Purification and characterization of aseanostatins: actinomycete-derived fatty acid inhibitors to myeloperoxidase release from human polymorphonuclear leukocytes.

We found inhibitors, designated aseanostatins P1 and P5, against myeloperoxidase (MPO) release from human polymorphonuclear leukocytes (PMN). Aseanostatins were extracted from an actinomycete isolated in Thailand and purified by a series of column chromatography of charcoal and silica gel, and HPLC. Physico-chemical characterization by gas liquid chromatography and GC-MS indicated that aseanostatins were fatty acids. The active forms of aseanostatins were recovered by hydrolyzing their methyl esters after HPLC. Two components P1 and P5 with the IC50 of 0.96 and 0.54 microgram/ml to the MPO release were obtained as pure forms, indicating aseanostatin P5 was higher activity than aseanostatin P1. The component P1 was identical with 12-methyltridecanoic acid and P5 was indistinguishable to 12-methyltetradecanoic acid (ante-i-15:0). Aseanostatin P5 (1 microgram/ml) did not inhibit beta-glucuronidase release, but O2- production a little. It has no effect on chemotaxis of PMN to fMet-Leu-Phe (10(-8)M), PMN adhesion or phosphorylation of a 64-kD protein in the PMN cell-lysate system.     

Influence of human plasma kallikrein on lysosomal enzyme release from polymorphonuclear leukocytes.

Stimulation of polymorphonuclear leukocytes with kallikrein demonstrated that enzyme acts selectively on the release of lysosomal enzymes of these cells. The release of collagenase, similarly to the release of lysozyme into the incubation medium increased proportionally to kallikrein concentration and the duration of incubation. Kallikrein had a small effect on beta-glucuronidase secretion. No effect on cytoplasm lactate dehydrogenase release was detected. These results suggest that kallikrein, as a soluble stimulus, predominantly induces degranulation of specific granules containing collagenase capable of degrading the connective tissue. Secretion of lysozyme and collagenase requires the presence of active kallikrein. Soybean trypsin inhibitor diminished the enzyme release.     

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.     

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.     

Tumour necrosis factor alpha augments the release of an endothelium-dependent vasoconstrictor from human polymorphonuclear leukocytes.

We have studied the effect of cytokine priming by recombinant human tumour necrosis factor alpha (rhTNF alpha) on the release of vasoactive mediators from human polymorphonuclear leukocytes (PMNs). Supernatants of human PMNs (100 microliters, 5 x 10(7) ml, from 12 donors) relaxed precontracted rabbit aortic rings. The relaxations were preceded by a transient contraction in four experiments. In contrast, supernatants of PMNs incubated with rhTNF alpha (0.3 nM, 30 min, 37 degrees C) elicited variable vascular responses that consisted of either a sustained contraction (n = 4), a transient contraction followed by a sustained relaxation (n = 2), or a sustained relaxation only (n = 6). The vascular tone produced by the rhTNF alpha-treated PMN supernatant was significantly greater over 30 min than that produced by the control supernatant (p < 0.01). Endothelium removal prevented the PMN-induced contractions and significantly decreased the vascular tone produced by all rhTNF alpha-treated PMN supernatants (p < 0.05). The PMN supernatants had no vasoactive effect on aortic rings at resting tension. Molecular size analysis indicated that the PMN-derived contractile mediator is > 30,000 Da. These results suggest that human PMNs, primed by rhTNF alpha, release a stable endothelium-dependent vasoconstrictor that opposes the action of a stable, spontaneously released direct vasodilator.     

Desensitization of substance P-induced K+ release in rat parotid

The angiotensin converting enzyme inhibitor enalapril (0.5 mg/kg i.v.) potentiated significantly the inhibitor effect of the thromboxane A₂ antagonist L-640,035 (1 mg/kg i.v.) on electrically induced platelet accumulation in the rabbit in vivo. Enalapril had no effect upon platelet accumulation when given alone. The hypotensive effects of enalapril did not account for the potentiation because a combination of hexamethonium (5 mg/kg i.v.) and hydralazine (1 mg/kg i.v.), which decreased blood pressure similarly to enalapril, did not augment the effect of L-640,035. Determination by radioimmunoassay of plasma levels of immunoreactive 6-keto-PGF₁, suggested that increases in PGI₂ levels after combined administration of enalapril and L-640,035 could explain the observed potentiation.     

