Effects of various drugs on superoxide generation, arachidonic acid release and phospholipase A2 in polymorphonuclear leukocytes

The effects of variety of drugs on metabolic burst and phospholipase A2 in polymorphonuclear leukocytes (PMNs) were investigated. The stimulation of PMNs by n-formyl-methionyl-leucyl-phenylalanine (FMLP) causes arachidonic acid (AA) to be released in the cells concomitantly with the generation of superoxide anion. These variables were effectively diminished with some clinically employed drugs including chlorpromazine, trifluoperazine, azelastine, clemastine and mepacrine at the lower concentration of 20 microM. In contrast, indomethacin and procaine were ineffective even at the higher concentration of 100 microM. Subcellular fractionation of PMNs revealed that phospholipase A2 activity was located both in the plasma membrane-rich fraction as well as the granule-microsome-rich fraction, and the potency of inhibition of membrane-bound phospholipase A2 by the above mentioned drugs was: indomethacin (IC50 = 3 microM) less than chlorpromazine less than azelastine and clemastine (IC50 greater than 100 microM). The low potency of antipsychotropic drugs and antihistaminic drugs in inhibiting the fractionated phospholipase A2 contrast with the high efficiency with which they inhibit the superoxide generation and the AA release from stimulated PMNs. The AA releases from the PMNs stimulated by FMLP or calcium ionophore (A23187) were almost equally diminished by various drugs at the lower concentration. From these observations, it appeared likely that these drugs might inhibit the metabolic stimulations of PMNs at the sites of the Ca2+-dependent activation processes of the enzymes responsible for the AA release and the superoxide generation.     

Inhibitory effect of disodium cromproxate on superoxide anion (O2-) generation and membrane potential changes in stimulated neutrophils

Disodium cromproxate is an antiallergic agent. This drug (0.5-2 mmol/L) inhibited O2- production by neutrophils induced by FMLP and PMA. However, the inhibition of FMLP-induced O2- generation was more pronounced than that induced by PMA. Disodium cromproxate also counteracted the changes in membrane potential in neutrophils induced by either FMLP or PMA. The actions of disodium cromproxate differed from those of propranolol, as propranolol had no antagonistic action on membrane potential changes induced by FMLP and PMA.     

Gold complexes and activation of human polymorphonuclear leukocytes. Dissociation of changes in membrane potential and oxidative burst.

The effects of the gold compounds on the alteration of membrane potential of polymorphonuclear leukocytes (PMN) in response to various stimulants have been compared with their effects on the oxidative burst. The present studies have shown that gold complexes [auranofin (AF), aurothiomalate (Autm), aurocyanide (Au(CN)2-)] have contrasting effects on the membrane potential of 3,3'-dipentyloxacarbocyanine [di-O-C5(3)] loaded PMN. Au(CN)2- at concentrations which inhibit the oxidative burst of PMN did not affect the membrane depolarization after activation of PMN by phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl phenylalanine (FMLP); Autm slightly stimulated the oxidative burst but had no effect on the depolarization of PMN. In contrast, AF inhibited the depolarization of stimulated PMN to an extent depending upon the concentration of AF, the time of preincubation and the stimulus. The membrane depolarization of PMN caused by PMA, FMLP and concanavalin A (ConA) was inhibited by AF (5 microM) but the depolarization induced by calcium ionophore (A23187) was not affected. AF at the same conditions inhibits the oxidative burst of PMN induced by all these single stimuli including the calcium ionophore. Dissociation of membrane depolarization and superoxide generation caused by AF was also seen in PMN activated by two stimuli. AF (5 microM) had little initial inhibitory effect on the oxidative burst of PMN stimulated by combinations of PMA and ConA or PMA and FMLP whereas it almost totally blocked the depolarization caused by these combinations. Preincubation of cells with 5 microM AF for less than 5 min prior to the addition of PMA allowed membrane depolarization which was followed rapidly by repolarization. None of the gold complexes studied had any effect on the resting membrane potential of PMN.     

