Oxidative Metabolism Of Human Neutrophils Research Articles

Baccharis dracunculifoliaDC (Asteraceae) selectively modulates the effector functions of human neutrophils

To examine whether the hydroalcoholic extract from Baccharis dracunculifolia leaves (BdE) modulates the human neutrophil oxidative metabolism, degranulation, phagocytosis and microbial killing capacity. In-vitro assays based on chemiluminescence, spectrophotometry, flow cytometry and polarimetry were used, as well as docking calculations. At concentrations that effectively suppressed the neutrophil oxidative metabolism elicited by soluble and particulate stimuli (<10 μg/ml), without clear signs of cytotoxicity, BdE (1) inhibited NADPH oxidase and myeloperoxidase activity; (2) scavenged H2 O2 and HOCl; (3) weakly inhibited phagocytosis; and (4) did not affect neutrophil degranulation and microbial killing capacity, the expression levels of TLR2, TLR4, FcγRIIa, FcγRIIIb and CR3 and the activity of elastase and lysozyme. Caffeic acid, one of the major B. dracunculifolia secondary metabolites, did not inhibit phagocytosis but interfered in the myeloperoxidase-H2 O2 -HOCl system by scavenging H2 O2 and HOCl, and interacting with the catalytic residues His-95, Arg-239 and Gln-91. BdE selectively modulates the effector functions of human neutrophils, inhibits the activity of key enzymes and scavenges physiological oxidant species. Caffeic acid contributes to lower the levels of oxidant species. Our findings help to unravel the mechanisms by which these natural products exert immunomodulatory action towards neutrophils.

Inhibition of the human neutrophil oxidative metabolism by Baccharis dracunculifolia DC (Asteraceae) is influenced by seasonality and the ratio of caffeic acid to other phenolic compounds

Ethnopharmacological relevanceThe great potential of phytotherapic drugs for treating and preventing inflammatory diseases mediated by increased neutrophil reactive oxygen species (ROS) generation has guided the search for new natural products with antioxidant and immunomodulatory properties. Baccharis dracunculifolia D.C. (Asteraceae), the main botanical source of Brazilian green propolis, is a native plant from Brazil widely used in folk medicine as anti-inflammatory. This study aims: (a) to determine the influence of seasonality on the chemical profile and biological activity of Baccharis dracunculifolia (Asteraceae) leaf extracts (BdE); (b) to analyze the correlation between the major compounds and the ability of BdE to modulate the superoxide anion and total ROS generation by human neutrophils. Materials and methodsThe extracts were obtained from leaf samples collected monthly during one year. The superoxide anion and total ROS generation were assessed by the lucigenin (CL-luc)- and luminol (CL-lum)-enhanced chemiluminescence assays. ResultsSeasonality influenced more the quantitative than the qualitative chemical profile of B. dracunculifolia, and affected its biological activity. The major compounds identified were caffeic acid, p-coumaric acid, aromadendrin-4′-methyl ether (AME), isosakuranetin and artepillin C. The IC50 values obtained for CL-lum and CL-luc inhibition by BdE ranged from 8.1–15.8 and 5.8–13.3µgmL−1, respectively, and correlated positively with caffeic acid concentration. CL-luc inhibition correlated negatively with the concentration of artepillin C, AME, isosakuranetin and total flavonoids. The BdE sample from May/07 inhibited CL-lum and CL-luc the most strongly (IC50=8.1±1.6 and 5.8±1.0µgmL−1, respectively), and contained the highest ratio of caffeic acid to the other isolated compounds; so, this ratio could be employed as chemical marker for this biological activity of B. dracunculifolia. ConclusionThe ability of B. dracunculifolia to inhibit the neutrophil ROS generation depends more on the type and ratio of phenolic compounds and flavonoids than on their high absolute concentrations. Together, our results help select the most appropriate plant material for the production of phytotherapic drugs to be used in the treatment of inflammatory diseases mediated by increased neutrophil activation.

