The activation of gold complexes by cyanide produced by polymorphonuclear leukocytes--I. The effects of aurocyanide on the oxidative burst of polymorphonuclear leukocytes

It has been suggested that the antiarthritic gold complex, aurothiomalate (Autm), is activated by its conversion to aurocyanide by polymorphonuclear leukocytes (PMN) which generate cyanide from thiocyanate. In an examination of this hypothesis, a study has been conducted on the effects of aurocyanide on the oxidative burst of polymorphonuclear leukocytes (PMN) and monocytes activated by phorbol myristate acetate (PMA). Aurocyanide produced delayed inhibition of the oxidative burst as shown by its effect on both lucigenin and luminol-dependent chemiluminescence and on the production of superoxide. It was a more potent inhibitor of luminol-dependent chemiluminescence than free thiomalate and other by-products of the reaction between Autm and cyanide. Aurocyanide had a biphasic effect on the PMA-stimulated hexose monophosphate shunt of PMN, with enhancement at 0.1 microM and inhibition at 10 and 100 microM. The activity of aurocyanide was also compared with that of auranofin, an orally active gold complex, which inhibits a variety of functions of PMN and monocytes. At low concentrations, auranofin produced delayed inhibition of chemiluminescence in a similar fashion to aurocyanide but at high concentrations was an immediate inhibitor of the oxidative burst.     

Effects of gold-chloroquine complexes on respiratory burst of polymorphonuclear leukocytes

The effects of gold-chloroquine derivatives with the formula [Au(PR3)(CQ)]PF6 (where R = Ph (1), Et or Me) on the superoxide anion production by human neutrophils (PMNs, polymorphonuclear cells) were investigated. When these complexes (0.1-3 mumol/l) were added to PMNs prior to the activators formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), they inhibited isoluminol-horseradish peroxidase-dependent chemiluminescence (CLisol). The inhibition was a direct result of effects on PMNs since chemiluminescence in the cell-free system (horseradish peroxidase-hydrogen peroxidase-isoluminol) was not affected. The above mentioned concentrations of the complexes did not show in vitro toxicity on the cells. On the other hand, when 1 mumol/l of complex 1 was added to cells after stimulation, the chemiluminescence of PMNs stimulated by PMA was inhibited, but not the chemiluminescence stimulated by fMLP. The gold-chloroquine binding was essential for the referred activity as chemiluminescence was not influenced by the precursors chloroquine (CAS 54-05-7) and AuCl(PPh3). Furthermore, the extent of inhibition of chemiluminescence in PMNs activated by PMA did not increase with the duration of preincubation in presence of 1 mumol/l of 1. Extensive washing of cells after preincubation with 1 mumol/l of 1 reversed the aforementioned inhibition. All these results show that the gold-chloroquine binding can lead to compounds with specific properties that could make them useful in the treatment of some inflammatory diseases.     

Inhibition of human platelet activation by polymorphonuclear leukocytes.

1. The effect of unstimulated human polymorphonuclear leukocytes (PMNs) on platelet activation was examined. 2. Human platelet aggregation and adenosine 5'-triphosphate (ATP) release induced by collagen (1-2 micrograms ml-1); thrombin (0.01-0.02 u ml-1) or arachidonic acid (AA) (0.1-0.2 mM) were markedly inhibited when conducted in the presence of unstimulated PMNs. 3. Platelet inhibition induced by PMNs was dependent on the number of PMNs and on the incubation time of the mixed cell suspension. 4. Platelet inhibition was not reversed in time when PMNs were depleted from the mixed-cell suspension. 5. PMN-mediated platelet-inhibition was not mediated by AA metabolites, oxygen reactive intermediates, nitric oxide or proteases. 6. The factor(s) accounting for the platelet inhibition mediated by PMNs are not yet characterized.     

Retinoids inhibit the respiratory burst and degranulation of stimulated human polymorphonuclear leukocytes.

