The effect of electromagnetic field on reactive oxygen species production in human neutrophils in vitro

The present study was undertaken in order to determine the effect of low frequency electromagnetic field (EMF) on reactive oxygen species (ROS) production in human neutrophils in peripheral blood in vitro. We investigated how differently generated EMF and several levels of magnetic induction affect ROS production. To evaluate the level of ROS production, two fluorescent dyes were used: 2′7′-dichlorofluorscein-diacetate and dihydrorhodamine. Phorbol 12-myristate 13-acetate (PMA), known as strong stimulator of the respiratory burst, was also used. Alternating magnetic field was generated by means of Viofor JPS apparatus. Three different levels of magnetic induction have been analyzed (10, 40 and 60 μT). Fluorescence of dichlorofluorescein and 123 rhodamine was measured by flow cytometry. The experiments demonstrated that only EMF tuned to the calcium ion cyclotron resonance frequency was able to affect ROS production in neutrophils. Statistical analysis showed that this effect depended on magnetic induction value of applied EMF. Incubation in EMF inhibited cell activity slightly in unstimulated neutrophils, whereas the activity of PMA-stimulated neutrophils has increased after incubation in EMF.     

Ca2+ ionophores inhibit superoxide generation by chemotactic peptide in rabbit neutrophils and the correlation with intracellular calcium

Summary Effects of Ca²⁺ ionophores, A23187 and lasalocid, on superoxide anion generation by chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine methyl ester, in rabbit peritoneal exudate neutrophils were studied. The ionophores by themselves did not activate superoxide anion generation in these neutrophils. When preincubated with the cells for 2 min, both the ionophores inhibited superoxide generation induced by chemotactic peptide. The inhibition was present even in the absence of extracellular Ca²⁺ and the inhibition was better then. Lasalocid produces a dose-dependent chlortetracycline fluorescence decrease response in neutrophils loaded with chlortetracycline. This response is independent of extracellular Ca²⁺ concentration and is related to release of Ca²⁺ from intracellular storage sites. The dose-range at which lasalocid gives this response is same as the dose-range at which it causes inhibition of superoxide response. It may be concluded that the inhibition of superoxide generation by these ionophores is correlated to intracellular Ca²⁺ modulation.Abbreviations FMLP Formyl-Methionyl-Leucyl-Phenylalanine methyl ester     

Stimulation of reactive oxidant production in neutrophils by soluble and insoluble immune complexes occurs via different receptors/signal transduction systems

Abstract Cell-free synovial fluid from patients with rheumatoid arthritis contains soluble and insoluble IgG-containing immune complexes which activate reactive oxidant production in human neutrophils. In this report we have measured the effects of inhibitors of signal transduction pathways on neutrophil activation by these complexes and also following activation by synthetic soluble and insoluble immune complexes made from human serum albumin (HSA) and anti-(HSA) antibodies. In all aspects studied, the soluble rheumatoid complexes and the soluble synthetic complexes were indistinguishable in the ways in which they activated neutrophils. Activation of reactive oxidant production in response to these soluble complexes was completely inhibited by pertussis toxin (indicating G-protein coupling of receptor occupancy), completely insensitive to staurosporine (indicating that oxidant production did not require protein kinase C activity), only marginally (<30%) inhibited by butanol (indicating that dependence upon activity of phospholipase D was minimal), and completely inhibited by chloracysine, an inhibitor of phospholipase A₂. In contrast, activation of reactive oxidant production in response to the insoluble rheumatoid or insoluble synthetic immune complexes was largely pertussis toxin insensitive, inhibited by >50% by staurosporine, inhibited by >50% by butanol, and completely inhibited by chloracysine. These results show that the receptor-mediated signal transduction systems activated by the soluble and insoluble immune complexes are different. Because the soluble complexes activate a transient burst of reactive oxidant secretion from primed neutrophils, the mechanisms regulating either the release or the intracellular production of oxidants within rheumatoid joints are distinct and hence may be pharmacologically modified independently of each other.     

