Claude Marquetty

Hydroxyl radical as a potential intracellular mediator of polymorphonuclear neutrophil apoptosis

We investigated reactive oxygen species (ROS) involvement in polymorphonuclear neutrophilic leukocyte (neutrophil) apoptosis triggering. Neutrophils were incubated with xanthine oxidase (XO), which produces superoxide anion (O2.-) and hydrogen peroxide (H2O2) or glucose oxidase (GO), which produces only H2O2. Both XO and GO accelerated apoptosis when compared to spontaneously aged neutrophils. Catalase inhibited both spontaneous apoptosis and XO- or GO-accelerated apoptosis, but superoxide dismutase did not. Hydrogen peroxide can enter the cell, thus generating intracellular oxidation, which was observed by flow cytometry. Furthermore, the intracellular reduced glutathione content fell in the presence of XO or GO; however, apoptosis was not accelerated in the presence of buthionine sulfoximine (BSO), suggesting that the fall in glutathione in the presence of XO or GO is a consequence of oxidative stress but not a trigger of apoptosis. Hydrogen peroxide can react with iron to form hydroxyl radicals (HO.); we observed that two iron chelators, deferoxamine and hydroxybenzyl ethylenediamine (HBED), both inhibited spontaneous and accelerated apoptosis, suggesting that HO. may mediate neutrophil apoptosis.

Inhibition of human neutrophil binding to hydrogen peroxide-treated endothelial cells by camp and hydroxyl radical scavengers

Hydrogen peroxide (H2O2) increases adherence of human polymorphonuclear neutrophils (PMN) to cultured human umbilical vein endothelial cells (HUVEC). Catalase and HO. scavengers did not affect the increased PMN adherence to HUVEC stimulated by other compounds such as phorbol myristate acetate (PMA) and thrombin, showing that the observed effect was H2O2- and HO.-specific. This effect was inhibited by hydroxyl radicals (HO.) scavengers and not by iron-chelators that do not penetrate the cells, suggesting the involvement of intracellular HO. in the increased adherence mechanism. An increase in cAMP inhibited H2O2-induced adherence, as observed with isoproterenol, isobutylmethylxanthine, and dibutyryl-cAMP. Similarly, pentoxifylline (Ptx), an HO. scavenger that also increases cAMP, inhibited H2O2-mediated adherence but had no effect on that induced by PMA or thrombin. PKA inhibitors cancelled the Ptx-induced inhibition of H2O2-mediated adherence. However, PKA inhibitors or atrial natriuretic peptide that decreases cAMP did not increase adherence, showing that decrease in cAMP is not responsible for increased adherence. HO. scavengers did not alter the H2O2-induced reduction in cAMP levels, but did inhibit the effect of H2O2 on adherence. We conclude that HO. mediates the H2O2-induced increased in PMN adherence to HUVEC, and that the increase in cAMP that mediates PKA activation downregulates this effect.

Luminol assay for microdetermination of superoxide dismutase activity: Its application to human fetal blood

A microtechnique for determining the superoxide dismutase activity in erythrocytes is described. This technique involves the inhibition of luminol-enhanced chemiluminescence of superoxide anion generated by xanthine-xanthine oxidase. Measurements required a steady-state chemiluminescence whether superoxide dismutase was present or absent; the level of luminescence was correlated to enzyme activity. Superoxide dismutase activity measured by this technique was 836 +/- 112 micrograms/g of hemoglobin for whole blood and 834 +/- 109 micrograms/g of hemoglobin for erythrocytes. When the reference technique was applied to larger amounts of blood, the results were 862 +/- 58 and 858 +/- 116 micrograms/g of hemoglobin for whole blood and washed erythrocytes, respectively. The enzymatic activity of superoxide dismutase from fetal blood (obtained by venipuncture in utero and of 19-26 weeks gestational age) was similar to that of adult blood, when measured by the new technique.

