Enhanced sensitivity of Streptomyces seoulensis to menadione by superfluous lipoamide dehydrogenase

Lipoamide dehydrogenase from Streptomyces seoulensis could facilitate menadione-mediated cytochrome c reduction, which was mostly inhibited by superoxide dismutase, indicating the obvious involvement of superoxide radical anion. In this reaction, the production of superoxide radical anion occurred via a menadione semiquinone radical anion. When exposed to menadione, lipoamide dehydrogenase-overexpressing cells showed a much lower survival rate with a concomitant decrease of intracellular protein thiol than the wild-type strain. These results suggest that lipoamide dehydrogenase is a facilitating agent in the redox cycling of quinone compounds in vivo as well as in vitro and could inevitably increase the potential toxicity of the compounds.     

An investigation of the transverse topology of bilirubin UDP-glucuronosyltransferase in rat hepatic endoplasmic reticulum.

Neutrophils stimulated with formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) in the presence of butanol and ethanol formed phosphatidyl alcohols through a phospholipase D mechanism. The alcohols inhibited phosphatidic acid and diradylglycerol (DRG) formation, but did not block inositol 1, 4, 5-trisphosphate release. fMet-Leu-Phe-stimulated superoxide production was inhibited by alcohol concentrations which blocked DRG formation, whereas opsonized-zymosan-stimulated superoxide production was only partially decreased. These results suggest that phospholipase D activation is functionally linked to superoxide production in the human neutrophil.     

Phe-D-Leu-Phe-D-Leu-Phe derivatives as formylpeptide receptor antagonists in human neutrophils: cellular and conformational aspects.

We synthesized several Phe-d-Leu-Phe-d-Leu-Phe analogues in which tert-butyloxycarbonyl and four different ureido substituents were included at the N-terminal of the peptides, obtained as free acid and methyl-ester derivatives. Their biological action was analysed on human neutrophil responses induced by N-formyl-Met-Leu-Phe (fMLF). Several in vitro assays were carried out: receptor binding, measurement of Ca2+ intracellular concentration, chemotaxis, superoxide anion production and enzyme release. A conformational investigation, using infrared absorption and circular dichroism, was also performed. Our results demonstrate that the compounds examined prefer an ordered conformation (beta-turn) in amphipathic environment, and are able to antagonize the neutrophil functions evoked by fMLF. Moreover, the extent of inhibition of Ca2+ intracellular enhancement, as well as of superoxide anion production and granule enzyme release, appears related to their affinity toward the formylpeptide receptor. The free acid peptide derivatives appear to be more active antagonists than the methyl-ester ones.     

Evaluation of synthetic sphingosine, lysosphingolipids and glycosphingolipids as inhibitors of functional responses of human neutrophils.

Human neutrophils, when exposed to soluble stimuli, aggregate, release oxygenated products of arachidonic acid and generate active oxygen species. Sphingolipid-derived products such as sphingosine and lysosphingolipids have been shown to exert selective actions on a variety of cell types, including neutrophils. Therefore, to determine the structural basis for selective inhibition of neutrophil responses by naturally occurring sphingolipids, seven compounds were prepared by total organic synthesis, and their impact on neutrophils in suspension has been studied. The compounds synthesized included sphingosine, psychosine, lactosyl lysosphingolipid, globotriaosyl (Gb3) lysosphingolipid, galactosyl cerebroside, lactosyl ceramide and Gb3 ceramide. The neutrophil responses studied were aggregation, leukotriene generation and superoxide anion production. When exposed to non-cytotoxic levels of the synthetic compounds, as monitored by exclusion of Trypan Blue, none of the synthetic sphingolipids inhibited A23187-induced aggregation of neutrophils. Only lactosyl lysosphingolipid, at a concentration of 1 microM, significantly inhibited aggregation induced by fMetLeuPhe; the other compounds in this series including sphingosine were without effect at equal molar concentrations (1 microM). Aggregation induced by phorbol 12-myristate 13-acetate (PMA) (0.1 microM) was significantly blocked by only two of the synthetic sphingolipids (1 microM). At concentrations below 1 microM, these inhibitory actions were not evident, nor was it possible to assign a structure-activity relationship for this series of compounds. None of the synthetic sphingolipids effectively inhibited the generation of superoxide anions induced by PMA. In addition, neither synthetic sphingosine nor psychosine affected either the formation or metabolism of leukotriene B4. Taken together, the results provide further evidence that sphingolipids, when added to intact cells, are not potent selective inhibitors of functional responses of human neutrophils.     