The effect of six prostaglandins, prostacyclin and iloprost on generation of superoxide anions by human polymorphonuclear leukocytes stimulated by zymosan or formyl-methionyl-leucyl-phenylalanine

Prostaglandins (PG) E2,E1,6-keto-E1 and D2 at concentrations of 0.15-0.80 microM inhibited by 25% the generation of superoxide anions (O2-) in human polymorphonuclear leukocytes (PMNs) stimulated with formyl-methionyl-leucyl-phenylalanine (FMLP). The potency of that inhibition by either PGD2 or PGE1 was the same when zymosan was used as a stimulator whereas PGE2 and 6-keto-PGE1 were by 13 and 21 times less potent inhibitors of O2-) in zymosan-stimulated as compared to FMLP-activated PMNs. PGF2 alpha inhibited the generation of O2- by activated PMNs only when used at the highest concentration studied (30 microM). Prostacyclin, 6-keto-PGF1 alpha and Iloprost (a carbacyclin analogue of prostacyclin) at concentrations up to 30 microM showed no significant inhibition of O2- in human PMNs stimulated either with FMLP or with zymosan. It is concluded that PGD2 and PGEs use a common basic mechanism for inhibition of the generation of O2- by PMNs activated with FMLP or zymosan. PGD2 is most generously furnished with these properties. In addition to this basic mechanism PGE2 and 6-keto-PGE1 abrogate the FMLP-induced response by occupation of formyl peptide receptor of PMNs. It is hypothesised that inhibition of the generation of O2- in PMNs and, possibly, in other cells by PGD2, PGE2 and by products of prostacyclin biotransformation might be responsible for their cytoprotective action in myocardial infarction, stroke, liver damage and peripheral vascular disease.     

Effects of non-steroidal anti-inflammatory agents on human neutrophil functions in vitro and in vivo

Human blood neutrophils exposed to appropriate stimuli aggregate, degranulate and generate superoxide anion (O2-). These responses are anteceded by mobilization of membrane-associated calcium, monitored as a decrease in fluorescence of cells preloaded with chlortetracycline (CTC). We studied the effects, both in vitro and in vivo, of non-steroidal anti-inflammatory agents (aspirin, indomethacin, ibuprofen and piroxicam) on these neutrophil responses to three stimuli: a chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (FMLP); a tumor promotor, phorbol myristate acetate (PMA); and a lectin, concanavalin A (Con A). The effects of these drugs were compared with those of two polyenoic inhibitors of arachidonate metabolism: eicosatrienoic acid (ETI) and eicosatetraynoic acid (ETYA). The pattern of inhibition of neutrophil functions varied both with inhibitor and the nature of the stimulus. Thus, aspirin, piroxicam, ETYA and ETI inhibited neutrophil aggregation, degranulation, and O2- generation in response to FMLP, whereas ibuprofen inhibited only aggregation and degranulation and indomethacin only inhibited aggregation. None of the agents inhibited aggregation or degranulation induced by PMA or Con A: only piroxicam inhibited O2- generation in response to PMA or Con A. ETI and ibuprofen inhibited decrements of CTC fluorescence induced by FMLP, but whereas ETI inhibited the CTC response to PMA or Con A, ibuprofen was without effect. The agents had varying effects on binding of the stimulus [( 3H]FMLP, [3H]Con A), but these did not correlate with neutrophil responses to the ligands. Neutrophils from subjects taking therapeutic doses of ibuprofen, indomethacin, or piroxicam showed profiles of inhibited responses to FMLP similar to those observed with these agents in vitro. These data suggest that, although non-steroidal anti-inflammatory agents may inhibit discrete neutrophil functions both in vitro and in vivo, their effects do not duplicate those of polyenoic inhibitors of arachidonate metabolism. Moreover, since the susceptibility of neutrophils differed not only with respect to each inhibitor, but also to the stimulus, it is unlikely that all neutrophil responses are necessarily linked by a common pathway that is blocked by inhibitors of arachidonic acid metabolism.     