Retinoids exhibit a wide spectrum of activities, including antiinflammatory properties. We have investigated the effect of retinoic acid (RA) and retinyl acetate (RAc) on the production of reactive oxygen metabolites and the release of lysosomal enzymes by human polymorphonuclear leukocytes (PMN). Incubation of PMN with RAc or RA (1-100 microM) caused a dose-dependent inhibition (upto 90%) in O2- production and chemiluminescence induced by phorbol myristate acetate (PMA), N-formyl-methionyl-leucyl-phenylanaline (fMLP), opsonized zymosan or ionophore A23187. Both retinoids (1-100 microM) also inhibited, in a dose-dependent way, degranulation induced by fMLP (upto 85% at the highest concentration of RA). These inhibitory effects appear irreversible, since they persist after the drugs are removed and the cells washed before stimulation. Inhibitors of cyclo-oxygenase activity such as acetylsalicyclic acid and indomethacin did not influence the effects of RAc. In contrast, BW755, an inhibitor of both cyclooxygenase and lipoxygenase, reversed the inhibitory action of RAc, suggesting that the effect of retinoids occurs possibly through the mediation of lipoxygenase products. The modulation of PMN oxidative metabolism and degranulation might help explain the antiinflammatory properties of retinoids.     

Effect of amphotericin B associated with a lipid emulsion on the oxidative burst of human polymorphonuclear leukocytes

• 1.1. Despite its toxicity, amphotericin B (AB) continues to be the drug of choice for the treatment of systemic fungal infection. The drug acts on several cell types, including polymorphonuclear leukocytes (PMN), where it inhibits the oxidative burst of cells submitted to several stimuli.
• 2.2. It was previously shown that the association of AB with a triglyceride-rich emulsion that physiologically mimics chylomicrons reduces toxicity.
• 3.3. We found that the association of AB with a triglyceride-rich emulsion reduces the loss of PMN viability produced by the drug.
• 4.4. The inhibition of the PMN oxidative burst triggered by phorbol 12-myristate 13-acetate (PMA) and opsonized zymosan (OZ) also was decreased by the association of the drug with this lipid emulsion.
• 5.5. Delivery of AB in a lipid emulsion may be of advantage in the treatment of immunosuppressed patients.

     

Mode of activation of the metabolic burst in polymorphonuclear leukocytes by calcium oxalate crystals

Microcrystals of calcium oxalate cause an activation of the metabolic burst in polymorphonuclear leukocytes, measured as NBT reduction. Crystal-induced NBT reduction, which was mainly due to superoxide release, was accompanied by enzyme release. Modulation of calcium oxalate-induced activation by several agents resembles the effect of these agents on activation by opsonized zymosan and by phorbol myristate acetate. Both the activation of the metabolic burst and concomitant enzyme release could be counteracted by certain anions, such as oxalate, pyruvate and citrate, indicating that positive charges on the crystals play an important role in crystal-cell interaction. Removal of the negatively charged sialic acid from the cell surface by neuraminidase did not affect the the action of the crystals. The mechanism, by which calcium oxalate activates polymorphonuclear leukocytes, is discussed.     

Low-dose sulfur mustard primes oxidative function and induces apoptosis in human polymorphonuclear leukocytes

Although considerable work has focused on understanding the processes of direct tissue injury mediated by the chemical warfare vesicant, sulfur mustard (2,2'-bis-chloroethyl sulfide; SM), relatively little is known regarding the mechanisms of secondary injury caused potentially by the acute inflammatory response that follows SM exposure. Polymorphonuclear leukocytes (PMNs) play a central role in the initiation and propagation of inflammatory responses that, in some cases, result in autoimmune tissue damage. The potential for PMN-derived tissue damage following SM exposure may, in part, account for the protracted progression of the injury before it resolves. The current study was undertaken to evaluate the priming, oxidative function, and viability of PMN following exposure to low doses of SM such as those that might remain in tissues as a result of topical exposure. Our results demonstrate that doses of SM ranging from 25 to 100 microM primed PMN for oxidative burst in response to activation by fMLP, and that doses of SM ranging from 50 to 100 microM induced PMN apoptosis. Understanding the mechanisms through which SM directly affects PMN activation and apoptosis will be of critical value in developing novel treatments for inflammatory tissue injury following SM exposure.     

