Effects of antioxidants on surfactant peroxidation by stimulated human polymorphonuclear leukocytes

Production of oxygen radicals by stimulated phagocytes followed by surfactant lipid peroxidation (LPO) and loss of surfactant function have all been implicated in the pathogenesis of acute lung injury. We studied the interactions between natural lung surfactant (Curosurf) and neutrophils in vitro , and compared various antioxidants; (superoxide dismutase (SOD), vitamin E, vitamin C, ebselen and melatonin), or combinations of them in duplicate and triplicate regarding their ability to decrease superoxide production and the peroxidation level of surfactant caused by activated phagocytes. The superoxide production of neutrophils activated by Candida albicans was measured with the nitroblue tetrazolium (NBT) test. The subsequent LPO was estimated as the content of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE). We found that lung surfactant decreased the superoxide production by activated neutrophils (29.7%) and that Curosurf was peroxidized with elevated MDA/4-HNE values. With supplements of antioxidants (except vitamin C), superoxide radical production and the surfactant LPO level fell in a dose-dependent manner. The protective effect of the antioxidants differed in each test. SOD had a slight effect in both tests. The findings with vitamin E, melatonin and ebselen were similar. The best combination was that of a natural and a synthetic antioxidant (melatonin-ebselen) with a 60% decrease in comparison to the corresponding control. These findings suggest that antioxidants, particularly in combination, prevent LPO of lung surfactant.     

Alterations in erythrocyte membrane fluidity by phenylhydrazine-induced peroxidation of lipids

The addition of phenylhydrazine (.05 – 0.5 mM) to hemoglobin-free human erythrocyte membranes results in the peroxidation of endogenous phospholipids as measured by the thiobarbaturic acid reaction. The incorporation of 1,6-diphenyl-1,3-5-hexatriene into these membranes revealed a decrease in bulk lipid fluidity. Additionally, the fluorescence intensity of 1-anilino-8-naphthalene sulfonate was decreased and red-shifted after phenylhydrazine treatment of the membranes. The results obtained are consistent with the view that changes in the physical state of plasma membranes subsequent to the peroxidation of membrane lipids may be a determinant of the mechanical properties of drug-treated as well as aging cells.     

Superoxide production by polymorphonuclear leukocytes

Summary Phagocytosis by polymorphonuclear leukocytes triggers a burst of oxidative metabolism resulting in hydrogen peroxide and superoxide production, and these active oxygen species function in the killing of microorganisms. A new cytochemical technique, based on a manganese dependent diaminobenzidine oxidation, has been developed to detect superoxide in these cells. It has been shown that superoxide generation is associated with the plasma membrane in cells activated by particulate (zymosan) and nonparticulate (phorbol myristate acetate) stimuli. This membraned activity is maintained during invagination such that reduced oxygen is generated within the endocytic vacuoles. Reaction product is absent from unstimulated cells; additionally, formation of precipitate is blocked by omission of Mn⁺⁺, low temperature, glutaraldehyde prefixation, and the presence of superoxide dismutase in the incubation medium.In honour of Prof. P. van Duijn     

Alterations in membrane fluidity of diabetic polymorphonuclear leukocytes.

Plasma membrane fluidity of polymorphonuclear leukocytes was investigated in 28 patients with insulin dependent diabetes mellitus and 30 healthy controls. Membrane fluidity was measured by steady-state fluorescence anisotropy of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated into the plasma membrane. The fluorescence anisotropy values in resting (unstimulated) polymorphonuclear leukocytes from diabetic subjects were significantly higher than those of controls (0.318 +/- 0.003 vs 0.287 +/- 0.003, P less than 0.001). The addition of the respiratory burst stimulus phorbol myristate acetate induced a stable increase in fluorescence anisotropy values in both groups. Fluorescence anisotropy values of stimulated polymorphonuclear leukocytes from the diabetic and control groups were not significantly different (P greater than 0.05). These data demonstrate a decrease in plasma membrane fluidity of resting polymorphonuclear leukocytes obtained from diabetic subjects. This finding could be in part explained by an increase in their basal respiratory burst activity.     