Leukotriene formation by human polymorphonuclear leukocytes from endogenous arachidonate. Physiological triggers and modulation by prostanoids

Human polymorphonuclear leukocytes (PMN) were isolated from freshly drawn venous blood by Dextran sedimentation and discontinuous Percoll gradient centrifugation. The effects of several putative triggers of the leukotriene formation such as C5a, PAF, FMLP, C3a, PMA, LTC4, LTD4, LTB4 or arachidonate were studied by RP-HPLC analysis. 280 nM C5a, 100 nM FMLP, 1 microM PAF or 20 microM arachidonate induced a marginal formation of 1.5-18 ng of LTB4 plus LTB4 metabolites/2 x 10(7) PMN. 560 nM C3a, 100 nM PMA, 1 microM LTC4, 1 microM LTD4 and 1 microM LTB4 each failed to induce any formation of 5-lipoxygenase products. Pretreatment of the cells with 40 microM ethylmercurithiosalicylate (merthiolate) enhanced the leukotriene formation by 100 nM FMLP about 40-fold, by 280 nM C3a about 120-fold and by 1 microM PAF about 14-fold. Merthiolate itself induced no leukotriene formation from human PMN and reduced the leukotriene formation by 20 microM arachidonate. The FMLP/merthiolate-induced activation of the PMN was concentration-dependent in respect to both FMLP and merthiolate. 1 microM LTC4, 1 microM LTD4 or 1 microM LTB4 also failed to trigger any LTB4 formation of merthiolate-treated PMN. 560 nM C3a or 100 nM PMA in combination with 40 microM merthiolate induced a slight formation of 28 ng and 10 ng of LTB4 plus LTB4 metabolites, respectively. The FMLP/merthiolate-induced leukotriene formation was modulated by prostanoids. PGE2, PGE1, PGD2 and 6-keto-PGE1 each evoked a concentration-dependent inhibition of the leukotriene formation with IC50 values of 0.07 microM, 0.18 microM, 0.27 microM and 6 microM respectively. In addition, significant inhibitory effects by PGI2, Iloprost (a carbacyclin analogue of prostacyclin), PGF2a or 6-keto-PGF1a were achieved; the corresponding IC50 values, however, amounted to 19-59 microM. Thus these compounds were about 500-fold less potent in comparison with PGE2 in inhibiting LTB4 formation by human PMN.     

The inhibitory effect of phenylpropanoid glycosides and iridoid glucosides on free radical production and beta2 integrin expression in human leucocytes

Rapid production of reactive oxygen species (ROS) and upregulation of beta2 integrin by leucocytes are two important inflammatory responses in human leucocytes. To evaluate whether three phenylpropanoid glycosides (acteoside, crenatoside, and rossicaside B) and two iridoid glucosides (boschnaloside and 8-epideoxyloganic acid) identified from two medicinal plants with similar indications (Orobanche caerulescens and Boschniakia rossica) exhibited anti-inflammatory activity, their effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol-12-myristate-13-acetate (PMA)-activated peripheral human neutrophils (PMNs) and mononuclear cells were examined. Pretreatment with 1-50 microM phenylpropanoid glycoside concentration-dependently diminished PMA- and fMLP-induced ROS production with IC50 values of approximately 6.8-23.9 and 3.0-8.8 muM, respectively. Iridoid glucoside was less effective than phenylpropanoid glycoside with an IC50 value of approximately 8.9-28.4 microM in PMA-activated PMNs and 19.1-21.1 microM in fMLP-activated mononuclear cells. Phenylpropanoid glycosides also effectively inhibited NADPH oxidase (NOX) and displayed potent free radical-scavenging activity, but did not interfere with pan-protein kinase C (PKC) activity. Furthermore, all compounds, except rossicaside B, significantly inhibited PMA- and fMLP-induced Mac-1 (a beta2 integrin) upregulation at 50 microM but not that of fMLP-induced intracellular calcium mobilization. These drugs had no significant cytotoxicity as compared with the vehicle control. Our data suggested that inhibition of ROS production, possibly through modulation of NOX activity and/or the radical scavenging effect, and beta2 integrin expression in leucocytes indicated that these compounds had the potential to serve as anti-inflammatory agents during oxidative stress.     