The effect of apomorphine on exocytosis and metabolic burst of polymorphonuclear leukocytes

1 In rabbit polymorphonuclear leukocytes (PMNLs) apomorphine at 10-100 microM inhibits fMet-Leu-Phe and A23187-induced exocytosis, and the phorbol myristate acetate- and fMet-Leu-Phe-induced activation of the metabolic burst. The secretory response was not restored by washing the cells after pretreatment with apomorphine. 2 The inhibitory effect of apomorphine was not prevented by the dopamine receptor antagonists haloperidol and pimozide, nor did dopamine itself inhibit fMet-Leu-Phe-induced exocytosis. It therefore seems unlikely that effects are mediated via dopamine receptors. However, sulphydryl reagents reduced the inhibitory effect of apomorphine, suggesting that it may depend upon interaction with susceptible sulphydryl groups, the intactness of which is required for exocytosis and other functions of PMNLs.     

Metabolic activation of eugenol by myeloperoxidase and polymorphonuclear leukocytes

Eugenol has recently been associated with the toxic effects of clove cigarettes on human lungs. We have studied the metabolism and adverse effects of eugenol on human polymorphonuclear leukocytes (PMNs). Myeloperoxidase, isolated and purified from human PMNs, catalyzed the oxidation of eugenol to a reactive intermediate which is likely to be a quinone methide. Eosinophil peroxidase, lactoperoxidase, prostaglandin H synthase, horseradish peroxidase, and rat intestinal peroxidase also supported this hydrogen peroxide dependent reaction. Glutathione inhibited the formation of this metabolite, resulting in the formation of glutathione disulfide and a small amount of eugenol-glutathione conjugates. In cellular incubations, phorbol ester stimulated PMNs catalyzed the covalent binding of [3H]eugenol to cellular protein, which was partially inhibitable by azide. Intracellular glutathione levels decreased by 90% over a period of 30 min in phorbol ester stimulated PMNs exposed to 100 microM eugenol compared with decreases of 30% (phorbol ester alone) or 5% (eugenol alone) in control incubations. In addition, eugenol was more cytotoxic to PMNs in the presence of phorbol ester than in its absence, and eugenol inhibited the phorbol ester stimulated oxidative burst in PMNs as reflected by a decrease in oxygen consumption, superoxide formation, and hydrogen peroxide formation. These results suggest that PMNs are capable of activating eugenol to a reactive intermediate and also suggest a mechanism whereby eugenol can potentially interfere with and adversely affect vital PMN functions.     

Effect of stobadine on human stimulated polymorphonuclear leukocytes.

The effect of stobadine (0.1-100 microM) on human polymorphonuclear (PMN) leukocytes stimulated with N-formyl-methionyl-leucyl-phenylalanine, a specific receptor activator, or with the calcium ionophore, A-23187 (receptor bypassing stimulus) was investigated with respect to: i) superoxide generation, ii) beta-glucuronidase release and iii) 3[H]-arachidonic acid liberation. Stobadine was found to exert an inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine but not on A-23187-stimulated PMN leukocytes. The effect was more intensive on superoxide generation and beta-glucuronidase release than on 3[H]-arachidonic acid liberation. These results indicate that the inhibitory effect of stobadine is most probably via a mechanism dependent on signal transduction across the plasma membrane. This effect may occur through inhibition of arachidonate signal transduction through a regulatory G-protein.     

Inhibition of oxidative metabolism in rabbit polymorphonuclear leukocytes by triphenyltin chloride

Treatment of rabbit polymorphonuclear leukocytes (PMN) with triphenyltin chloride (TPTC1) inhibited chemiluminescence generation stimulated by particulate stimulus, zymosan, or soluble stimuli, concanavalin A + cytochalasin D. Superoxide anion (O ₂ ^– ) production was also inhibited, indicating that the inhibition involved inhibition of early oxidative metabolic process(es). The direct inhibition of the activation process of the oxidative burst was established by the experiments showing that a) chemiluminescence generated by xanthine oxidase-acetaldehyde system was not inhibited by TPTC1, b) washing the PMN after the treatment with TPTC1 did not affect the results of chemiluminescence, and c) there was no change in cell viability after the treatment with TPTC1.     