An inhibitor of cyclic AMP-dependent protein kinase enhances the superoxide production of human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine

Intact human neutrophils produced superoxide (O₂^–) by the stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) even when the extracellular Ca²⁺ was absent (0.56±0.13 nmol/min per 10⁶ cells). The production by fMLP was enhanced more than twice in the presence of the extracellular Ca²⁺. Moreover, the O₂^– production by fMLP in the presence of extracellular Ca²⁺ was enhanced nearly three times by the treatment of cells with H-89, an inhibitor of cyclic AMP-dependent protein kinase (PKA). The enhancement was not observed when the extracellular Ca²⁺ was depleted from the reaction mixture. In addition, H-89 did not enhance fMLP-induced O₂^– production of electropermeabilized neutrophils in which the intracellular Ca²⁺ concentration was fixed to about 100 nM. These observations suggest that not only Ca²⁺ influx but the inhibition of PKA is necessary for the maximum O₂^– production by fMLP and that the O₂^– production is partially suppressed by the activation of PKA induced by fMLP.     

Rhythmic patterns in phagocytosis and the production of reactive oxygen species by zebrafish leukocytes

Multiple components of vertebrate immune systems have been shown to exhibit circadian fluctuations. While the zebrafish is currently generating a wealth of information on the molecular pacemakers that may control circadian rhythms, there have been no reports of rhythmic activity in zebrafish leukocytes. In this study, we found that phagocytosis and the production of reactive oxygen species by zebrafish leukocytes varied significantly throughout twenty-four hour periods. A distinct peak in cellular ROS levels occurred before dawn, while the kinetics of respiratory burst responses were least rapid at this time of day. Phagocytosis of E. coli peaked late in the day, whereas there was no daily variation in phagocytosis of S. aureus. As seen in other species, the number of bacteria ingested per cell peaked during the night. These data provide direct evidence of rhythmic immune system activity, and demonstrate that zebrafish can be a valuable model in which to study the relationships between circadian gene expression, systemic pacemakers, and the activity of vertebrate immune system cells.     

Cannabinoid receptor-independent suppression of the superoxide generation of human neutrophils (PMN) by CP55 940, but not by anandamide

Cannabinoids have been shown to affect various immune functions. To date, almost no data exist on PMN, which provide the first line antimicrobial defense. The objective of the present study was to investigate the effects of the synthetic dibenzopyrane ligand CP55 940, the endogenous cannabinoid anandamide and methanandamide on the "respiratory burst" of isolated human PMN in vitro. After preincubation with high micromolar concentrations of CP55 940, fMLP-stimulated PMN showed a reduction in superoxide production, whereas the spontaneous burst activity of resting PMN remained unaffected. This inhibitory effect of CP55 940 was not CB-receptor-mediated. In contrast, anandamide and methanandamide did not alter the oxidative microbicidal PMN function.     

Influence of 4-hydroxynonenal on chemiluminescence production by unstimulated and opsonized zymosan-stimulated human neutrophils

The lipid peroxidation product 4-hydroxy-2, 3-trans-nonenal (HNE) has a spectrum of biological effects on different cell types depending on the concentrations tested. In particular micromolar HNE concentrations stimulate neutrophil migration and polarization whereas higher doses inhibit. In our experimental conditions, fMet-Leu-Phe (fMLP) increased CL production of both unstimulated and zymosan-stimulated neutrophils, whereas cell stimulation with low HNE concentrations as well as zymosan addition to HNE incubated cells did not enhance light emission. In contrast 10(-4) M HNE reduced CL emission by unstimulated cells nearly to background values, completely depressed CL production by zymosan-stimulated cells and reduced phagocytosis. Cysteine was found to be able to counteract the HNE effect by about 70 per cent. The possibility that this aldehyde could exert its inhibitory effect through the alkylation of NADPH-oxidase SH-groups is postulated. Moreover, our present data on differences observed between fMLP and HNE indicate a different chemotactic mechanism induced by these two classes of compounds and lead to the conclusion that the local functional features of the attracted cells may be different.     

The metabolism of arachidonic and eicosapentaenoic acids in human neutrophils stimulated by A23187 and FMLP