Priming effect of recombinant human interleukin-2 and recombinant human interferon-gamma on human neutrophil superoxide production

The effect of recombinant human interleukin-2 (rhIL-2) and recombinant human interferon-gamma (rhIFN-gamma) were evaluated on superoxide (O2-) production of human polymorphonuclear neutrophils (PMNs). Ten minutes incubation with rhIL-2 showed a dose-dependent enhancement of n-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced O2-production of human PMNs, and the rate of enhancement reached 49.6% at the concentration of 3000 U/ml rhIL-2. Same pretreatment with rhIFN-gamma also showed a dose-dependent enhancement of FMLP-induced O2-production of human PMNs, and the maximal rate of enhancement was 47.0% at the concentration of 3000 U/ml rhIFN-gamma. Any cytokines given alone did not induce O2- production. These cytokines showed no enhancement of phorbol myristate acetate (PMA)-induced O2- production, neither. The effects of these cytokines to FMLP-induced O2- production were kept in calcium-free medium. Moreover, incubation with these cytokines caused no elevation of intracellular free calcium concentration [( Ca++]i) in resting PMNs. Incubation with them did not change the increase of [( Ca++]i) of PMNs induced by FMLP significantly, neither. Recombinant forms of cytokines are used clinically, now. These results may be helpful for the use of them.     

Effect of nisoldipine on priming and activation of the human neutrophil respiratory burst

Nisoldipine inhibits calcium (Ca++) influx in human neutrophils: Preincubation with the dihydropyridine, nisoldipine (1.5 microM) resulted in a 30% decrease in [45]Ca++ influx during formyl-methionine-leucine-phenylalanine (FMLP) stimulation in primed as well as resting cells. Although the drug does not inhibit Ca++ dependent effector functions elicited by FMLP, e.g. superoxide (O2-) production, it inhibits FMLP priming, a phenomenon that is independent of extracellular Ca++. Nisoldipine exhibited a narrow dose response with an ED50 of ca. 1 microM and total inhibition of primed O2- response at 1.5 microM. Nisoldipine (1.5 microM) also abolished the incremental rise of Ca++i in primed neutrophils stimulated with FMLP. The dissociation of nisoldipine inhibitory effects on cell effector function and Ca++ transport were corroborated in studies with neutrophils stimulated with influenza virus and phorbol myristate acetate (PMA), stimuli which do not exhibit an extracellular Ca(++)-dependence in their elicited responses. Unlike in FMLP-stimulated cells, nisoldipine reduced influenza virus and PMA initiated respiratory burst, indicating that this drug has inhibitory effects on neutrophil function independent of its effect on Ca++ metabolism. Possible sites of action are postulated at phospholipase A2 or calmodulin-regulated activities. Caution is thus required in interpreting the effects of dihydropyridine on cell function, when the drug is used at micromolar concentration.     

Evaluation of in-vitro anti-inflammatory activity of some 2-alkyl-4,6-dimethoxy-1,3,5-triazines

The ability of some 2-alkyl(aryl)-4,6-dimethoxy-1,3,5-triazine derivatives to interfere with production of reactive oxygen species (ROS) by human phagocytes was evaluated in an in-vitro cell model. Superoxide anion (O(2)(-*)) production by human polymorphonuclear cells (PMNs), challenged by the chemotactic agent N-formylmethionyl-leucyl-phenylalanine (FMLP), was inhibited in a dose-dependent manner by all the compounds tested, compounds 3, 4 and 5 being statistically the most active. Adhesion of PMNs to vascular endothelial cells (ECs) is a critical step in recruitment and infiltration of leucocytes into tissues during inflammation, and the effects of 1,3,5-triazine derivatives on PMN adhesion to ECs from the human umbilical vein (HUVEC) were also investigated. Triazines were incubated with PMNs and HUVEC; adhesion was quantitated by computerized micro-imaging fluorescence analysis. The 1,3,5-triazines tested inhibited the adhesion evoked by pro-inflammatory stimuli, such as platelet activating factor (PAF), FMLP, phorbol myristate acetate (PMA), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta(IL-1beta) in a dose-response manner over the concentration range 10(-9) to 10(-4)M, compounds 5 and 6 being the most active. Both of these compounds inhibited PMN adhesion to HUVEC, even when endothelial or PMN stimuli were used. Indeed, when both cell populations were activated contemporarily, the anti-adhesive effect was enhanced. The study suggests that 2-aryl-4,6-dimethoxy-1,3,5-triazines deserve further evaluation as anti-inflammatory agents.     