4-Methylcoumarin Derivatives Inhibit Human Neutrophil Oxidative Metabolism and Elastase Activity

Increased neutrophil activation significantly contributes to the tissue damage in inflammatory illnesses; this phenomenon has motivated the search for new compounds to modulate their effector functions. Coumarins are natural products that are widely consumed in the human diet. We have evaluated the antioxidant and immunomodulator potential of five 4-methylcoumarin derivatives. We found that the 4-methylcoumarin derivatives inhibited the generation of reactive oxygen species by human neutrophils triggered by serum-opsonized zymosan or phorbol-12-myristate-13-acetate; this inhibition occurred in a concentration-dependent manner, as revealed by lucigenin- and luminol-enhanced chemiluminescence assays. Cytotoxicity did not mediate this inhibitory effect. The 7,8-dihydroxy-4-methylcoumarin suppressed the neutrophil oxidative metabolism more effectively than the 6,7- and 5,7-dihydroxy-4-methylcoumarins, but the 5,7- and 7,8-diacetoxy-4-methylcoumarins were less effective than their hydroxylated counterparts. An analysis of the biochemical pathways suggested that the 6,7- and 7,8-dihydroxy-4-methylcoumarins inhibit the protein kinase C-mediated signaling pathway, but 5,7-dihydroxy-4-methylcoumarin, as well as 5,7- and 7,8-diacetoxy-4-methylcoumarins do not significantly interfere in this pathway of the activation of the human neutrophil oxidative metabolism. The 4-methylcoumarin derivatives bearing the catechol group suppressed the elastase and myeloperoxidase activity and reduced the 1,1-diphenyl-2-picrylhydrazyl free radical the most strongly. Interestingly, the 5,7-dihydroxy-4-methylcoumarin scavenged hypochlorous acid more effectively than the o-dihydroxy-substituted 4-methylcoumarin derivatives, and the diacetoxylated 4-methylcoumarin derivatives scavenged hypochlorous acid as effectively as the 7,8-dihydroxy-4-methylcoumarin. The significant influence of small structural modifications in the inhibitory potential of 4-methylcoumarin derivatives on the effector functions of neutrophil makes them interesting candidates to develop new drugs for the treatment of inflammatory diseases mediated by increased neutrophil activation.

Inhibitory activity of liposomal flavonoids during oxidative metabolism of human neutrophils upon stimulation with immune complexes and phorbol ester

Context and objective: The massive production of reactive oxygen species by neutrophils during inflammation may cause damage to tissues. Flavonoids act as antioxidants and have anti-inflammatory effects. In this study, liposomes loaded with these compounds were evaluated as potential antioxidant carriers, in attempt to overcome their poor solubility and stability. Materials and methods: Liposomes containing quercetin, myricetin, kaempferol or galangin were prepared by the ethanol injection method and analyzed as inhibitors of immune complex (IC) and phorbol ester-stimulated neutrophil oxidative metabolism by luminol (CLlum) and lucigenin-enhanced (CLluc) chemiluminescence (CL) assays. The mechanisms involved this activity of liposomal flavonoids, such as cytotoxicity and superoxide anion scavenging capacity, and their effect on phagocytosis of ICs were also investigated. Results and discussion: The results showed that the inhibitory effect of liposomal flavonoids on CLlum and CLluc is inversely related to the number of hydroxyl groups in the flavonoid B ring. Moreover, phagocytosis of liposomes by neutrophils does not seem to necessarily promote such activity, as the liposomal flavonoids are also able to reduce CL when the cells are pretreated with cytochalasin B. Under assessed conditions, the antioxidant liposomes are not toxic to the human neutrophils and do not interfere with IC-induced phagocytosis. Conclusion: The studied liposomes can be suitable carriers of flavonoids and be an alternative for the treatment of diseases in which a massive oxidative metabolism of neutrophils is involved.