Aluminium salts accelerate peroxidation of membrane lipids stimulated by iron salts

Aluminium salts do not themselves stimulate peroxidation of ox-brain phospholipid liposomes, but they greatly accelerate the peroxidation induced by iron(II) salts at acidic pH values. This effect of Al(III) is not seen at pH 7.4, perhaps because Al(III) salts form insoluble complexes at this pH in aqueous solution. Peroxidation of liposomes in the presence of Al(III) and Fe(II) salts is inhibited by the chelating agent desferrioxamine, and by EDTA and diethylenetriaminepentaacetic acid at concentrations greater than those of Fe(II) salt. Aluminium salts slightly stimulate the peroxidation of peroxide-depleted linolenic acid micelles, but they do not accelerate the peroxidation induced by addition of iron(II) salts to the micelles at acidic pH. Aluminium salts accelerate the peroxidation observed when human erythrocytes are treated with hydrogen peroxide at pH 7.4. Desferrioxamine decreases the peroxidation. We suggest that Al(III) ions produce an alteration in membrane structure that facilitates lipid peroxidation, and that the increased formation of fluorescent age pigments in the nervous system of patients exposed to toxic amounts of Al(III) may be related to this phenomenon. The ability of desferal to bind both iron (III) and aluminium(III) salts and to inhibit lipid peroxidation makes it an especially useful chelating agent in the treatment of 'aluminium overload'.     

Binding of the lipid peroxidation product 4-hydroxynonenal to human polymorphonuclear leukocytes

4-Hydroxynonenal (HNE) is produced during peroxidation of polyunsaturated fatty acids. It exerts a chemokinetic effect on human polymorphonuclear leukocytes (PMN). Investigations of this mechanism were performed. The results indicate that [³H]-HNE binding to PMN results both in non-specific bonds to the numerous SH groups of the cells and in binding to a saturable, reversible and specific HNE site. Scatchard analysis revealed that this is a single site with an apparent affinity constant of 319 nM and a density of 1·57 pmol (10⁶)^?1 cells. This specific binding site may be involved in the chemokinetic effect of HNE.     

Calcium potentiates the peroxidation of erythrocyte membrane lipids

A clonal cell line from rat osteosarcoma was found to possess parathyroid hormone and isoproterenol sensitive adenylate cyclase. This study examines the relationship between the two hormones and triphosphoguanine nucleotide with respect to enzyme activation. Concentration-dependence curves, analyzed by computer-aided curve fitting, revealed: (1) in the presence of 5 microM GTP there were two apparent affinities for parathyroid hormone (Km 9 and 89 nM) and isoproterenol (Km 72 and 340 nM; (2) and two affinities for guanosine-5' (beta, gamma-imido)triphosphate (Km 0.25 and 1.3 microM); (3) hormones and guanine nucleotides reciprocally shifted each other's concentration dependence curve to the high affinity sites; (4) parathyroid hormone and isoproterenol interacting with high affinity sites competed for the same adenylate cyclase; (5) parathyroid hormone and isoproterenol, acting on low affinity sites had additive effects and also stimulated adenylate cyclase in the absence of added guanine nucleotides. The findings are consistent with (i) competition of parathyroid hormone and isoproterenol for the activation of the high (hormone) affinity complex containing: receptors, nucleotide subunit, triphosphoguanine nucleotide, catalytic unit (ii) the apparent presence of receptor-nucleotide sub-unit GDP-catalytic unit complexes with low hormone affinity which are stimulated by parathyroid hormone and isoproterenol separately.     

Oxygen-derived free radicals producing activity and survival of activated polymorphonuclear leukocytes

Summary Activation of polymorphonuclear (PMN) leukocytes is known to generate oxygen free radicals (OFR). However the fate of activated PMN leukocytes is not known. We investigated the OFR producing (chemiluminescence) activity and the survival of the activated PMN leukocytes. The study was divided into two groups. Group I, In vivo study (n = 7): zymosan (8.4 mg/kg) was administered intravenously in the anesthetized dogs and the blood samples were collected before and after 5, 15, 30, 60 and 120 min of zymosan administration. This group represents the in vivo pre-stimulated PMN leukocytes; Group II, In vitro study (n = 7): the blood were collected from dogs and further divided into two groups. Group A (n = 7): non-stimulated, without any added zymosan and group B (n = 7): zymosan was added to stimulate PMN leukocytes. Blood samples from group A and B were also collected at various time intervals similar to in vivo studies. Oxygen free radical producing activity of PMN leukocytes was monitored by measuring luminoldependent chemiluminescence (CL). Opsonized zymosan was used to activate PMN leukocytes. The studies in which the PMN leukocytes were stimulated in in vivo, both oxygen derived free radicals and superoxide dismutase (SOD) inhibitable oxygen free radical CL decreased significantly for 60 min and tended to reach thereafter to the pre-stimulated values. The resting chemiluminescence (chemiluminescence without zymosan stimulation in the assay medium) increased significantly for 15 min reaching to pre-stimulated values at 30 min and thereafter. In in vitro studies, oxygen derived free radicals CL of pre-stimulated PMN leukocytes (Group B) was depressed for the whole duration of investigation while SOD inhibitable CL was depressed for only 60 min. There was approximately a two-fold increase in the resting CL within 5 min of PMN leukocyte activation and it remained high for the whole duration of study. The chemiluminescence of non-stimulated PMN leukocytes in vitro (group A) remained practically normal throughout the period of observation. In in vivo studies, total white blood cells (WBC) and PMN leukocyte counts decreased initially and tended to approach towards pre-stimulated values at the end of the protocol. There were no changes in these counts in in vitro studies. These results indicate that the capacity to generate OFR is decreased in the in vivo and in vitro pre-stimulated PMN leukocytes. However this activity recovers with time. This study also suggests that the activated PMN leukocytes are not destroyed.     

