Involvement of leukotriene B4 and platelet-activating factor in cytokine priming of human polymorphonuclear leucocytes.

Recombinant human (rh) tumour necrosis factor (TNF) alpha and rh granulocyte-macrophage colony-stimulating factor (GM-CSF) prime human polymorphonuclear leucocytes (PMN) for increased superoxide anion (O2-) generation and for increased platelet-activating factor (PAF) biosynthesis and leukotriene B4 (LTB4) release. Both PAF and LTB4 are candidate mediators for the enhanced O2- generation in cytokine-primed PMN, since exogenous PAF or LTB4 primes PMN. We measured the generation and release of these mediators and examined their potential roles in cytokine priming using the PAF receptor antagonist, WEB 2086, and the inhibitor of 5-lipo-oxygenase, CGS 8515.rhTNF-alpha or rhGM-CSF, alone, increased PAF levels in PMN, but did not cause PAF release or LTB4 synthesis. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated the release of detectable and biologically active amounts of both LTB4 and PAF in primed, but not in non-primed PMN. However, neither blockade of PAF receptors, nor inhibition of LTB4 synthesis influenced the priming of O2- generation by rhTNF-alpha or rhGM-CSF. Simultaneous pretreatment of PMN with WEB 2086 and CGS 8515 also failed to inhibit priming. Our results do not exclude a role for cell-associated PAF in the priming response, but indicate that the release of PAF and LTB4 do not mediate this phenomenon. The ability of cytokines to amplify the production and release of lipids may represent a mechanism to attract and localize the pro-inflammatory actions of stimulated PMN to regions where cytokine levels are also elevated.     

Orally administered tolfenamic acid inhibits leukotriene synthesis in isolated human peripheral polymorphonuclear leukocytes

Special interest has been focused on the development of dual inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism. In contrast to other classic NSAIDs, some fenamates in clinically achievable concentrations have been shown to inhibit synthesis of 5-lipoxygenase productsin vitro. In the present work, we studied the effect of orally administered tolfenamic acid (600 mg) on Ca ionophore A 23187-induced leukotriene synthesis in isolated human polymorphonuclear leukocytes. Leukotriene production was reduced in all 14 subjects studied, the mean inhibition of LTB₄ synthesis being 16±3% and that of LTC₄ 33±7%. The inhibition correlated positively with serum tolfenamic acid concentrations. We suggest that inhibition of leukotriene synthesis is an additional mechanism of the anti-inflammatory, antimigraine and antidysmenorrhoeic effects of tolfenamic acid, and a possible explanation for its rare gastric and bronchoconstrictive side-effects.     

Characterization of effect ofN-formyl-methionyl-leucyl-phenylalanine on leukotriene synthesis in human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMNLs) were exposed toN-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) in the presence or absence of exogenous arachidonic acid. Analysis of incubation mixtures by high-performance liquid chromatography showed that f-Met-Leu-Phe stimulated the synthesis of 5-hydroxy-eicosatetraenoic acid (HETE) and of leukotriene B₄ (LTB₄) which was rapidly metabolized into 20-OH-LTB₄ and 20-COOH-LTB₄. The stimulatory effect of f-Met-Leu-Phe was dose and time dependent. The tripeptide showed maximum stimulatory activity at the concentration of 1 M and after 20–30 min of incubation. Addition of arachidonic acid to the f-Met-Leu-Phe-stimulated PMNLs resulted in an increase in the synthesis of LTB₄ and 5-HETE. Pretreatment of the PMNLs with cytochalasin B strongly potentiated (up to six-fold) the stimulatory effect of f-Met-Leu-Phe on 5-lipoxygenase product synthesis, whereas cytochalasin B alone or with arachidonic acid had no significant effect. The tripeptide did not increase the synthesis of plateletderived 12-HETE, and 12-hydroxyheptadecatrienoic acid, or of PMNL-derived 15-HETE, suggesting that its action was selective for PMNL 5-lipoxygenase. The present data indicate that f-Met-Leu-Phe causes the release of arachidonic acid from cellular lipids and activates the 5-lipoxygenase.     

Comparative studies of human eosinophil migration towards platelet-activating factor and leukotriene B4

Peripheral-blood polymorphonuclear cells from 36 donors with or without eosinophilia were studied for their in vitro responsiveness towards a wide range of concentrations of leukotriene B4 (LTB4) and platelet-activating factor (PAF). The mean percentage of migrated eosinophils was 17.6 for PAF, 21.1 for LTB4, 20.1 for buffer controls with cells from eosinophil patients, and 1.1 for PAF, 8.9 for LTB4 and 3.2 for buffer control with noneosinophil donors. The quantitative response of eosinophils towards PAF was lower than that towards LTB4 on a molar basis. The data showed high interindividual variations for eosinophil responsiveness towards mediators and buffer and suggest disease-dependent alterations of receptor expression or of basic metabolic activity of eosinophils as possible reasons for this variability.     

