Ascorbate-mediated enhancement of reactive oxygen species generation from polymorphonuclear leukocytes: modulatory effect of nitric oxide

Abstract

Recent studies from our laboratory have demonstrated that ascorbate potentiated enzymatic synthesis of nitric oxide (NO) from polymorphonuclear leukocytes (PMNs). NO is known to modulate various function of PMNs such as chemotaxis, adherence, aggregation, and generation of reactive oxygen species (ROS). The role of ascorbate in the PMN phagocytosis, ROS generation, and apoptosis was thus evaluated in the present study. Ascorbate and its oxidized and cell-permeable analog, dehydroascorbate (DHA), did not affect the phagocytosis but enhanced ROS generation and apoptosis following treatment with Escherichia coli or arachidonic acid. A detailed investigation on the DHA-mediated response indicated that inhibitors of DHA uptake, reduced nicotinamide adenine dinucleotide phosphate oxidase, NO synthase, or ROS scavengers attenuated ROS generation. In DHA-treated cells, enhanced generation of peroxynitrite was also observed; thus, ascorbate-mediated ROS and reactive nitrogen species generation might mediate cytotoxicity toward the ingested microbes and subsequently, augmented PMN apoptosis. Results of the present study have helped in delineating the role of ascorbate in the modulation of NO-mediated ROS generation from PMNs.

Nitric oxide‐mediated augmentation of neutrophil reactive oxygen and nitrogen species formation: Critical use of probes

“…3). DCF is a commonly used probe to detect the oxidative stress in various types of cells including neutrophils (22,28). DCF was initially used to detect hydrogen peroxide (H 2 O 2 ) formation (33); however, peroxynitrite, hypochlorous acid, and peroxidases also oxidized DCF (34,35).…”

“…To determine the effect of various interventions, cells were incubated at 378C with NADPH oxidase inhibitor (DPI, 10 lM), MPO inhibitor (ABAH, 100 lM), or NO synthase (NOS) inhibitor 7-NI (1 mM)/L-NAME (1 mM) for 15 min or 5 min with ROS scavenger (NAC, 5 mM) and ONOO 2 scavenger (ebselen, 10 lM) before loading with the dye. Reactive species generation from the PMNs has been reported as mean fluorescence or mean stimulation index (MSI) (28). MSI is ratio of mean fluorescence of the stimulated versus unstimulated cells.…”

“…A flow cytometry-based method was used to study the bacterial phagocytosis by the human PMNs as described previously (28). Bacteria (Escherichia coli) were heat-inactivated at 608C for 30 min before the labeling with FITC (50 lg/ml) in the dark with continuous stirring for 60 min at room temperature.…”

Evaluation of Vitamin C for Adjuvant Sepsis Therapy

“…Hydrogen peroxide is generated in the interstitial fluid by oxidation of large amounts of exogenous ascorbate (10) and this may alter the function of some cells. Another source of hydrogen peroxide is extravasated neutrophils, because ascorbate enhances reactive oxygen species production by neutrophils exposed to E. coli (52). Indeed, ascorbate kills cancer cells in culture and the underlying mechanism involves promotion hydrogen peroxide formation in the culture medium, because the cell killing can be suppressed by extracellular catalase (46).…”

“…Another possible source of hydrogen peroxide is extravasated neutrophils because this cell type accumulates ascorbate and raises the extracellular concentration of reactive oxygen specis (9). Ascorbate and DHAA (300 lM) do not alter phagocytosis of E. coli by isolated neutrophils but do enhance subsequent generation of reactive oxygen species (52), which may include hydrogen peroxide. Further investigation is required to determine if bacteriostatic levels of hydrogen peroxide are produced in the interstitial fluid after injection of ascorbate in patients whose rate of ascorbate oxidation is accelerated, as it is in septic patients (19).…”