Auranofin inhibits the activation pathways of polymorphonuclear leukocytes at multiple sites.

In order to characterize the mechanism by which the anti-rheumatic gold complex auranofin (AF) affects the functions of resting and activated polymorphonuclear leukocytes (PMN) the following studies were performed: (1) The effect of AF on the major processes involved in the respiratory burst of PMN: glucose transport and phosphorylation; hexose monophosphate (HMP) shunt activity in intact cells and in a cell-free system; superoxide production by particulate fractions and intact PMN measured as lucigenin-dependent chemiluminescence. (2) A comparison of the effects of AF added to the PMN before, at the time of, or subsequent to the stimulants [N-formyl-methionyl-leucyl phenylalanine (FMLP), concanavalin A (ConA), calcium ionophore (A23187) and phorbol myristate acetate (PMA)]. (3) The effect of AF on PMN activated by two stimulates (PMA, ConA) added sequentially. AF (0.1-10 microM) caused a dose-dependent inhibition of lucigenin-dependent chemiluminescence regardless of the activator (FMLP, ConA, A23187, PMA) when AF was added before the activator. In contrast, when AF was added to PMN after stimulation, it inhibited only the chemiluminescence of PMN stimulated by PMA. Furthermore, the chemiluminescence was largely unaffected by AF in sequentially activated PMN. The relative sensitivity to AF of the various processes studied indicates that blockade of the activation signal appears to be responsible for inhibition of the respiratory burst of PMN.     

Characteristics and mechanisms of azithromycin accumulation and efflux in human polymorphonuclear leukocytes.

Azithromycin achieves prolonged, high tissue concentrations in spite of low serum levels and obviously must be active at tissue sites of infection to be effective. These unique features prompted us to evaluate the interactions of azithromycin and human polymorphonuclear leukocytes (PMN). Uptake of radiolabeled antibiotic by PMN was determined by a velocity-gradient centrifugation technique and expressed as the ratio of cellular to extracellular drug concentration (C/E). Azithromycin was massively accumulated by human PMN (C/E=387.2 at 2 h). Uptake was not influenced by inhibitors of cellular metabolism, but phagocytosis slightly inhibited the entry of azithromycin into PMN. After removal of extracellular drug, the release (efflux) of azithromycin from PMN was extremely slow. Agents which neutralize lysosomal pH, preventing protonation and trapping of azithromycin, markedly increased antibiotic efflux. Active concentration and prolonged retention of azithromycin by phagocytic cells should allow delivery and subsequent release of accumulated drug at sites of infection.     

Suppression of respiratory burst of polymorphonuclear leukocytes by Azelastine hydrochloride (Azeptin)

The inhibitory action of azelastine hydrochloride (Azeptin) on the respiratory burst in peripheral polymorphonuclear leukocytes (PMN) and pulmonary alveolar macrophages (PAM) has been studied. Azeptin in vitro suppressed chemiluminescence and superoxide (O ₂ ^- ) generation by human PMN in a dose-and time-dependent manner. Phorbol myristyl acetate (PMA) and formyl-methionyl-leucyl-phenylalanine (FMLP)-induced O ₂ ^- generation were strongly suppressed by 10^–6M and 10^–5M Azeptin, respectively. PMN and PAM from rabbits injected with Azeptin 0.2 mg·kg^–1 for 5 days showed lower chemiluminescence and O ₂ ^- generation than cells from untreated rabbits. Nitroblue tetrazolium reduction activity in human PMN was suppressed by treatment of PMN with 10^–6M Azeptin for 6 h. Inositol trisphosphate, intracellular free calcium, and protein kinase C activity were decreased by 10^–6M to 10^–5M Azeptin. The tyrosine phosphorylation of many proteins, especially a 115 kDa protein, was suppressed by 10^–5M Azeptin. However, superoxide dismutase activity in PMN, PAM, and lung tissue samples was only slightly decreased, even when the rabbits were treated with 1.0 mg·kg^–1 Azeptin for 5 days. The results suggest that Azeptin suppresses multiple signal transduction steps in the respiratory burst of PMN. This suppressive action should be very useful in the prevention and treatment of reactive oxygen-associated disorders.Abbreviations Azeptin azelastine hydrochloride - PMN polymorphonuclear leukocytes - PAM pulmonary alveolar macrophages - PMA phorbol myristyl acetate - FMLP formyl-methionyl-leucyl-phenylalanine - PKC protein kinase C - ROS reactive oxygen species - O ₂ ^- superoxide - IP₃ inositol 1,4,5-trisphosphate - DG diacylglycerol - NBT nitroblue tetrazolium - SOD superoxide dismutase - TNF- tumor necrosis factor-alpha     

