suggesting its origin from an intracellular pool shared by several pathways for H202 catabolism. When cells were pretreated with cytochalasin B and opsonized bacteria added, reduced oxygen consumption was observed, but this was in parallel to a reduction in specific binding of organisms to the cells when compared to normal. Under the influence of inhibited phagosome formation by cytochalasin B, the cells released an increased amount of superoxide and peroxide into the extracellular medium relative to oxygen consumption, and all detectable peroxide release could be inhibited by the addition of ferricytochrome c. Decreased H202 production in the presence of this compound could not be ascribed to diminished bacterial binding, decreased oxidase activity, or increased H202 catabolism and was reversed by the simultaneous addition of SOD. Furthermore, SOD and ferricytochrome c had similar effects on another H202-dependent reaction, protein iodination, in both normal and cytochalasin B cells. When oxygen consumption, 02., and H202 release were compared in the presence of azide under identical incubation conditions, the molar relationships for normal cells were 1.00:0.34:0.51 and for cytochalasin B-treated cells 1.00:0.99:0.40, respectively. Nonopsonized, or opsonized but disrupted, bacteria did not stimulate any of these metabolic functions.The results indicate that with normal cells approximately 50% of H202 released during phagocytosis is derived directly from 02: by dismutation, the remainder appearing from an (intra)cellular source shared by azide-inhibitable heme enzymes. With cytochalasin B treatment the evidence is consistent with the derivation of all H202 from an 02: precursor which is released from the cell surface. Furthermore, when activated by phagocytic particle

The antioxidant action of Polypodium leucotomos extract and kojic acid: reactions with reactive oxygen species

“…Hydrogen peroxide (H 2 O 2 ) was detected by the scopoletin peroxidase assay (27,32). Scopoletin (5 µM) was added to a 1-cm light path cuvette containing 50 µl of 1.0 mg/ml HPO and the antioxidants PL and KA (0.0 to 0.1 mg/ml) in 150 mM KH 2 PO 4 -KOH buffer, pH 7.4.…”

“…Scopoletin (5 µM) was added to a 1-cm light path cuvette containing 50 µl of 1.0 mg/ml HPO and the antioxidants PL and KA (0.0 to 0.1 mg/ml) in 150 mM KH 2 PO 4 -KOH buffer, pH 7.4. The cuvettes were maintained for 5 min at 37ºC (27), amounts of H 2 O 2 (0.1 mM) were added and H 2 O 2 was quantified after 5 min of incubation using a fluorescence spectrophotometer (32). The excitation light was set at 350 nm.…”

“…The intensity of the emission fluorescence at 460 nm was monitored and recorded continuously with a HITACHI F-4500 spectrofluorometer. The measurement was obtained in triplicate and the system was standardized in the absence of antioxidants with a known amount of peroxide (27,32).…”

The Effect of an SOD Derivative (SM-SOD) Administration in a Burned Rat Model.

“…Histological examination revealed a fairly large number of neutrophils not only in the burned skin, but also in such remote organs as the lung and kidney, particularly 3 hr after burn injury (data not shown). In the target organs, oxygen-free radicals may be produced by the invading leukocytes, which, upon activation, release large amounts of the superoxide radical (02-) (Root and Metcalf 1977).…”

Ligation of CR1 attenuates Fc receptor-mediated myeloperoxidase release and HOCl production by neutrophils

“…Hydrogen peroxide generation was determined by activating neutrophils with or without SAIgG in the presence of 1.0 mM sodium azide, 50 µM scopoletin, and 5 µg/mL horseradish peroxidase [17]. The supernatants were analyzed in a spectrophotofluorometer with an excitation wavelength of 350 nm and the change in emitted 460-nm wavelength was measured.…”