Modulation of human neutrophil oxidative metabolism and degranulation by extract of Tamarindus indica L. fruit pulp

The tamarind ( Tamarindus indica L .) is indigenous to Asian countries and widely cultivated in the American continents. The tamarind fruit pulp extract (ExT), traditionally used in spices, food components and juices, is rich in polyphenols that have demonstrated anti-atherosclerotic, antioxidant and immunomodulatory activities. This study evaluated the modulator effect of a crude hydroalcoholic ExT on some peripheral human neutrophil functions. The neutrophil reactive oxygen species generation, triggered by opsonized zymosan (OZ), n-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), and assessed by luminol- and lucigenin-enhanced chemiluminescence (LumCL and LucCL, respectively), was inhibited by ExT in a concentration-dependent manner. ExT was a more effective inhibitor of the PMA-stimulated neutrophil function [IC 50 (in μg/10 6cells) = 115.7 ± 9.7 (LumCL) and 174.5 ± 25.9 (LucCL)], than the OZ- [IC 50 = 248.5 ± 23.1 (LumCL) and 324.1 ± 34.6 (LucCL)] or fMLP-stimulated cells [IC 50 = 178.5 ± 12.2 (LumCL)]. The ExT also inhibited neutrophil NADPH oxidase activity (evaluated by O 2 consumption), degranulation and elastase activity (evaluated by spectrophotometric methods) at concentrations higher than 200 μg/10 6 cells, without being toxic to the cells, under the conditions assessed. Together, these results indicate the potential of ExT as a source of compounds that can modulate the neutrophil-mediated inflammatory diseases.

Inhibitory Effects of Human Neutrophil Functions by the 45-kD Glycoprotein Derived from the Parasitic Nematode Trichinella spiralis

Aim: We evaluated the effect of the 45-kD protein of Trichinella spiralis (gp45), purified by affinity chromatography, on random migration and chemotaxis, the oxidative metabolism of human neutrophils and on the CD11b upregulation induced by formyl-methionyl-leucyl-phenylalanine (f-MLP). Methods: Donor neutrophils incubated with different amounts of gp45 (0.5, 1, 1.5, 2 μg/ml) or buffer and the random migration and chemotaxis, evaluated by means of a special technique of image analysis, and the chemiluminescence response to f-MLP or phorbol myristate acetate (PMA) were analyzed. The effect on CD11b upregulation was assessed incubating cells with the protein, when activating them with f-MLP. Results: The results showed that gp45 inhibited both random and stimulated migrations, and reduced the response to f-MLP and PMA. Furthermore, gp45 significantly reduced the upregulation of the CD11b induced by f-MLP. Conclusion: The results show that gp45 inhibits PMN in different functions, suggesting an anti-inflammatory action.

Effects of homœopathic preparations of organic acids and minerals on the oxidative metabolism of human neutrophils

A number of different potencies of commercially available homœopathic preparations in saline solution were tested for their ability to regulate the oxidative metabolism (superoxide production) and adhesion function of human neutrophils in vitro. 15% to 30% inhibition of oxidative metabolism was caused by Sulphur 6x, Manganum phosphoricum 6x and 8x, and Magnesium phosphoricum 6x and 8x. Phosphorus slightly reduced superoxide production, with varying results in a series of experiments. Using Magnesium phosphoricum and Phosphorus, small inhibitory effects (8–11%) were noted event a high potencies. Among the organic acids, a group ( Acidum malicum 4x and Acidum fumaricum 4x) enhanced superoxide production, while others either inhibited the response ( Acidum citricum and Acidum succinicum, 3x and 4x) or had no effect ( Acidum α-ketoglutanicum and Acidum cis-aconitum). Attempts to reproduce these effects using solutions prepared in the laboratory confirmed the inhibitory effects of Managanum phosphoricum 6x and of organic acids in the 3x, while other data indicated that critical factors in the methodology of preparation may affect the results.

Sulfite stimulates NADPH oxidase of human neutrophils to produce active oxygen radicals via protein kinase C and Ca 2+/calmodulin pathways