A23187 stimulates the metabolism of endogenous as well as exogenous arachidonic acid (AA) and eicosapentaenolc acid (EPA) to their corresponding leukotrienes in human neutrophils. In contrast, conflicting results have been obtained concerning the effect of FMLP on the metabolism of these fatty acids. In the present study we compared the effect of A23187 and FMLP on the release and metabolism of these fatty acids in neutrophils. Stimulation of neutrophils with A23187, but not with FMLP, resulted in detectable levels of AA in the presence or absence of BW755C (a dual inhibitor of cyclooxygenase and lipoxygenase). The absolute amount of nonesterified AA in the extracts of neutrophils exposed to the agonist A23187 in the presence of BW755C was 20% higher than that obtained in the absence of BW755C, indicating that only a small fraction of the released AA was converted to lipoxygenase products. Furthermore, significant quantities of AA and EPA metabolites were detected only after treatment of neutrophils with A23187, but not with FMLP. Both A23187 and FMLP stimulated the conversion of exogenous EPA to 5-lipoxygenase products, with A23187 being somewhat more effective. In addition, significant differences were noted on the effect of EPA and DHA on the conversion of AA to its metabolites in A23187-stimulated neutrophils. Our results provide strong evidence that the amounts of eicosanoid precursors mobilized in response to FMLP are extremely small, if any, and this appears to be the likely explanation for the lack of eicosanoid detection by HPLC in FMLP-stimulated neutrophils.     

Relationship between the binding of N-formylmethionylleucylphenylalanine and the respiratory responses in human neutrophils

The results presented in this paper demonstrate that the chemotactic peptide N-formylmethionylleucylphenylalanine (-Met-Leu-Phe) is rapidly inactivated by the products of the respiration of human neutrophils stimulated by the peptide itself. The process of inactivation is impeded by the addition of inhibitors of myeloperoxidase (KCN, NaN₃), of catalase, of methionine but not by the addition of superoxide dismutase, indicating that the mechanism of inactivation is the oxidation of methionine residue by myeloperoxidase-H₂O₂-halide system. The oxidation of the peptide causes the rapid cessation of the respiratory burst, since the sulfoxide derivative loses its ability to bind the specific receptors of neutrophil surface and, hence, its biological activity. The comparison between the time course of the binding of f-Met-Leu-[³H]Phe to the specific receptors and the rate of the respiratory response of neutrophils in the presence and in the absence of the process of peptide oxidation was used to investigate the mechanism of the activation of the respiratory burst by the peptide-receptor complexes. In conditions where the inactivation of the stimulatory agent takes place the stimulated respiration slows down and resumes the resting state shortly after the cessation of the binding, although a substantial amount of the peptide remains bound to the specific receptors. In conditions where the degradation of the peptide does not occur the binding of the peptide and the respiratory burst continue for a longer period of time, but the rate of the respiration, calculated in terms of the instantaneous velocity (V_ist), is not correlated to the amount of the ligand bound to the membrane receptors measured at various times, indicating that a summation of the effects ofthe ligand-receptor complexes does not occur as they form. These findings demonstrate, as far as the respiratory response is concerned, that the bioogical activity of the peptide-receptor complexes is short-lived and that continuous de-novo receptor occupancy is necessary for the maintenance of the activated respiration.     

Production of chemokines and reactive oxygen species by human neutrophils stimulated by Helicobacter pylori

Background. Bacteria have different characteristics in stimulation of human neutrophils to produce reactive oxygen species (ROS) and chemokines. This study examined the ability of Helicobacter pylori to induce production of ROS and chemokines by human neutrophils. Methods. H. pylori strains (1.5 × 10⁸ CFU/ml) were cocultured with 5 × 10⁴ neutrophils isolated from healthy subjects. Samples were incubated with human serum with or without IgG antibodies to H. pylori. ROS production was measured using luminol‐dependent chemiluminescence (LmCL), and the concentrations of chemokines (IL‐8, RANTES, MIP‐1α and MCP‐1) were measured by ELISA. Results. The mean of the highest LmCL (peak height; PH) value stimulated by H. pylori was 3318 in the absence of serum. PH increased to 4687 when incubated with anti‐H. pylori antibody‐positive sera (p < .001) but antibody‐negative sera did not affect LmCL response. The mean final concentration of IL‐8 produced in the absence of serum was 142.6 pg/ml. Increased IL‐8 production was seen by addition of antibody positive serum (p < .01). IL‐8 production was not significantly correlated with production of ROS. On the other hand, H. pylori stimulation did not induce neutrophil production of RANTES, MIP‐1α or MCP‐1. Conclusions. H. pylori was capable of inducing IL‐8 production by human neutrophils, but not C‐C chemokines. Production of C‐X‐C dominant chemokine by neutrophils is consistent with the pathological characteristics of H. pylori‐induced gastritis, where persistent neutrophil infiltration is present.     