Auranofin affects early events in human polymorphonuclear neutrophil activation by receptor-mediated stimuli

Auranofin, a new oral antirheumatic gold compound, in concentrations achieved therapeutically, inhibits neutrophil phagocytosis, chemotaxis, chemiluminescence, reduction of cytochrome c, and release of lysosomal enzymes. To further characterize the mechanism by which auranofin affects neutrophils, we studied the effects of auranofin on unstimulated properties and functions of neutrophils as well as on rapidly stimulated functions. When examined by electron microscopy, 4 micrograms/ml of auranofin significantly decreased the number of visualized centriole-associated microtubules in resting cells. Furthermore, auranofin inhibited neutrophil spreading on glass and caused a decrease in negative surface charge (electrophoretic mobility). In addition, auranofin inhibited several fmet-leu-phe-stimulated responses such as shape change, increases in centriole-associated microtubules, decreases in surface charge, and elicited membrane potential changes (di-O-C5(3) dye response). Auranofin (1 micrograms/ml) inhibited fmet-leu-phe-stimulated superoxide and hydrogen peroxide production by 80% (p less than 0.05), and also increased the affinity of receptors for fmet-leu-phe (from Ka 0.035 to Ka 0.48, p less than 0.001). Auranofin also affected neutrophil responses to phorbol myristic acetate (PMA). The total amount of PMA-stimulated superoxide production was suppressed by as little as 0.4 micrograms/ml of auranofin, but the lag time for activation was shortened by low concentrations of auranofin (0.5 to 1 microgram/ml). Four micrograms per milliliter of auranofin suppressed the decrease in surface charge induced by PMA. However, auranofin did not influence superoxide production elicited by the ionophore A23187. The results indicate that auranofin affects the earliest detected responses in neutrophil activation by certain receptor-mediated stimuli.     

Apolipoprotein A-I decreases neutrophil degranulation and superoxide production.

Neutrophils participate in the acute phase response and are often associated with tissue injury in a number of inflammatory disorders. The acute phase response is accompanied by alterations in the metabolism of apolipoprotein A-I and high density lipoprotein (HDL). Structural considerations led to studies investigating the effect of purified HDL and apolipoprotein A-I on neutrophil degranulation and superoxide production. Apolipoprotein A-I but not HDL inhibited IgG-induced neutrophil activation by about 60% as measured by degranulation and superoxide production. This suggests that the lipid-associating amphipathic helical domains of apolipoprotein A-I mediate this effect. In support of this was finding inhibitory effects with two synthetic model lipid-associating amphipathic helix peptide analogs. Apolipoprotein A-I, containing tandem repeating amphipathic helical domains, was approximately ten times more effective than the two peptide analogs and inhibited neutrophil activation at well below physiologic concentrations. Competitive binding studies indicate that resting neutrophils have approximately 190,000 (Kd = 1.7 x 10(-7)) binding sites per cell for apolipoprotein A-I, consistent with a ligand-receptor interaction. These observations suggest that apolipoprotein A-I may play an important role in regulating neutrophil function during the inflammatory response.     

The chymotrypsin inhibitor carbobenzyloxy-leucine-tyrosine-chloromethylketone interferes with the neutrophil respiratory burst mediated by a signaling pathway independent of PtdInsP2 breakdown and cytosolic free calcium.