The effect of sulfite on the oxidative metabolism of human neutrophils was studied in vitro. Superoxide anion production of PMN was determined using superoxide dismutase-inhibitale lucigenin-dependent CL. The addition of sulfite in concentration of 0.01 mM–1 mM results in an up to 6-fold increase in CL of nonstimulated neutrophils at 37°C and pH 7. Neutrophils stimulated with zymosan or PMA have an additional 2-fold stimulation when sulfite is added. Higher sulfite concentrations (2 mM –10 mM) decrease the CL of both nonstimulated and stimulated cells. The activity of NADPH oxidase, responsible for O 2 .− production, is significantly increased in neutrophils incubated with 1 mM sulfite. Neutrophils from patients with chronic granulomatous disease, which are cytochrome b 558 negative or have p47 phox deficiency, exhibit no significant NADPH oxidase activity and show no increase in CL by sulfite. Inhibitors of protein kinase C, H7, and calphostin C, as well as inhibitors of CA 2+- and calmodulin-dependent processes, W7 and R 24 571, completely inhibited the increased CL of sulfite-treated neutrophils. These findings indicate that sulfite in low concentrations stimulates neutrophils to produce superoxide anions by activation of NADPH oxidase through a signal transduction pathway involving protein kinase C and Ca 2+/calmodulin.

Heat-aggregated IgA prepared from patients with IgA nephropathy increases calcium mobilization and superoxide production of human neutrophils in vitro.

IgA nephropathy, characterized by predominant mesangial IgA deposition, is the commonest glomerulonephritis worldwide. It is envisaged that circulating IgA plays a primary role in the glomerular injury of IgA nephropathy. In this study, we examined the pathophysiologic effect of IgA and IgG isolated from IgA nephritic patients on the signal transduction and oxidative metabolism of human neutrophils. Heat-aggregated forms, monomers, and F(ab')2 fragments of IgA and IgG were prepared from sera of 11 IgA nephritic patients and 11 healthy controls. Signal transduction was studied by measuring calcium mobilization and oxidative metabolism by measuring superoxide production. Different forms of IgA and IgG from patients with IgA nephropathy did not induce a significant increase in calcium mobilization directly. Nonetheless, neutrophils preincubated with heat-aggregated IgA or IgG from IgA nephritic patients demonstrated a significant rise in calcium mobilization upon subsequent stimulation by a chemotactic peptide, FMet-Leu-Phe (FMLP). Heat-aggregated IgA or IgG pretreatment of neutrophils increased FMLP-induced calcium mobilization in a dose-dependent manner. Aggregated IgA or IgG prepared by heat aggregation from IgA nephritic patients induced a significantly greater superoxide production from neutrophils than immunoglobulins from healthy controls. Similarly, heat-aggregated IgA and IgG induced superoxide production in a dose-dependent manner. Our data suggest that heat-aggregated forms of IgA and IgG exert an upregulatory effect on signal transduction and oxidative metabolism in human neutrophils. These findings indirectly support the view that neutrophils could be activated in IgA nephropathy and may potentially be participating in the inflammatory process of glomerular and interstitial injury.

Stimulation of human neutrophil oxidative metabolism by nonopsonized Neisseria gonorrhoeae.

Nonopsonized gonococci possessing opacity-associated (Opa; previously PII) outer membrane proteins stimulate neutrophils to undergo a vigorous oxidative response when measured by luminol-dependent chemiluminescence (LDCL). In these studies, we characterized the mechanism of this stimulation. No gonococci that we tested induced measurable release of neutrophil superoxide anion (O2-) or hydrogen peroxide (H2O2) as measured by reduction of cytochrome c or the oxidation of scopoletin, respectively. Neutrophils pretreated with gonococci and then exposed to phorbol myristate acetate, the chemotactic peptide formylmethionylleucylphenylalanine, or opsonized zymosan released levels of neutrophil O2- and H2O2 comparable to controls, indicating that gonococci were not preventing or inhibiting neutrophil O2- or H2O2 release. To ascertain a possible explanation for these seemingly contradictory observations (i.e., induction of LDCL, but no release of O2- or H2O2), we further characterized the ability of Opa+ gonococci to stimulate LDCL. By using 1 mM azide and 4 U of horseradish peroxidase to monitor extracellular LDCL selectively and 2,000 U of catalase to monitor intracellular LDCL selectively, we determined that greater than 80% of total gonococcus-induced neutrophil LDCL occurred intracellularly. In addition, neutrophils stimulated with Opa+ gonococci showed a marked increase in O2 uptake and hexose monophosphate shunt activity. We conclude that Neisseria gonorrhoeae induces neutrophil oxidative metabolism without causing release of detectable amounts of reactive oxygen intermediates into the surrounding milieu. The gonococcus apparently directs oxidase assembly and activity to the phagolysosomal membrane. This could be a mechanism by which extracellular gonococci persist for extended periods in vivo in the presence of high concentrations of neutrophils.