Lactoferrin inhibits the binding of lipopolysaccharides to L-selectin and subsequent production of reactive oxygen species by neutrophils

The activation of leukocytes by lipopolysaccharides (LPS), resulting in the oxidative burst, contributes to the pathogenesis of septic shock. The binding of LPS to L-selectin, which was reported as a serum-independent LPS receptor on neutrophils, induces the production of oxygen free radicals. Human lactoferrin (hLf), an anti-inflammatory glycoprotein released from neutrophil granules during infection, binds to LPS. In this study, we investigated the capacity of hLf to inhibit the L-selectin-mediated activation of neutrophils. Our experiments revealed that hLf prevents the binding of LPS to L-selectin in a concentration-dependent manner. Inhibition was maximum (87.7+/-0.5%) at a concentration of 50 microg/ml of hLf. Furthermore, hLf inhibited up to 55.4+/-0.5% of the intracellular hydrogen peroxide production induced by LPS in neutrophils. These findings suggest that the anti-inflammatory properties of hLf are due, at least in part, to their ability to prevent the binding of LPS to neutrophil L-selectin.     

Induction of apoptosis by Meretrix lusoria through reactive oxygen species production, glutathione depletion, and caspase activation in human leukemia cells

Apoptosis-induced directed fractionation and purification was used to identify the bioactive components of hard clams (HC), Meretrix lusoria. Two stereoisomers of epidioxysterol were previously identified as the active compounds in the ethyl acetate fraction (HC-EA). The molecular mechanism of HC-EA-induced apoptosis was also investigated in this study. Dissipation of mitochondrial membrane potential, release of mitochondrial cytochrome c into cytosol, and subsequent induction of pro-caspase-9 and -3 processing preceded apoptosis in HL-60 cells, confirmed by DNA fragmentation, chromatin condensation, changes in the cell membrane and the appearance of a sub-G1 DNA peak. Furthermore, treatment with HC-EA caused a rapid loss of intracellular glutathione content and stimulation of reactive oxygen species (ROS). Antioxidants such as catalase, N-acetylcysteine, pyrrolidine dithiocarbamate, and superoxide dismutase, but not allopurinol and diphenylene iodonium, significantly inhibited HC-EA-induced cell death. Apoptosis was completely prevented by a pan-caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone (z-VAD-FMK). The induction of apoptosis by M. lusoria may prove to be a pivotal mechanism for its cancer chemopreventive action.     

Chemiluminescence of human bloodstream monocytes and neutrophils: An unusual oxidant(s) generated by monocytes during the respiratory burst

Maximal rates of O and H₂O₂ production by human bloodstream monocytes activated during the respiratory burst by phorbol ester were only about 10% of those of neutrophils. Furthermore, monocytes possess only about 5% of the myeloperoxidase activity of neutrophils and so can only produce low levels of HOCI and related compounds. These combined reductions in O generating ability and lower myeloperoxidase levels result in low luminol chemiluminescence stimulated during the respiratory burst of monocytes. However, although monocytes generate much lower levels of O and H₂O₂ than neutrophils, these cells produce comparable rates of PMA-stimulated lucigenin chemiluminescence. Hence, this assay does not accurately reflect the production of either of these two oxidants by activated phagocytes, and further lucigenin must react with some other oxidant(s) via a process which leads to photon emission. This oxidant(s) is not O, H₂O₂, · OH, ¹O₂ or NO, but is derived from O generated during the respiratory burst and is generated in greater quantities by activated monocytes compared with neutrophils. Thus, lucigenin chemiluminescence is an indirect measure of superoxide release.     

Studies on stimulus-response coupling in human neutrophils: I. Role of monovalent cations in the respiratory and secretory response to N-formylmethionylleucylphenylalanine