The effects of carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK), an inhibitor of chymotrypsin, were investigated on the activation pathways of the human neutrophil respiratory burst. At 10 microM zLYCK, a parallel inhibition was observed of superoxide production stimulated with the chemo-attractant FMLP and of chymotrypsin-like activity of human neutrophils. By contrast, superoxide production induced by PMA was minimally affected by zLYCK. The known transduction pathways triggered by FMLP were analyzed. zLYCK did not affect either the FMLP-induced cytosolic free calcium transient, inositol 1,4,5 trisphosphate formation, nor the PMA-induced phosphorylation of the 47-kDa substrate of protein kinase C. zLYCK did not affect the activity of protein kinase C extracted from neutrophils. In Ca(2+)-depleted cells, in which phosphatidylinositol 4,5-biphosphate breakdown does not occur, zLYCK inhibited the FMLP-induced respiratory burst in cells primed by low doses of PMA. The activity of the NADPH oxidase tested with active membranes from stimulated neutrophils or in a cell-free system was not inhibited by zLYCK. We conclude that: 1) zLYCK inhibits superoxide production through the inhibition of a chymotrypsin-like protease of the neutrophil, 2) zLYCK inhibits FMLP-induced activation of NADPH oxidase through a pathway independent of PtdInsP2 breakdown and cytosolic free calcium, and 3) zLYCK may prove a useful probe for the characterization of its target protease in neutrophil activation.     

Effects of leukocyte inhibitory factor (LIF) on neutrophil mediated antibody-dependent cellular cytotoxicity

The human lymphokine, leukocyte inhibitory factor (LIF), was investigated for its effect on neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) for K562 targets. Highly purified LIF (0.5 to 2 U/ml) induced a significant dose-dependent potentiation of neutrophil ADCC by up to 54.9% (p less than 0.001). Higher concentrations of LIF inhibited cytotoxicity. The degree of cytotoxicity was found to correlate (r = 0.99) with the increased secretion of superoxide after neutrophil-target cell interaction. Anaerobic conditions inhibited cytotoxicity mediated by both control and LIF-treated neutrophils. The latter observation lends support to the concept that enhanced ADCC was mediated through increased superoxide production and not through the induction of a separate pathway. Increased superoxide production may have resulted from an upregulation of the transduction mechanism leading to neutrophil stimulation through the Fc receptor. In addition, we demonstrated an increased capacity of the neutrophil to adhere to its target (average 3.3:1 effector:target ratio in untreated cells to 4.8:1 after treatment with LIF), and this may also have been responsible for the increase in the respiratory burst and subsequent enhanced ADCC. These observations provide potential support for an in vivo role for LIF in tumor immunity.     

Unsaturated phosphatidylcholines inhibit superoxide production in human neutrophils.

Unsaturated long chain phosphatidylcholines such as phosphatidylcholine dioleoyl and phosphatidylcholine dilinoleoyl in micromolar concentrations inhibited the superoxide production in neutrophils stimulated by the activators of protein kinase C, phorbol 12-myristate 13-acetate and 1,2-dioctanoyl-sn- glycerol. In contrast, the superoxide production induced by surface receptor agonist, formyl-methionyl-leucyl-phenylalanine, was unaffected by the phospholipids. These data suggest that surfactant phosphatidylcholines may have a modulatory role on neutrophil oxidative burst in the lung during inflammation where there is a preponderance of unsaturated phosphatidylcholines.     

Selective inhibition of human neutrophil chemotaxis to N-formyl-methionyl-leucyl-phenylalanine by sulfones

The therapeutic efficacy of the sulfones, dapsone, and sulfoxone in neutrophilic dermatoses may be related to the effects of these drugs on neutrophil function. Therefore we determined whether neutrophil chemotactic migration to various chemoattractants could be inhibited by sulfones in vitro. The chemotactic responses of human neutrophils from healthy donors were tested by using N-formyl-methionyl-leucyl-phenylalanine (F-met-leu-phe), purified human C5a, and leukocyte-derived chemotactic factor (LDCF). Therapeutic concentrations of sulfones selectively inhibited neutrophil chemotaxis to F-met-leu-phe, but did not affect neutrophil chemotaxis to LDCF or C5a. Inhibition of neutrophil chemotaxis to F-met-leu-phe was induced by both dapsone and sulfoxone at a concentration of 10 micrograms/ml without affecting random migration, and the inhibition was reversed by washing the neutrophils. When dapsone- and sulfoxone-treated neutrophils (100 micrograms/ml) were stimulated with F-met-leu-phe, neutrophil superoxide generation was not inhibited. Sulfapyridine (10 micrograms/ml) also selectively inhibited neutrophil chemotaxis to F-met-leu-phe; however, sulfamethoxazole and sulfisoxazole did not affect chemotaxis. The inhibitory effects of dapsone, sulfoxone, and sulfapyridine could not be demonstrated with granulocytes from rabbits or guinea pigs nor with human monocytes. Experiments with radiolabeled dapsone showed rapid, nonspecific, and reversible binding of dapsone to human neutrophils. These data suggest that a mechanism of action of sulfones in neutrophilic dermatoses may be a selective inhibition of neutrophil migration to as yet undefined chemoattractants in the skin.     