Studies on the antioxidative activity of phloroglucinol derivatives isolated from hypericum species

Twenty-one phloroglucinol derivatives, belonging to 8 different carbon skeletons, were tested for their ability to influence the oxidative burst of polymorphonuclear cells (PMNs) after stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). Results revealed a strong reduction of oxygen production by PMNs after stimulation with FMLP for the compounds ialibinone E ( 5), hyperguinone B ( 15) and hyperforin ( 21). The IC 50 values obtained were 2.5 microM ( 5), 3.3 microM ( 15) and 1.8 microM ( 21), respectively. Slight modifications of the substituents or variation of the stereochemistry resulted in a significant loss of activity. None of the active compounds showed antioxidative activity after stimulation with OZ. The influence of compounds 5, 15 and 21 on the production of oxygen radicals in an H 2 O 2 /horseradish peroxidase system was investigated and revealed potent activity only for compound 5 (IC 50 1 microM). The superoxide-scavenging properties of ialibinone E ( 5) and hyperguinone B ( 15) were additionally tested in a cytochrome c assay and only ialibinone E ( 5) was found to be significantly active at lower micromolar concentrations. Ialibinone E ( 5) was not active in a xanthine oxidase assay (urate formation) in concentrations up to 100 microM and its activity is therefore not attributable to the inhibition of this enzyme. It can be assumed that the activity of compounds 5 and 15 in the different cellular and enzymatic assays, is most likely caused by different and maybe specific mechanisms and cannot be explained by a radical scavenger activity alone. None of the active phloroglucinols showed cytotoxic effects against the PMNs.     

Enhancing and inhibitory effects of nitric oxide on superoxide anion generation in human polymorphonuclear leukocytes.

1. The effects of sodium nitroprusside (SNP, a nitric oxide donor) and authentic nitric oxide (NO) on superoxide anion (O2-) generation were investigated in human polymorphonuclear leukocytes (PMNs). 2. Neither SNP (10 nM to 10 microM) nor NO (40 nM to 40 microM) alone induced O2- generation or change of intracellular Ca2+ concentration ([Ca2+]i) in human PMNs. 3. Pretreatment with SNP or NO at the concentrations used (SNP, 10 nM to 10 microM: NO, 40 nM to 40 microM) showed a biphasic concentration-dependent effect on O2- generation induced by f-methionyl-leucyl-phenylalanine (FMLP). Low concentrations of SNP (10 nM to 100 nM) and NO (400 nM) did not affect either basal cyclic GMP levels or cyclic GMP levels stimulated by FMLP, but enhanced FMLP-induced O2- generation and [Ca2+]i elevation. On the other hand, high concentrations of SNP (10 microM) and NO (40 microM) alone elevated cyclic GMP levels and inhibited FMLP-induced O2- generation and [Ca2+]i elevation. 4. 8-Bromo-cyclic GMP (8-Br-cyclic GMP) at concentrations ranging from 1 microM to 1 mM did not induce O2- generation on its own and had little effect on FMLP-induced O2- generation and [Ca2+]i elevation. 5. Addition of a high concentration of NO (40 microM) decreased authentic O2- formation by pyrogallol in a cell-free system, but a low concentration of NO (400 nM) had no effect on this. On the other hand, addition of SNP in the concentration-ranges used had no effect on authentic O2- formation by pyrogallol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of SCA40 on human isolated bronchus and human polymorphonuclear leukocytes: comparison with rolipram, SKF94120 and levcromakalim.