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 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.     

N-phenyllinoleamide metabolism by human polymorphonuclear leukocytes

1. N-phenyllinoleamide (NPLA), the anilide of linoleic acid, has been associated with the epidemiology of Toxic Oil Syndrome, but so far data available on its metabolism are scarce. On account of the similarities in chemical structure between linoleic acid and NPLA, the objective here has been to investigate the oxidative metabolism of this xenobiotic by human polymorphonuclear leukocytes.

2. Human polymorphonuclear leukocytes were incubated with 0.1 mM NPLA spiked with NPLA labelled either on the aniline or the fatty acid moieties. The metabolites were separated by high-performance liquid chromatography and individually collected prior to gas chromatography-mass spectrometry analysis.

3. Identification of the metabolites as N-phenyl-9-hydroxy- and N-phenyl-13-hydroxy-10,12-octadecenamide (9-HNPLA and 13-HNPLA) and their corresponding non-amidated metabolites, the 9-hydroxy- and 13-hydroxyoctadecenoic acids (9-HODE and 13-HODE), suggests that NPLA can be metabolized via the same hydroperoxidative processes acting upon linoleic acid.

4. Identification of free aniline as a NPLA metabolite suggests an amidase-like activity with liberation of aniline and the free fatty acid moieties.     

Carnosic acid and carnosol potently inhibit human 5-lipoxygenase and suppress pro-inflammatory responses of stimulated human polymorphonuclear leukocytes

Carnosic acid (CA) and carnosol (CS) are phenolic diterpenes present in several labiate herbs like Rosmarinus officinalis (Rosemary) and Salvia officinalis (Sage). Extracts of these plants exhibit anti-inflammatory properties, but the underlying mechanisms are largely undefined. Recently, we found that CA and CS activate the peroxisome proliferator-activated receptor gamma, implying an anti-inflammatory potential on the level of gene regulation. Here we address short-term effects of CA and CS on typical functions of human polymorphonuclear leukocytes (PMNL). We found that (I), CA and CS inhibit the formation of pro-inflammatory leukotrienes in intact PMNL (IC(50)=15-20 microM [CA] and 7 microM [CS], respectively) as well as purified recombinant 5-lipoxygenase (EC number 1.13.11.34, IC(50)=1 microM [CA] and 0.1 microM [CS], respectively), (II) both CA and CS potently antagonise intracellular Ca(2+) mobilisation induced by a chemotactic stimulus, and (III) CA and CS attenuate formation of reactive oxygen species and the secretion of human leukocyte elastase (EC number 3.4.21.37). Together, our findings provide a pharmacological basis for the anti-inflammatory properties reported for CS- and CA-containing extracts.     

E-type prostaglandins but not iloprost inhibit platelet activating factor-induced generation of leukotriene B4 by human polymorphonuclear leukocytes.

1. Platelet-activating factor (Paf) was found to stimulate leukotriene B4 release from human polymorphonuclear leukocytes (PMN). This stimulation was considerably enhanced in the presence of small amounts of exogenous arachidonic acid. 2. Prostaglandin E2 (PGE2) and PGE1 (0.1-10 microM) inhibited this reaction dose-dependently. No inhibition was seen with iloprost at these concentrations. 3. The data suggest that E prostaglandins are potent inhibitors of Paf-induced leukotriene generation by human PMNs and thus may serve as local factors controlling Paf-mediated inflammatory reactions.     

Effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on substance P-induced histamine release from rat peritoneal mast cells.

12-O-tetradecanoylphorbol-13-acetate (TPA, 1 to 30 ng/ml) produced a dose-related inhibition of substance P (SP)-induced histamine release from rat peritoneal mast cells. TPA itself induced some histamine release over this concentration range (maximum release about 20% of total). Maximum inhibition of SP-induced release by TPA required preincubation with TPA for at least 10 min. The inhibitory action of TPA was observed in the absence as well as in the presence of extracellular calcium (0.4 mM). Inhibition of diacylglycerol kinase by R 59022 or of diacylglycerol lipase by RHC 80267 reduced SP-induced histamine release. Oleolylacetylglycerol (OAG, 50 microM) inhibited histamine release induced by SP but was less potent than TPA. It is concluded that protein kinase C activation in rat peritoneal mast cells is associated with inhibition of SP-induced histamine release.     