Absence of effect of chloroquine in vivo on neutrophil oxidative metabolism in human subjects

High levels of antimalarial drugs have been shown to be inhibitory to the oxidative metabolism of human neutrophils in vitro. In this study, chemiluminescence of, and chloroquine concentrations in, neutrophils from volunteers treated with prophylactic or therapeutic malaria regimens were assessed. Although detectable chloroquine levels were found in neutrophils, their chemiluminescence was not reduced at prophylactic or at therapeutic drug levels, in comparison with controls without chloroquine treatment. At these clinically achievable concentrations, chloroquine was not found to interfere with the oxidative metabolism of neutrophils.

Gamma interferon is able to enhance the oxidative metabolism of human neutrophils

The oxidative metabolism of human neutrophils has been studied after incubation of the cells with recombinant interferon-y. Neutrophils incubated for 2-4 hours with 2-50 U/ml recombinant interferon-y undergo a higher respiratory burst measured both as Oz consumption and Oz- production when stimulated with formyl-methionyl-leucyl-phenylalanine, Concanavalin A or phorbol myristate acetate. The potentiating effect of interferon-y requires more than one hour of incubation, is optimal at 20-50 U/ml and depends on the presence of serum in the incubation medium. The interferon effect depends on new protein synthesis. Cycloheximide at doses which do not alter the respiratory response of normal cells completely prevents the potentiating effect of interferon.

Antimalarial drugs and human neutrophil oxidative metabolism

The effect of several commonly used antimalarial drugs on human peripheral blood neutrophil oxidative metabolism was studied. The following drugs were tested: chloroquine diphosphate, quinine HCl, mefloquine, proguanil HCl, cycloguanil, pyrimethamine, sulphadoxine, and tetracycline HCl. It was found that none of the antimalarial drugs examined, at clinically obtainable concentrations, had any inhibitory effect on neutrophil oxygen consumption, glucose oxidation, superoxide production, NBT reduction, and chemiluminescence. However, at higher concentrations chloroquine, quinine, mefloquine, and proguanil inhibited neutrophil oxidative burst. There was a slight enhancing effect on neutrophil oxidative metabolism by pyrimethamine, combination of pyrimethamine-sulphadoxine, cycloguanil and tetracycline at concentrations lower than the clinical levels.

Effect of saturated and unsaturated fatty acids on the oxidative metabolism of human neutrophils. The role of calcium ion in the extracellular medium

The ability of fatty acids to stimulate the generation of superoxide anion (O 2 −) by human neutrophils was investigated with respect to their Krafft points. Saturated (myristic acid) and unsaturated (elaidic and oleic acid) induced a marked O 2 − generation and release from human neutrophils at pH 7.4 in the absence of Ca 2+, while 0.3 mM Ca 2+ inhibited both myristic acid and elaidic acid-induced O 2 − release. [ 14C]Myristic acid association with neutrophils was reduced by addition of Ca 2+, whereas oleic acid association was not affected. When the pH of the reaction mixture was lowered to 6.4, 0.6 mM Ca 2+ did not inhibit the O 2 − generation by human neutrophils. These results indicate that the inhibitory effect of Ca 2+ on the fatty acid-induced O 2 − generation might be due to the ionic interaction between the carboxyl group of the fatty acid and Ca 2+. Furthermore, 11-methyltridecanoic acid, a branched isomer of myristic acid, which showed the low Krafft point even in the presence of Ca 2+, stimulated O 2 − generation by human neutrophils not only in the absence but also in the presence of 0.6 mM Ca 2+. The effect of Ca 2+ on the fatty acid-induced O 2 − generation by neutrophils was discussed with reference to its possible relationship to the Krafft point.

Effect of Staphylococcus aureus enterotoxin F on human neutrophil oxidative metabolism.