Neutrophil activation by surface bound IgG: pertussis toxin insensitive activation

Surface bound IgG induces neutrophil degranulation and production of superoxide radicals by a mechanism that is not inhibited by either pertussis toxin or cholera toxin, whereas these functions induced by soluble mediators such as FMLP and soluble aggregates of IgG are profoundly inhibited by pertussis toxin. Interaction of neutrophils with surface bound IgG triggers the loss of 32P labeled PIP2 and PIP and the influx of extracellular calcium. Neither of these cellular events when induced by surface bound IgG is inhibited by pertussis toxin. These observations suggest that neutrophil activation induced by surface bound IgG proceeds along a pathway which is not regulated by proteins which are inhibited by either pertussis or cholera toxins.     

Effects of two leukotriene B4 (LTB4) receptor antagonists (LY255283 and SC-41930) on LTB4-induced human neutrophil adhesion and superoxide production

Leukotriene B4 (LTB4) induces a number of functional changes in human neutrophils, including both superoxide release and CD11b/CD18 (Mo1)-mediated adherence to various substrates, such as keyhole limpet hemocyanin (KLH). These effects are both time- and concentration-dependent. Neutrophil adhesion was at least 10-fold more sensitive to the stimulatory action of LTB4 than superoxide production. Two LTB4 receptor antagonists, LY255283 (1-(5-ethyl-2-hydroxy-4-(6-methyl-6-(1H-tetrazol-5-yl)heptyloxy )- phenyl)ethanone) and the sodium salt of SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8- propyl-2H- 1-benzopyran-2-carboxylic acid) were evaluated for effects on human neutrophil superoxide production and adhesion. Despite being more sensitive to LTB4-induced stimulation, neutrophil adhesion was at least 100-fold less sensitive to inhibition by LY255283 and SC-41930 than superoxide production. Both LTB4 receptor antagonists behaved similarly in these models. These compounds did not inhibit neutrophil responses induced by granulocyte/macrophage colony-stimulating factor (GM-CSF).     

Superoxide enhances hypochlorous acid production by stimulated human neutrophils

Stimulated neutrophils undergo a respiratory burst discharging large quantities of superoxide and hydrogen peroxide. They also release myeloperoxidase, which catalyses the conversion of hydrogen peroxide and Cl- to hypochlorous acid. Human neutrophils stimulated with opsonized zymosan promoted the loss of monochlorodimedon. This loss was entirely due to hypochlorous acid, since it did not occur in Cl(-)-free buffer, was inhibited by azide and cyanide, and was enhanced by adding exogenous myeloperoxidase. It was not inhibited by desferal, diethylenetriaminepentaacetic acid, mannitol or dimethylsulfoxide, which excluded involvement of the hydroxyl radical. Approx. 30% of the detectable superoxide generated was converted to hypochlorous acid. As expected, formation of hypochlorous acid was completely inhibited by catalase, but it was also inhibited by up to 70% by superoxide dismutase. Superoxide dismutase also inhibited the production of hypochlorous acid by neutrophils stimulated with phorbol myristate acetate. Our results indicate that generation of superoxide by neutrophils enables these cells to enhance their production of hypochlorous acid. Furthermore, inhibition of neutrophil processes by superoxide dismutase and catalase does not necessarily implicate the hydroxyl radical. It is proposed that superoxide may potentiate oxidant damage at inflammatory sites by optimizing the myeloperoxidase-dependent production of hypochlorous acid.     