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.     

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.     

Histamine release from human peripheral blood leukocytes analyzed by histamine radioimmunoassay.

Histamine release from human peripheral blood leukocytes, after in vitro challenge with allergen extracts, is usually measured by fluorometry. In the present study we compared the results of the automated fluorometric histamine assay (Siraganian) with those of the Immunotech histamine radioimmunoassay. Histamine release dose-response curves obtained after histamine measurement by both methods were superimposable.     

Cyclic GMP mediates SIN-1-induced inhibition of human polymorphonuclear leukocytes.

Different nitrovasodilators were used to assess the role of cyclic GMP in the regulation of polymorphonuclear leukocyte (PMN) function. Molsidomine and its metabolites, 3-morpholinosydnonimine (SIN-1) and N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A) at 0.01-1 mM, inhibited lysosomal enzyme release from PMN stimulated by 30 nM formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). At 1 mM, molsidomine, SIN-1 and SIN-1A decreased beta-glucuronidase release by 19, 37 and 46% of the control, respectively. Glyceryl trinitrate (GTN) and sodium nitroprusside (SNP) showed no effect on beta-glucuronidase release from PMN. At 1 mM, SIN-1A, SIN-1 and SNP in the presence of 0.5 mM isobutylmethylxanthine (IBMX) stimulated cyclic GMP 21-, 9- and 14-fold, respectively, demonstrating a relation between cyclic GMP stimulation and neutrophil inhibition by the molsidomine metabolites. GTN and unmetabolized molsidomine were without effect on cyclic GMP levels. The hypothesis of an inhibitory effect of cyclic GMP on neutrophil function was further supported by the attenuation of SIN-1-induced inhibition of enzyme release by methylene blue (10 microM), an inhibitor of soluble guanylate cyclase. Moreover, 8-bromo cyclic GMP and dibutyryl cyclic GMP, 1 mM, decreased beta-glucuronidase release from FMLP-stimulated PMN by 12 and 44% of the control, respectively. These data demonstrate that cyclic GMP is an inhibitory second messenger in human PMN and suggest that this action of SIN-1 may be of considerable interest under conditions of platelet/PMN activation, e.g. during myocardial ischemia.     

In vitro effects of ureidopenicillins on human polymorphonuclear leukocytes

The in vitro effects of mezlocillin, azlocillin and piperacillin on chemotaxis and adhesivity of human leukocytes were comparatively studied. After incubation with all these antibiotics, chemotactic and adhesivity counts were similar to those of the antibiotic-free cells. Scanning electron microscope examination showed enlargement of surface and length measurements after incubation with azlocillin (P less than 0.005 and P less than 0.001) and mezlocillin (P greater than 0.05 and P less than 0.05), while piperacillin produced no alteration. These findings could provide additional information in the study of leukocyte/antibiotics interactions.     

Effect of tumor necrosis factor on granule release and LTB4 production in adherent human polymorphonuclear leukocytes

Tumor necrosis factor (rTNF) has previously been shown to induce PMN chemotaxis, stimulate PMN adhesion to vascular endothelium and stimulate hydrogen peroxide secretion from PMNs adhered to biological surfaces. We investigated the activity of both rTNF alpha and rTNF beta on adherent and suspension cultures of human PMNs. rTNF alpha selectively stimulated the release of the specific granule in a dose dependent manner. Exocytosis of the specific granule was measured with an enzyme-immunoassay for lactoferrin and a radioassay for vitamin B12-binding protein. Adherent PMNs released up to 60% of the total lactoferrin content of the cells with no increase in myeloperoxidase (MPO) secretion when stimulated with 0.1-10 nM rTNF alpha. The PMNs in suspension cultures also selectively released the specific granule, although total release was reduced suggesting that adherence of PMNs increased their ability to respond to physiological stimuli. When PMNs in suspension cultures or adherent cells were stimulated with rTNF alpha, no LTB4 production was detectable, yet the cells retained the ability to synthesize LTB4 when stimulated with calcium ionophore A23187. Neither rTNF alpha or rTNF beta stimulated the release of the azurophilic granule, measured by the secretion of MPO and neutrophil elastase activity. These results suggest that a function of rTNF alpha and rTNF beta on PMNs is the release of the contents within the specific granule.