Leukotriene formation by bovine polymorphonuclear leukocytes

The leukotriene production by bovine polymorphonuclear leukocytes isolated from peripheral blood has been studied. Cells were incubated in the presence of arachidonic acid, glutathione, calcium ionophore A23187 and Ca2+. The leukotrienes then formed are leukotriene C4, leukotriene B4, two all-trans isomers of leukotriene B4 and the double dioxygenation product 12-epi-6-trans-8-cis-leukotriene B4. Leukotriene C4 is formed in such a large quantity by the bovine polymorphonuclear leukocyte that it might constitute an excellent and inexpensive source for the biosynthetic preparation of this spasmogenic leukotriene.     

The uptake of amines by polymorphonuclear leukocytes

Muscarinic and β-adrenergic ligands associate with polymorphonuclear leukocytes to show high-affinity, saturable accumulation. This association can be distinguished from specific receptor binding by its temperature dependence, sensitivity to pH, requirement of an energy source, inhibition by ionophores, and inhibition by a variety of permeable basic amines.Our results suggest that these amines accumulate in acidic lysosomes which are plentiful in these cells. This permeable amine effect can be inhibited without affecting specific receptor binding.     

Preparation and characterization of plasma membrane vesicles from human polymorphonuclear leukocytes

It would be advantageous to prepare models of the neutrophil plasma membrane in order to examine the role of the plasma membrane in transmembrane signal transduction in the human neutrophil and to dissect ligand-receptor interactions and structural changes in the cell surface upon stimulation. A number of investigators have prepared neutrophil membrane vesicles by homogenization, sonication, or centrifugation--techniques that can result in the loss of substantial amounts of surface membrane material, disruption of lysosomes causing proteolysis of membrane proteins, and contamination of the plasma membrane fraction by internal membranes. These limitations have been overcome in the present studies by employing a modification of the method previously developed in this laboratory. Human neutrophils were suspended in a buffer simulating cytoplasmic ionic and osmotic conditions and disrupted by nitrogen cavitation. The resultant cavitate was freed of undisrupted cells and nuclei and then centrifuged through discontinuous isotonic/isoosmotic Percoll gradients, which resolved four fractions: alpha (intact azurophilic granules), beta (intact specific granules), gamma (membrane vesicles), and delta (cytosol). The gamma fraction was highly enriched in alkaline phosphatase, a marker of the plasma membrane. In addition, this fraction contained less than 5% of the amounts of lysosomes (indicated by lysozyme activity) and nuclei (indicated by DNA content) found in intact cells or in unfractionated cavitate. Furthermore, the gamma fraction contained less than 10% of the levels of endoplasmic reticulum, Golgi, mitochondrial, and lysosomal membranes in cells or cavitates, as determined by assays for glucose 6-phosphatase, galactosyl transferase, monoamine oxidase, and Mo1 (CD11b/CD18; Mac-1), respectively. Finally, 75% of the membrane vesicles were sealed, as indicated by assay of ouabain-sensitive (Na+,K+) ATPase activity, and 55% were oriented right-side-out, as determined by exposure of concanavalin A (ConA) receptors and sialic acid residues on the surfaces of the vesicles. These heterogeneous preparations could be enriched for right-side-out vesicles by their selective adherence to ConA-coated plates and subsequent detachment by rinsing the surfaces of the plates with alpha-methylmannoside. This enrichment protocol did not affect the integrity of the vesicles and resulted in populations in which greater than 85% of the vesicles were oriented right-side-out. This procedure thus permits the preparation of sealed, right-side-out membrane vesicles that may be used as valid experimental models of the neutrophil plasma membrane in a variety of functional studies.     