1. SCA40 (0.1 nM-0.1 mM) produced concentration-dependent suppression of the spontaneous tone of human isolated bronchus (-log EC50 = 6.85 +/- 0.09; n = 10) and reached a maximal relaxation similar to that of theophylline (3 mM). The potency (-log EC50 values) of SCA40 compared to other relaxants was rolipram (7.44 +/- 0.12; n = 9) > SCA40 > or = levcromakalim (6.49 +/- 0.04; n = 6) > SKF94120 (5.87 +/- 0.10; n = 9). 2. When tested against the activity of the isoenzymes of cyclic nucleotide phosphodiesterase (PDE) isolated from human bronchus, SCA40 proved highly potent against PDE III (-log IC50 = 6.47 +/- 0.16; n = 4). It was markedly less potent against PDE IV (4.82 +/- 0.18; n = 4) and PDE V (4.32 +/- 0.11; n = 4). 3. Human polymorphonuclear leukocytes (PMNs) stimulated with N-formylmethionyl-leucyl-phenylalanine (FMLP) produced a concentration-dependent superoxide anion generation and elastase release. SCA40 (1 nM-10 microM) produced a concentration-related inhibition of FMLP (30 nM approximately EC50)-induced superoxide production (-log IC50 = 5.48 +/- 0.10; n = 6) and elastase release (-log IC50 = 5.50 +/- 0.26; n = 6). Rolipram was an effective inhibitor of superoxide generation and elastase release (-log IC50 values approximately 8) while SKF94120 and levcromakalim were scarcely effective. 4. FMLP (30 nM) and thimerosal (20 microM) induced leukotriene B4 production and elevation of intracellular calcium concentration in human PMNs. The production of leukotriene B4 was inhibited by SCA40 in a concentration-related manner (-log IC50 = 5.94 +/- 0.22; n = 6) but SCA40 was less effective against the elevation of intracellular calcium. Rolipram was an effective inhibitor of leukotriene B4 synthesis (-log IC50 approximately 7) and intracellular calcium elevation (-log IC50 approximately 6) while SKF94120 and levcromakalim were scarcely effective. 5. It is concluded that SCA40 is an effective inhibitor of the inherent tone of human isolated bronchus. The bronchodilatation produced by SCA40 appears mainly related to PDE inhibition since the potency of SCA40 as a relaxant of human isolated bronchus was found to be close to its potency as inhibitor of PDE III activity isolated from human bronchus. In addition, SCA40 exhibited inhibitory effects on human PMN function stimulated by FMLP. These effects may be related to the ability of SCA40 to inhibit PDE IV from human PMNs while the contribution of PDE V inhibition is uncertain. We found no evidence of a role for levcromakalim-sensitive plasmalemmal K+-channels in human PMNs.     

Dieldrin activates rat neutrophils in vitro

Suppression of phagocytic cell function has been proposed as a possible mechanism for the enhanced sensitivity to certain infectious agents exhibited by animals exposed to the organochloride insecticide, dieldrin. In the present study, we examined the effects of dieldrin on superoxide production by glycogen-elicited peritoneal neutrophils (PMNs) from the rat. Dieldrin caused a concentration-dependent increase in superoxide production by PMNs incubated in vitro at 37 degrees C. Superoxide release was increased significantly with 10 microM dieldrin and reached a maximum of 17 nmol/10 min/2.0 X 10(6) PMNs at a dieldrin concentration of 35 microM. Preincubation of PMNs for 5 min at room temperature with a barely suprathreshold concentration of either phorbol 12-myristate 13-acetate (PMA) or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) enhanced dieldrin-stimulated superoxide release by as much as ninefold or threefold, respectively. Maximum enhancement was obtained with 10 microM dieldrin for both PMA and FMLP. Time course studies with PMA-pretreated cells revealed that the rate of superoxide release was dependent on the concentration of dieldrin. Extracellular calcium played an important role in dieldrin-stimulated superoxide release, since PMNs treated with dieldrin in the absence of extracellular calcium did not release superoxide. Also, pretreatment with calcium ionophore A23187 greatly enhanced superoxide release from dieldrin-stimulated PMNs. These results show that dieldrin has a stimulatory effect on superoxide release from rat PMNs in vitro and that this stimulation is dependent on extracellular calcium.     

Antioxidant properties of dipyridamole as assessed by chemiluminescence

The ability of dipyridamole (DIP) to scavenge oxygen metabolites generated by either activated human neutrophils (PMNs) or cell-free systems using luminol(s)- and lucigenin-enhanced chemiluminescence was investigated. In the presence of DIP (15-50 microM) a dose-dependent inhibition period was seen in phorbol myristate acetate (PMA)-stimulated PMNs as assayed by isoluminol-enhanced chemiluminescence (ILCL) with horseradish peroxidase (HRP). Although such a lag period was not observed in the absence of HRP, 50 microM DIP inhibited extracellular ILCL by more than 50%. Intracellular luminol-enhanced chemiluminescence (LCL) as assayed in either PMA- or in ionomycin-activated PMNs was not affected by dipyridamole (15-50 microM). In cell-free systems, DIP produced concentration-dependent inhibition in H2O2-(45% at 50 microM), OH- (40%, at 0.1 microM) and HOCl-(20% at 10 microM). Both absorbance and fluorescence scans revealed that DIP is able to react with equimolar quantities of either H202 or HOCl. These results suggest that DIP scavenges reactive oxygen species (ROS) presumably secreted by activated human PMNs in the following decreasing order: *OH > HOCl > H2O2 > O2-.     