Inhibition of active oxygen generation by dipyridamole in human polymorphonuclear leukocytes.

The effect of dipyridamole on active oxygen generation by human polymorphonuclear leukocytes (PMN) was investigated. Dipyridamole inhibited the production of oxidative metabolites from human PMN stimulated by opsonized zymosan and formyl-methionyl-leucyl-phenylalanine dose and time dependently. To determine whether dipyridamole directly inhibits the production of oxygen metabolites by human PMN, human PMN were preincubated with dipyridamole washed prior to stimulation. Dipyridamole was found to directly inhibit human PMN from generated active oxygen metabolites at therapeutic concentrations. Dipyridamole may possibly be a potential scavenger of active oxygen metabolites since it inhibited active oxygen metabolite production from human PMN very rapidly. Dipyridamole was also found to directly affect the scavenging of active oxygen metabolites generated by opsonized zymosan-stimulated human PMN at therapeutic concentrations. This action of dipyridamole was also noted to be exerted against hydroxyl radicals and superoxide anions produced biochemically by an electron spin resonance spectrometer. It thus follows that dipyridamole may inhibit human PMN active oxygen metabolite generation and affect directly the scavenging of active oxygen metabolites at therapeutic concentrations.     

Inhibitory effect of furosemide on activation of human peripheral blood polymorphonuclear leukocytes stimulated with n-formyl-methionyl-leucyl-phenylalanine

The clinical efficacy of inhalatory furosemide (Fu) has been extensively studied in bronchial asthma patients but there are only a few studies addressing its action on cells participating in the underlying inflammatory process. Therefore, we investigated the effect of Fu on human peripheral blood polymorphonuclear leukocytes (PMNL) at concentrations that can be achieved in the bronchial lining fluid by inhalation, i.e. 10(-5), 10(-4) and 10(-3) M. The influence of Fu on the following PMNL parameters was investigated: intracellular calcium changes ([Ca2+]i) as a part of signal transduction and luminol dependent chemiluminescence (LCL) as an indirect measure of NADPH-oxidase activation upon n-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation; chemotaxis to fMLP, phagocytosis and intracellular killing of Staphylococcus aureus. Incubation with Fu resulted in a concentration dependent reduction of Ca2+ influx and Fu (10(-3) M) decreased the main Ca2+ parameters to one half of the control values and to the level obtained in calcium-free buffer. In contrast, Fu had no effect if preincubated with the cells and then removed by washing. The LCL signal was reduced by Fu (10(-3) M) from 2000 +/- 870 to 550 +/- 440 arbitrary units [aU] (p<0.05). In contrast to the [Ca2+]i measurements, a slightly diminished LCL was also observed following preincubation with Fu and washing. No effect of Fu was found on phagocytosis and intracellular killing of St. aureus. Fu diminished chemotaxis to fMLP but at 10(-3) M it also displayed weak chemoattractant properties. The differential action of Fu on human PMNL may add to the understanding of its topical and restricted efficacy in bronchial asthma.     