Radiation-induced lipid peroxidation and the fluidity of erythrocyte membrane lipids

The effect of radiation-induced peroxidation on the fluidity of the phospholipids of the erythrocyte membrane was studied using both erythrocyte ghosts and liposomes formed from the polar lipids of erythrocytes. In liposomes, the oxidation of the phospholipids increased with radiation dose, but there was no change in the fluidity of the lipids as measured by spin-label motion. Under the same conditions of irradiation, no oxidation of phospholipid was detected in erythrocyte ghosts, although changes occurred in the motion of spin labels intercalated with the membrane. These changes were attributed to radiation-induced alterations in the membrane proteins. It is concluded that alterations in motion of spin labels, observed with intact membranes after irradiation, are most likely the result of changes in the structure of membrane proteins rather than the lipids.     

Differences in the susceptibility of plant membrane lipids to peroxidation

Peroxidation of three membrane lipid preparations from plants was initiated using Fe-EDTA and ascorbate and quantified as the production of aldehydes and loss of esterified fatty acids. Using liposomes prepared from commercial soybean asolecithin, the degree of peroxidation was shown to be dependent on: the free radical dose, which was varied by the ascorbate concentration; the presence of tocopherol in the liposome; the configuration, of the liposome, multilamellar or unilamellar; and time after initiation. There were dramatic interactions among these factors which led to the conclusion that in comparing the susceptibility of different membrane preparations it is essential to examine the kinetics of the peroxidation reactions. The composition of the liposome was a major determinant of the degree of peroxidation and of the type of degradative reactions initiated by the oxygen free radicals. A fresh polar lipid extract from Typha pollen had very similar fatty acid composition to the soybean asolecithin, but was more resistant to peroxidation as shown by less aldehyde production and increased retention of unsaturated fatty acids after treatment. Similarly, microsomal membranes from the crowns of non-acclimated and cold acclimated winter wheat (Triticum aestivum L.) seedlings had a much higher linolenic acid content than soybean asolecithin but was much more resistant to peroxidation. In the winter wheat microsomes, the loss of esterified fatty acids was not selective for the unsaturated fatty acids; consequently, even with 40% degradation, the degree of unsaturation in the membrane did not decrease. These different reaction mechanisms which occur in plant membranes may explain why measurements of fatty acid unsaturation fail to detect peroxidative reactions during processes such as senescence, aging and environmental stress.     

Modifying effect of vinblastine on superoxide anion production by membrane perturbing agents in polymorphonuclear leukocytes

Superoxide anion production in peritoneal polymorphonuclear leukocytes obtained from guinea pigs was stimulated by in vitro treatment with membrane-perturbing agents, such as cytochalasin D, concanavalin A, phorbol myristate acetate, myristate, digitonin, and NaF. Vinblastine modified these stimulating effects on the superoxide anion production, but its modifying effect was not uniform. The effect of cytochalasin D was stimulated by vinblastine at the concentration of 10(-5)-10(-7) M, whereas it was inhibited at the concentration of 10(-4) M. At 10(-4)-10(-5) M, vinblastine was inhibitory to the effect of concanavalin A, and lower concentrations had no significant effect. Stimulation of the superoxide anion production by phorbol myristate acetate and myristate was further enhanced by vinblastine at any concentration in the range of 10(-4)-10(-8) M with peaks at 10(-6) and 10(-5) M, respectively. Vinblastine had little effect on the stimulation of the superoxide anion production by digitonin and NaF throughout the concentration range examined. The mechanism of the interaction of these membrane-perturbing stimulants and vinblastine is discussed.     

Egestion of degraded meningococci by polymorphonuclear leukocytes.

Quantitative studies were carried out on the in vitro phagocytosis of 14C-labeled Neisseria meningitidis by mouse polymorphonuclear leukocytes. Intact, "loaded" leukocytes were found to excrete radioactive bacterial products back into supernatant fluids. Morphological events associated with the exocytosis events revealed a fusion between the phagocytic vacuole and plasma membranes of the leukocyte followed by an emptying of the vacuole contents. Egested materials were free from whole meningococci and consisted mainly of membranous vesicles.