Effect of phosphotyrosine proteins on phorbol myristate acetate-induced NADPH oxidase activation in guinea pig peritoneal polymorphonuclear leukocytes

Phorbol myristate acetate (PMA)-induced superoxide radical (O(2)(-))-production in guinea pig peritoneal polymorphonuclear leukocytes (PMNs) was significantly lower than that in peripheral cells. To determine the role of phosphotyrosine proteins in the lower O(2)(-) production, the effect of ST638 and genistein, tyrosine kinase inhibitors, on PMA-induced O(2)(-) production in peritoneal PMNs was examined. PMA-induced O(2)(-)-production of the cells was increased by the pretreatment with ST638 or genistein, the increment depending on the inhibitor concentration. The p47phox level in the plasma membrane of PMA-stimulated PMNs was increased by the pretreatment with ST638, although the phosphorylated p47phox level in the cells was not altered by ST638. On the other hand, PMA-induced O(2)(-)-production of peripheral PMNs was not affected by the pretreatment with ST638, but that of cytochalasin B (CB)-primed peripheral PMNs significantly increased by further treatment with ST638. The phosphotyrosine protein level of peritoneal PMNs was higher than that of the peripheral cells, especially in cytosolic proteins including 50-60 and 70-85 kDa proteins, and that of the CB-primed peripheral cells was also higher than that of the intact cells in similar cytosolic proteins to those above. Further treatment of CB-primed peripheral cells with ST638 resulted in a lower level of phosphotyrosine proteins. These findings suggest that phosphorylation of some protein(s) at specific tyrosine residues inhibits the translocation of p47phox to the plasma membrane from the cytosol, resulting in lower O(2)(-)-generation in casein-induced peritoneal exudate PMNs.     

Effects of antiallergic agents on polymorphonuclear leukocytes. The inhibition of arachidonic acid release and superoxide production

The aim of this study was to examine the effects of antiallergic agents on the functions of polymorphonuclear leukocytes (PMNs) in terms of its arachidonic acid release and superoxide-anion generation. The stimulations of arachidonic acid release by formyl-methionyl-leucyl-phenylalanine (FMLP) were effectively diminished by 20 microM of azelastine as well as clemastine. Challenges of 20 microM and 50 microM of these agents inhibited approximately 50% and 100% of the arachidonic acid release, respectively. On the contrary, inhibitions of over 50% were not caused by cromoglycate, chlorpheniramine and diphenhydramine at concentrations up to 50 microM. The potency of the above examined drugs on the superoxide generations from PMNs were similar to the effects of arachidonic acid release. Ketotifen, however, showed intermediate effects indicating that a challenge of 50 microM ketotifen inhibited approximately 50% of the arachidonic acid release without having an effect on the superoxide generation. These experimental observations suggested that one of the important roles of the antiallergic agents including azelastine (known as a chemical mediator release inhibitor) and clemastine (known as a histamine H1 receptor antagonist) could be an inhibition of the first step of the arachidonic acid cascade.     

Carvedilol, a new beta-adrenoceptor antagonist and vasodilator antihypertensive drug, inhibits superoxide release from human neutrophils.

Carvedilol produced a dose-dependent inhibition of superoxide (O2-) release from human neutrophils (PMNs) (IC50 = 28 microM) and scavenged O2- generated during dihydroxyfumaric acid (DHF) autooxidation (IC50 = 41 microM). Other beta-blockers, such as celiprolol, labetalol and atenolol, or the antioxidant, 'lazaroid', U74500A had no effect on O2- either released from PMNs or generated during DHF autooxidation. Propranolol, at 0.3 mM, inhibited O2- release from PMNs (73%) but failed to scavenge O2- generated from DHF. The novel free radical-scavenging effect of carvedilol may contribute to the cardioprotective activity of the compound.     