Epidemiologic and animal challenge studies have suggested that SEF may play a critical role in toxic-shock syndrome [1]. However, the pathogenic mechanism of SEF activity is not known. One means by which this toxin could elicit the widespread organ dysfunction observed in toxic-shock syndrome would be by directly promoting PMN production of toxic oxygen species and the resultant secondary endothelial cell damage [2]. To evaluate this possibility, we assayed the effect of SEF on PMN oxidative metabolism. SEF at 0.01-100 ng/ml did not stimulate O2- release or O2 consumption by inactive PMNs. Similarly, incubation of PMNs with 10 ng of SEF/ml for 1 hr neither potentiated nor inhibited cellular O2 consumption stimulated by optimal (10 mg/ml) or suboptimal (0.1 mg/ml) concentrations of opsonized zymosan. Finally, SEF had no effect on O2- release by PMNs stimulated by PMA. PMN viability, as assessed by trypan blue exclusion, was unaffected by SEF. This study did not address the possibility that SEF might indirectly activate PMN oxidative metabolism by promoting leukocytic pyrogen production by monocytes and macrophages [3]. SEF neither directly activated PMN oxidative activity nor potentiated the cellular oxidative response to particulate or soluble stimuli. Consequently, direct stimulation of PMN-derived, O2- mediated damage to endothelial cells is not a tenable hypothesis to explain the mechanism of SEF toxicity.

Inhibition of Zymosan Activation of Human Neutrophil Oxidative Metabolism by a Mouse Monoclonal Antibody

Abstract We have studied a neutrophil-specific murine monoclonal antibody, PMN7C3 (IgG3), which specifically alters PMN oxidative metabolism stimulated by serum-opsonized zymosan (STZ) or Candida albicans (STC). Polymorphonuclear cells (PMNs) exposed to PMN7C3 show a significant depression in O2- release (52.8% +/- 2.5% of control), H2O2 release (44.4% +/- 6.0% of control), and O2 consumption (73.9% +/- 2.6% of control) in response to STZ. O2 release in response to phorbol myristate acetate (PMA) was modestly reduced (78.4% +/- 3.7%) by PMN7C3 treatment, but not to the extent seen with STZ or STC. PMN7C3 did not affect O2 release by PMNs stimulated by zymosan opsonized with IgG or by S. aureus, A 23187, or FMLP. PMN7C3 was not cytotoxic, did not trigger oxidative metabolism when used as a stimulus, did not alter STZ- induced degranulation, and did not interfere with binding or uptake of STZ by PMNs. Exposure of PMNs to PMN7C3 decreased PMN rosette formation with erythrocytes coated with C3b (54% of control) or C3bi (63% of control), but had no affect on rosette formation with IgG-coated erythrocytes. PMN7C3 does not bind to monocytes and had no affect on rosette formation by this cell type. Binding of antibody PMN7C3 to the neutrophil surface inhibits the oxidative response to opsonized STZ or STC, possibly in part by altering the function or expression of C3b and C3bi receptors. Monoclonal antibodies such as PMN7C3 provide highly specific probes that may be used to define the molecular features of the stimulus-coupled response of PMN activation.

Inhibition of zymosan activation of human neutrophil oxidative metabolism by a mouse monoclonal antibody

We have studied a neutrophil-specific murine monoclonal antibody, PMN7C3 (IgG3), which specifically alters PMN oxidative metabolism stimulated by serum-opsonized zymosan (STZ) or Candida albicans (STC). Polymorphonuclear cells (PMNs) exposed to PMN7C3 show a significant depression in O2- release (52.8% +/- 2.5% of control), H2O2 release (44.4% +/- 6.0% of control), and O2 consumption (73.9% +/- 2.6% of control) in response to STZ. O2 release in response to phorbol myristate acetate (PMA) was modestly reduced (78.4% +/- 3.7%) by PMN7C3 treatment, but not to the extent seen with STZ or STC. PMN7C3 did not affect O2 release by PMNs stimulated by zymosan opsonized with IgG or by S. aureus, A 23187, or FMLP. PMN7C3 was not cytotoxic, did not trigger oxidative metabolism when used as a stimulus, did not alter STZ-induced degranulation, and did not interfere with binding or uptake of STZ by PMNs. Exposure of PMNs to PMN7C3 decreased PMN rosette formation with erythrocytes coated with C3b (54% of control) or C3bi (63% of control), but had no affect on rosette formation with IgG-coated erythrocytes. PMN7C3 does not bind to monocytes and had no affect on rosette formation by this cell type. Binding of antibody PMN7C3 to the neutrophil surface inhibits the oxidative response to opsonized STZ or STC, possibly in part by altering the function or expression of C3b and C3bi receptors. Monoclonal antibodies such as PMN7C3 provide highly specific probes that may be used to define the molecular features of the stimulus-coupled response of PMN activation.