Human blood neutrophils suspended in Na⁺-free, high-K⁺, phosphate-buffered solution exhibit respiratory and secretory responses to N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) much higher than those suspended in phosphate-buffered solution containing physiological concentration of K⁺ and Na⁺. The differences between the responses are very marked at low doses of fMet-Leu-Phe (10^?9, 10^?8 M), progressively decrease at higher doses, and disappear at the maximal stimulatory concentration of the peptide (10^?6 M). The higher responses of human neutrophils to fMet-Leu-Phe are not dependent on the membrane depolarization, that occurs when the cells are suspended in high-K⁺ buffered solution, but on the absence, or on the low concentration, of Na⁺ in the suspending medium. In fact: (i) the higher respiratory and secretory responses progressively decrease by substituting K⁺ with Na⁺ in the suspending solution, without change of the state of depolarization; (ii) the replacement of extracellular Na⁺ with choline ions does not affect the transmembrane potential of neutrophils but induces higher respiratory and secretory responses to fMet-Leu-Phe; (iii) the membrane depolarization induced by gramicidin and by ouabain does not result in a higher respiratory response to chemotactic peptide. These results indicate that in human neutrophils Na⁺ plays a regulative role in the stimulation of the respiratory burst and in the secretion induced by the chemotactic peptide. This regulation does not influence the maximal responses, but the threshold of the responses. K⁺ is also involved at least in the respiratory response, since the effect of the absence of Na⁺ is potentiated when the concentration of K⁺ of the suspending solution is high. Furthermore, the finding that a very high respiratory burst and the secretion of β-glucuronidase and vitamin B-12-binding protein can be induced by fMet-Leu-Phe in human neutrophils in the absence of external Na⁺ indicates that the entry of this cation and the consequent decrease in transmembrane potential are not necessary events for the activation of respiration and secretion by the peptide. The mechanism underlying the effect of the modification of ionic composition of the external medium is discussed in terms of the molecular events triggered by the stimulus at the level of the plasma membrane and of the recognition phenomena at the cell surface, that are common steps for the induction of the respiratory and secretory responses in neutrophils.     

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

Abstract

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H₂O₂ were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H₂O₂ in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H₂O₂ via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.     

Reaction products of 1-naphthol with reactive oxygen species prevent NADPH oxidase activation in activated human neutrophils, but leave phagocytosis intact

Activation of human neutrophils with opsonized particles in the presence of a nontoxic dose of 1-naphthol resulted in inhibition of superoxide anion production but not of the phagocytotic activity of the cells. In this study we have investigated the mechanism of action of 1-naphthol. The inhibition is not at the level of cellular activation since the FMLP-induced rise of intracellular free calcium was unaffected. Our results show that the (metabolic) activation of 1-naphthol to 1,4-naphthoquinone by reaction with H2O2 from the oxidative burst is a necessary event for the inhibition to occur. The study provides evidence that by its reactivity with essential thiol groups 1,4-naphthoquinone (1,4-NQ) prevents the assembly of a functional NADPH-oxidase in the neutrophil membrane.     

Effect of phenolic acids of microbial origin on production of reactive oxygen species in mitochondria and neutrophils

Cardiovascular death is frequently associated with atherosclerosis, a chronic multifactorial disease and a leading cause of death worldwide. Genetically engineered mouse models have proven useful for the study of the mechanisms underlying cardiovascular diseases. The apolipoprotein E-deficient mouse has been the most widely used animal model of atherosclerosis because it rapidly develops severe hypercholesterolemia and spontaneous atherosclerotic lesions similar to those observed in humans. In this review, we provide an overview of the cardiac and vascular phenotypes and discuss the interplay among nitric oxide, reactive oxygen species, aging and diet in the impairment of cardiovascular function in this mouse model.     

Stimulation of mitochondrial reactive oxygen species production by unesterified,unsaturated fatty acids in defective human spermatozoa

Male infertility is a relatively common condition affecting 1 in 20 men of reproductive age. The etiology of this condition is thought to involve the excessive generation of reactive oxygen species by human spermatozoa; however, the cause of this aberrant activity is unknown. In this study we demonstrate that defective human sperm populations are characterized by high cellular contents of both esterified and unesterified fatty acids and a decrease in the proportion of the total fatty acid pool made up by docosahexaenoic acid. The free unsaturated fatty acid content of these cells was positively correlated with the induction of mitochondrial superoxide generation (P < 0.001). This relationship was causal and mediated by the range of unesterified, unsaturated fatty acids that are present in human spermatozoa. Thus direct exposure of these cells to free unsaturated fatty acids stimulated mitochondrial superoxide generation and precipitated a loss of motility and an increase in oxidative DNA damage, two key attributes of male infertility. We conclude that defective human spermatozoa are characterized by an abnormally high content of fatty acids that, in their unesterified, unsaturated form, promote ROS generation by sperm mitochondria, creating a state of oxidative stress and a concomitant loss of functional competence.