Flow cytometric evaluation of human neutrophil apoptosis during nitric oxide generation in vitro: the role of exogenous antioxidants

Among numerous inflammatory mediators a nitric oxide molecule is supposed to be important in the modulation of neutrophil survival in vivo and in vitro. The effect of exogenous supply of NO donors such as SNP, SIN-1, and GEA-3162 on the course of human neutrophil apoptosis and the role of extracellular antioxidants in this process was investigated. Isolated from peripheral blood, neutrophils were cultured in the presence or absence of NO donor compounds and antioxidants for 8, 12, and 20 hours. Apoptosis of neutrophils was determined in vitro by flow cytometric analysis of cellular DNA content and Annexin V protein binding to the cell surface. Exposure of human neutrophils to GEA-3162 and SIN-1 significantly accelerates and enhances their apoptosis in vitro in a time-dependent fashion. In the presence of SNP, intensification of apoptosis has not been revealed until 12 hours after the culture. The inhibition of GEA-3162- and SIN-1-mediated neutrophil apoptosis by superoxide dismutase (SOD) but not by catalase (CAT) was observed. Our results show that SOD and CAT can protect neutrophils against NO-donors-induced apoptosis and suggest that the interaction of NO and oxygen metabolites signals may determine the destructive or protective role of NO donor compounds during apoptotic neutrophil death.     

Effect of lipoproteins on macrophage superoxide generation

Superoxide production by macrophages and leukocytes may have an important role in atherogenesis. Whether lipoproteins modulate the superoxide production of these cells is not clear. Therefore, the effect of lipoproteins on the production of superoxide by rat peritoneal macrophages was tested. VLDL and LDL inhibited digitonin-stimulated superoxide production in a dose-dependent manner. Maximum inhibition was observed at 10 μg ml^?1 of VLDL protein and 50 μg ml^?1 of LDL protein respectively. In contrast, HDL (40 μg protein ml^?1) enhanced digitoninstimulated superoxide production (by 47 per cent). Macrophage superoxide production induced by arachidonic acid was enhanced by both VLDL (130 per cent) and HDL (84 per cent), whereas LDL had no effect. The lipoproteins had no effect on macrophage superoxide stimulated by other agonists such as phorbol myristate 13-acetate, sodium fluoride or the calcium ionophore, A23187. The effect of lipoproteins was also tested on human polymorphonuclear leukocyte superoxide generation, stimulated by digitonin and PMA. Ten μg of VLDL, 50 μg of LDL and 50 μg of HDL proteins ml^?1, inhibited digitonin-induced superoxide production by 50, 100 and 33 per cent respectively. Lipoproteins had no effect on PMA stimulated superoxide generation by human polymorphonuclear leukocytes. The stimulatory and inhibitory effects of lipoproteins on macrophage and neutrophil superoxide generation could be important in the understanding of oxidation-mediated development of atherosclerosis.     

Effects of some isoxazolpyrimidine derivatives on nitric oxide and eicosanoid biosynthesis

The inhibitory effect of some isoxazolpyrimidine derivatives on iNOS and COX-2 endotoxin induction in mouse peritoneal macrophages has been studied. Three of these compounds inhibited nitrite and PGE2 accumulation in a concentration dependent-manner at microM range. None of these active compounds affected iNOS, COX-2, COX-1 or PLA2 activities, although some reduced iNOS or COX-2 expression. Besides, no effect was observed on human neutrophil inflammatory responses (LTB4 biosynthesis and superoxide or elastase release). Active compounds were assayed by oral administration in the mouse air pouch model, where they inhibited nitrite accumulation without affecting PGE2 levels or leukocyte migration.     