Histamine modulation of human neutrophil oxidative metabolism, locomotion, degranulation, and membrane potential changes.

Previous reports have suggested that histamine modulates neutrophil chemotaxis, but this has not been observed by all laboratories. We have re-addressed this controversial point and demonstrate that histamine and H1- and H2-receptor-specific agonists cause limited inhibition of chemotaxis while stimulating chemokinesis. Furthermore, using the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) as a stimulus, we demonstrate that histamine and H1/H2 agonists inhibit f-met-leu-phe-stimulated changes in membrane potential (monitored with the cyanine dye dipentyloxacarbocyanine), superoxide anion production (cytochrome c reduction), hydrogen peroxide formation (scopoletin fluorescence), and degranulation of granule contents (lysozyme and beta-glucuronidase) in a dose-dependent manner but have no effect on neutrophil functions stimulated by the secretagogues phorbol myristate acetate or A23187. All inhibitory effects of histamine and the H1/H2 agonists are reversed in a competitive manner by the H2 antagonist cimetidine. In addition, structure-activity studies using H1 and H2 receptor agonists and antagonists indicate that a single site with specificity for both H1 and H2 analogue structures modulates the various f-met-leu-phe-stimulated functions studied. Kinetic studies demonstrate that the inhibitory effects of histamine on neutrophil function are only observed when histamine is added before f-met-leu-phe and that inhibition occurs within 10 to 20 sec of histamine addition, does not persist after its removal, and is reversed by addition of cimetidine 10 to 20 sec before stimulation with f-met-leu-phe. Although the inhibitory effects of histamine are exerted early in the sequence of PMN activation by f-met-leu-phe, histamine does not affect the binding or internalization of f-met-leu-[3H]phe. The ability of histamine to modify the variety of neutrophil responses demonstrated in this report suggests an important and direct role for histamine in the regulation of inflammatory reactions in acute allergic settings or other disease states in which histamine release may occur.

Effect of respiratory syncytial virus and virus-antibody complexes on the oxidative metabolism of human neutrophils.

The effect of respiratory syncytial virus (RSV) or mixtures of RSV and its specific antibody on the oxidative metabolic activity of human polymorphonuclear leukocytes was studied by the technique of luminol-dependent chemiluminescence. Peripheral blood neutrophils obtained from normal healthy donors were used. RSV alone failed to induce any chemiluminescent response by the neutrophils. However, mixtures of RSV and RSV antibody-positive serum regularly elicited significant neutrophil chemiluminescence. Ultracentrifugation, electron microscopy, and Raji cell immune complex assays of virus-antibody mixtures suggested that the neutrophil chemiluminescent response was related to the presence of specific immune complexes of RSV antigen-antibody. Heat inactivation of the serum significantly reduced the polymorphonuclear leukocyte chemiluminescence, and the response also appeared to be dependent on the dose of the virus and the antibody in the reaction mixture. It is proposed that interaction between the neutrophil and RSV-specific immune complexes may contribute to the pathogenesis of RSV infection via the possible release of metabolic products from the activated neutrophils.

Effects of fluoride on the oxidative metabolism of human neutrophils

Although 20 m m fluoride stimulates neutrophils to produce superoxide anion, the kinetics of production are dissimilar to those observed with other stimuli. Further, fluoride was shown to inhibit oxidation of [1- 14C] glucose, and incubation of neutrophils with fluoride did not result in significant generation of chemiluminescence or iodination of cellular material, as did other soluble stimuli. These results indicate a discordance between the effect of fluoride on superoxide generation and other metabolic parameters associated with the respiratory burst. The data suggest that fluoride might not be a useful probe for investigating the molecular basis of the respiratory burst in human neutrophils.