Spectroscopic interference of hemoglobin with neutrophil cytochrome b-245 and its elimination by carbon monoxide

A cytochrome b, designated as cytochrome b-245, exists in neutrophils and is probably involved in their stimulated oxidative burst. As a rule, its concentration is spectroscopically measured by the height of its alpha-peak at 558-559 nm (dithionite-reduced minus oxidized). Hemoglobin (Hb), which usually contaminates neutrophils isolated from blood, interferes with the spectroscopic measurement of the cytochrome. Hb contamination from less than 0.4 red blood cells per 100 neutrophils leads to over-estimation of the cytochrome by approximately 50%. This interference can be overcome by bubbling CO through neutrophil homogenates heavily contaminated by Hb, prior to the conventional spectroscopic procedure. The cytochrome B-245 concentration obtained in neutrophils by CO bubbling is 7.2 +/- 1.28 pmoles per 10(6) polymorphonuclear neutrophils.

Role of cytochrome b-559 in arachidonic acid activation of resting human neutrophils.

Whether or not cytochrome b-559 is a necessary component of NADPH oxidase activity in neutrophils is still controversial. In highly purified plasma membranes isolated from resting neutrophils and lacking cytochrome b, addition of arachidonic acid induced an NADPH oxidase activity. This activity was similar to that of plasma membranes isolated from phorbol myristate acetate (PMA)-stimulated cells which possessed cytochrome b. Addition of arachidonic acid to the latter plasma membranes did not alter the oxidase activity. It can be concluded that plasma membranes isolated from resting neutrophils have, in the presence of arachidonic acid, an NADPH oxidase activity similar to that of PMA-stimulated cells, except that it is independent of cytochrome b-559.

Plasma membranes of human neutrophils: A one-step isolation procedure by cell disruption in paraffin oil

Plasma membranes of high purity and good yield have been prepared from human polymorphonuclear neutrophils by a one-step procedure involving disruption of cells suspended in paraffin oil and forced by pressure through an annular slit. This results in a band floating above the oil which is composed of large sheets of plasma membranes. Enrichment values for the plasma membrane marker alkaline phosphatase and 125I-labeled protein after surface labeling performed at the whole cell level were 23-fold and 22-fold, respectively. Contamination of the plasma membrane by other organelles was negligible and approximately 2 mg of membrane protein was obtained from 10(9) neutrophils. The procedure is very fast and the use of paraffin oil avoids lengthy high-speed centrifugation. The technique also allows isolation of granules devoid of plasma membrane and can probably be applied to other cell types.

Effect of degranulation on superoxide dismutase activity in human neutrophils

Resting neutrophils possess cytosolic cyanide-sensitive (CNs) superoxide dismutase (SOD) and cyanide-insensitive (CNi) SOD, located in an undefined organelle of the 27,000 g sedimentable fraction of its homogenate. Stimulated neutrophils generate large amounts of superoxide anion, part of which is released in the extracellular medium and contributes to changes that occur in inflammatory foci. Our purpose was to assess whether or not the neutrophil upon stimulation secreted either or both CNs and CNi SOD activity, because the process could protect against the release of superoxide anion. Human neutrophils stimulated in vitro with phorbol myristate acetate released 32.6% and 53% of their content in myeloperoxidase (an azurophilic granule marker) and vitamin B12 binding activity, respectively. The CNi SOD was not secreted at all, whereas 16% and 23% of CNs SOD were released by resting and stimulated neutrophils, respectively. In contrast, lactate dehydrogenase, a cytosolic marker, was released by both resting and stimulated cells (approximately equal to 9%). These results suggest that CNi SOD is not located in the granules but in another organelle that does not degranulate upon stimulation and consequently does not protect against superoxide anion formed by neutrophils in the extracellular medium. In contrast, CNs SOD is slightly but significantly released (P less than .02) and may be protective. Neutrophils from two patients with chronic granulomatous disease behaved similarly to control neutrophils but their content of both types of SOD was higher than that of the controls.