The evaluation and structure-activity relationships of 2-benzoylaminobenzoic esters and their analogues as anti-inflammatory and anti-platelet aggregation agents

Forty-seven 2-benzoylaminobenzoic esters were synthesized and evaluated in anti-platelet aggregation, inhibition of superoxide anion generation, and inhibition of neutrophil elastase release assays. Most 2-benzoylamino-4-chlorobenzoic acid derivatives showed selective inhibitory effects on arachidonic acid (AA)-induced platelet aggregation. Among them, compounds 6b and 7b exhibited more potent inhibitory effects (ca. 200-fold) than aspirin. Additionally, compounds 1a and 5a showed strong inhibitory effects on neutrophil superoxide generation with IC(50) values of 0.65 and 0.17 microM, respectively. However, compounds 6d and 6e exhibited dual inhibitory effects on platelet aggregation and neutrophil elastase (NE) release; therefore, these two compounds may be new leads for development as anti-inflammatory and anti-platelet aggregatory agents.     

Inhibition of human neutrophil superoxide generation by alpha 1-antichymotrypsin.

Reactive oxygen intermediates and serine proteases are important components of host defense systems but can produce host injury if not tightly regulated. To determine whether these components can be coordinately controlled, we investigated regulation of superoxide generation by physiologically relevant concentrations of a) highly purified serum-derived antichymotrypsin (ACT), b) recombinant, wild-type ACT, c) rACT in which amino acid substitutions were engineered into the reactive center, and d) chymotrypsin/ACT complexes. These proteins and protein complexes inhibited superoxide anion production in neutrophils stimulated by f-Met-Leu-Phe, Con A, or PMA. In contrast, ligand-stimulated degranulation was not inhibited. In addition, using the recombinants and complexes, the region of ACT involved in inhibiting superoxide anion production was shown to be structurally distinct from the reactive center of the protein. The results indicate that functional domains of ACT corresponding to different biological activities can be decoupled and suggest that three species of ACT (intact ACT, a complexed protease/ACT form, and a partially denatured or proteolyzed form of ACT) that can exist in the microenvironment of an activated neutrophil may play an important role in regulating neutrophil function.     

Partial dependency of 1-oleoyl-2-acetyl-glycerol-induced superoxide production by human neutrophils on calcium ions and cytochalasin B

Superoxide production by neutrophils induced by 1-oleoyl-2-acetyl-sn-glycerol at concentrations below 100 microM was enhanced by extracellular calcium ions, while that of phorbol myristate acetate was unaffected. Verapamil, a calcium-channel blocker, more effectively inhibited the superoxide production induced by 1-oleoyl-2-acetyl-sn-glycerol than that of phorbol myristate acetate. Cytochalasin B at 5 micrograms/ml significantly potentiated superoxide production by 1-oleoyl-2-acetyl-sn-glycerol at concentrations below 100 microM, but not that of phorbol myristate acetate. It is suggested that neutrophil activation induced by the former have different features from that of the latter.     

Receptor-specific threshold effects of cyclic AMP are involved in the regulation of enzyme release and superoxide production from human neutrophils

We have investigated the sequence of events leading from the activation of adenylate cyclase and increases in intracellular cyclic AMP to the modulation of enzyme release and superoxide production in human neutrophils. In the isolated plasma membrane, adenylate cyclase is activated by both prostaglandin E1 and isoproterenol. In the whole cell only a small increase in cyclic AMP is observed, though in the presence of the phosphodiesterase inhibitor, methylisobutylxanthine a substantial amplification in intracellular cyclic AMP is observed with both isoproterenol and prostaglandin E1. These conditions are relevant to the regulation of cell function, since fMet-Leu-Phe-stimulated superoxide production is inhibited by either prostaglandin E1 or isoproterenol in the absence of methylisobutylxanthine, while enzyme release is inhibited only via the prostaglandin E1 receptor and then only in the presence of methylisobutylxanthine. For enzyme release and superoxide production, the order of potency for three prostaglandins tested was prostaglandin E1 greater than prostaglandin D2 much greater than prostaglandin F2 alpha. Our results suggest that (a) superoxide production is more sensitive to regulation by cyclic AMP than enzyme release, (b) the type of receptor occupied as well as the threshold level of cyclic AMP attained are important to the regulation of enzyme release, and (c) although elevation in cyclic AMP is inhibitory to neutrophil function, phosphodiesterase inhibition is required in addition to adenylate cyclase activation to effect maximal inhibition.