Localization of 5-lipoxygenase within human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMN) stimulated with the Ca-ionophore A23187 or opsonized zymosan not only release marker enzymes of specific granules but secrete 5-lipoxygenase activity as well. In the presence of BSA cells incubated with [14C]AA were able to synthetize 5-HPETE but failed to produce 5-HETE, LTB4, and its omega-oxidation metabolites. Subcellular fractionation studies by differential and isopycnic equilibrium density centrifugation demonstrated main lipoxygenase activity in particulate fractions consisting of specific granules, but not in cytosolic fractions. These results suggest the association of 5-lipoxygenase with specific granules. 5-lipoxygenase released from the cells upon appropriate stimulation reached its peak activity after 10 min and was then rapidly inactivated. It appears that the intermediate 5-HPETE may be generated extracellularly but has to re-enter the intracellular space for further metabolization.     

Sulfur mustard primes phagocytosis and degranulation in human polymorphonuclear leukocytes

Sulfur mustard (2,2'-bis-chloroethyl-sulfide; SM) is a chemical warfare vesicant that causes debilitating skin lesions. Although a great deal of work has focused on the direct effects of SM exposure on the epithelium, it is unclear how much the inflammatory response, induced by exposure, contributes to lesion pathogenesis. Keratinocytes exposed to SM express a number of inflammatory mediators and elicit a cellular infiltrate consisting largely of polymorphonuclear leukocytes (PMN). PMN infiltration into SM lesions occurs as early as 30 min and peaks after several hours postexposure, and, despite the relatively short half-life of SM, PMN infiltrating a lesion could be exposed to micromolar concentrations of the agent. Previously, we have shown that exposure to low doses of sulfur mustard prime oxidative function in human PMN. The current study was undertaken to evaluate the effects of low-dose SM exposure on PMN phagocytosis, degranualtion and chemotaxis. PMN exposed to low doses of SM (50-200 microM) showed a dose-dependent enhancement of phagocytic function. Exocytosis of PMN azurophilic and specific granules [determined by analysis of granule-specific intravesicular receptors, Interleukin 10 receptor (IL-10R) and CD63] was also enhanced by SM exposure. Finally, we examined the effect of SM as a chemoattractant for PMN and show that SM is not itself a chemotaxin. These results suggest that SM injury may, in part, be caused by normal inflammatory function, and that therapeutic strategies aimed at down-regulating PMN activation could lessen the severity of SM injury and the time required for its resolution.     

Dissociation of phagocytosis, metabolic stimulation and lysosomal enzyme release in human leukocytes.

In this paper studies are reported concerning the relationship between particle binding to the plasma membrane of human polymorphonuclear leukocytes (PMN's), phagocytosis, generation of oxidative metabolites, and the release of lysosomal enzymes by these cells. Superoxide (O2-) generation by, and lysosomal enzyme release from, normal PMN's and cytochalasin B-treated cells were measured. We have found that neither phagocytosis nor lysosomal degranulation are prerequisites for enhanced O2- generation. Cytochalasin B-treated PMN's, incapable of ingesting particles but still able to bind particles to membrane receptors, generated enhanced amounts of O2- when treated with serum-treated zymosan (STZ), a C3b receptor stimulus, or with aggregated IgG (agg IgG), an Fc receptor stimulus. Moreover, the soluble stimulators complement component C5a, phorbol myristate acetate (PMA), and calcium ions in the presence of the ionophore A23187, also increased the O2- production of these cells. In all cases a time and dose-dependent stimulation was found of both the O2- generation and the lysosomal enzyme release, but there was no correlation between ability of any stimulus to provoke enzyme release and its ability to stimulate O2- generation. When PMN's were preincubated with 5 X 10(-4) M hydrocortisone-Na-succinate, lysosomal enzyme exocytosis with the immune reactants was inhibited 16-35%. Hydrocortisone also inhibited O2- generation, except when STZ was used as the stimulus. Thus, in the case of stimulation of functional processes of PMN's via the C3b receptor, hydrocortisone inhibits membrane fusion without interfering with one of the early biochemical events (O2-production).