Bile and bile salts potentiate superoxide anion release from activated, rat peritoneal neutrophils

Certain bile salts cause hepatotoxicity as well as injury to extrahepatic organs when administered to animals. Activated neutrophils (PMNs) may cause tissue injury by releasing reactive oxygen species and other products. Since PMNs may come in contact with biliary components, such as bile salts, following chemical insult to the liver or during cholestasis, we examined the capacity of bile and bile salts to stimulate superoxide anion (O2-) release from rat peritoneal PMNs in vitro. Neither bile nor bile salts, with the exception of lithocholate, could by themselves stimulate O2- release from PMNs. Lithocholate (32 microM) caused small but statistically significant release of O2- from PMNs. When PMNs were primed with a barely suprathreshold concentration of 12-O-tetradecanoyl-phorbol-13-acetate (PMA), a classic stimulus for PMNs, the addition of bile and certain bile salts markedly enhanced O2- release from PMNs. The monohydroxy bile salt, lithocholate, had the greatest stimulatory activity toward PMA-primed PMNs, causing approximately an eightfold increase in O2- release. The enhancing effect of lithocholate was maximal between 10 and 32 microM, and it also occurred with PMNs isolated from rat blood. Dihydroxy bile salts, deoxycholate and chenodeoxycholate (100 microM), caused more modest enhancement of O2- release (two- to threefold) from primed PMNs. Cholate, a trihydroxy bile salt, was not active at these concentrations. Conjugation of either lithocholate or chenodeoxycholate with either glycine or taurine markedly reduced the ability of the bile salt to enhance O2- release from primed PMNs. Structural alterations on the hydrophilic side chain or within the planar, hydrophobic portion of the bile salt molecule reduced the capacity to enhance O2- release from PMA-primed PMNs. These results indicate that bile salts can potentiate the respiratory burst in PMNs and suggest a role for this interaction in toxicoses or disease states characterized by elevated serum bile salts.     

Platelet-leukocyte interaction: activation of rabbit platelets by FMLP-stimulated neutrophils.

1 The effect of the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) was studied on cells in whole rabbit blood or on a mixture of purified rabbit platelets and neutrophils. 2 In blood, FMLP triggered cell aggregation (measured by electrical impedance) which was dependent upon the concentration of FMLP (9.9 +/- 0.7 and 5.2 +/- 1.2 ohms at 1 and 0.01 microM FMLP respectively). This aggregation was accompanied by a strong decrease in platelet counts (54.6 +/- 6.0 and 45.6 +/- 3.8% for 1 and 0.01 microM FMLP respectively) and by a smaller decrease in neutrophil counts (25.0 +/- 1.9 and 12.9 +/- 1.7% at 1 and 0.01 microM FMLP respectively). 3 When purified platelets and neutrophils were co-incubated, the addition of 0.1 microM induced a marked aggregation (50.0 +/- 1.6 vs. 19.5 +/- 1.6% of light transmission, n = 8, P less than 0.001), ATP secretion (8.4 +/- 1.0 vs. 0.1 +/- 0.1 nmol ml-1, n = 6, P less than 0.001) and a decrease in platelet counts. FMLP induced aggregation of purified neutrophils and release of lysozyme but lacked direct platelet-stimulating effects. The release of lactate dehydrogenase, a cytoplasmic marker and lysozyme were unchanged under the interaction conditions. 4 Platelet activation was reduced by about 30% with 100 microM aspirin or indomethacin and by about 70% with 100 microM BW 755C. Two Paf-acether antagonists, BN 52021 (100 microM) and WEB 2086 (1 microM) suppressed platelet activation by 70-80%. 5 The supernatant of FMLP-stimulated neutrophils induced platelet activation only when bovine serum albumin was present. Rabbit neutrophils stimulated in the presence of serum albumin by 1 microM FMLP formed 2 nM Paf-acether of which half was released to the extracellular medium. 6 Our results indicate that the stimulation of neutrophils by FMLP induces platelet activation in whole blood and on isolated cells and that both arachidonic acid-metabolites and Paf-acether participate in platelet activation.