Inhibition of leukotriene B4-INDUCED increase in intracellular calcium ion level of human peripheral blood polymorphonuclear leukocytes by Y-24180, an antagonist of platelet-activating factor receptor

Y - 24180 ( (±) - 4 - (2-chlorophenyl ) - 2 - [2 - (4-isobutylphenyl ) ethyl] - 6,9 - dimethyl - 6H - thieno [3,2 - f ] [1,2,4] triazolo [4,3-a] [1,4] diazepine) , an antagonist of platelet-activating factor (PAF) receptor, has already been reported to inhibit leukotriene B₄ (LTB₄) -induced activation of polymorphonuclear leukocytes. In this article, to clarify the mechanism of inhibition of LTB₄-induced activation by Y-24180, we examined the effect of Y-24180 on LTB₄-induced increase in intracellular calcium ion ( [Ca²] _i) level of human polymorphonuclear leukocytes at a single cell level using a laser scanning confocal microscope. Preincubation with Y-24180 significantly inhibited the increase in [Ca²] _i level at 0.3–3 μM, while WEB 2086 (4- [3- [4- (2-chlorophenyl ) -9-methyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a] [1,4] diazepin-2-yl] propionyl] morpholine) , another PAF receptor antagonist, did not show the inhibitory effect at 3–30 μM, indicating that the difference in potency between these compounds was more than 100-fold. The LTB₄-induced increase in [Ca²] _i level was suppressed by microinjection of anti-PAF antibody, but not control antibody, into leukocytes. Microinjection of Y-24180 at 0.003–0.03 μM or WEB 2086 at 0.03–0.3 μM into leukocytes also inhibited the LTB₄-induced increase. The microinjection of WEB 2086 at the 10-fold greater concentrations than those of Y-24180 inhibited to the almost equal level to that obtained by Y-24180. In addition, microinjection of PAF into leukocytes induced the increase in [Ca²] _i level. Preincubation with Y-24180 at 1 or 3 μM significantly attenuated the PAF microinjection-induced increase, but WEB 2086 showed little effect at 3–30 μM. These results indicate that Y-24180 inhibits LTB₄-induced activation of leukocytes by suppressing the increase in [Ca²] _i level and the inhibitory effect is mediated by antagonistic action against intracellular PAF-induced up-regulation of [Ca²] _i level. The difference in inhibitory activity for the increase in [Ca²] _i level between Y-24180 and WEB 2086 when these are added in the culture medium may depend upon their aptitude for the transmembrane influx.     

Human polymorphonuclear leukocytes release leukotriene B4 during phagocytosis ofStaphylococcus aureus

We studied release of leukotriene B₄ (LTB₄) by human polymorphonuclear leukocytes (PMNs) during phagocytosis of staphylococci in the presence or absence of arachidonic acid. The 12×10⁷ PMNs incubated with 3×10⁹ opsonizedS. aureus and 50M arachidonic acid released 1.45±0.42 nmol LTB₄. No LTB₄ was detected after stimulation of PMNs withS. aureus or arachidonic acid by themselves. However, by increasing the concentration of arachidonic acid to 200 or 400M, 1.22±0.45 and 1.98±0.49 nmol LTB₄, respectively, was released by PMNs. The effect of different bacteria-PMN ratios on LTB₄ production was also studied. LTB₄ varied from 0.3 to 2.0 nmol when bacteria/PMN ratios increased from 5 to 50 (respectively) in the presence of 50 M arachidonic acid. Thus, phagocytizing PMNs produce LTB₄ in the presence of arachidonic acid, and its production is dependent on the number of bacteria phagocytized.     

Involvement of endogenous leukotriene B4 and platelet-activating factor in polymorphonuclear leucocyte recruitment to dermal inflammatory sites in rats

A critical role for leukotriene B(4) (LTB(4)) and/or platelet-activating factor (PAF) in regulating polymorphonuclear cell (PMN) trafficking to inflammatory sites has been reported in a number of experimental inflammatory models. In vitro, newly synthesized LTB(4) and PAF were shown to act in an autocrine/paracrine or intracrine fashion to enhance intracellular arachidonic acid availability and leukotriene biosynthesis. This suggested potentially cooperative effects of these lipid mediators in regulating PMN extravasation. The present study aimed to elucidate whether endogenous LTB(4) and PAF may both act to regulate plasma extravasation and PMN trafficking to inflammatory sites in experimental inflammation. With this aim, we have used selective and potent PAF and LTB(4) receptor antagonist pretreatments in dermal and pulmonary inflammation models in rats. Our results show additive inhibitory effects of dual LTB(4) and PAF receptor blockade in either PAF- or LTB(4)-elicited cutaneous PMN accumulation compared to single-drug administration. Furthermore, the combined administration of the drugs inhibited the PMN accumulation induced by the chemically unrelated soluble agonists tumour necrosis factor-alpha and C5a. Finally, in a model of pulmonary inflammation induced by the intravenous injection of Sephadex beads, lung neutrophilia was reduced by 63% following the administration of LTB(4) and PAF antagonists, in contrast with the lack of effect of single drug administration. Our results strongly support a role of both endogenous LTB(4) and PAF in regulating PMN trafficking to inflammatory sites in various experimental conditions.     

Synthesis of platelet-activating factor from human polymorphonuclear leukocytes: regulation and pharmacological approaches

Human polymorphonuclears release a platelet-activating factor (PAF-acether) when challenged with various stimuli. PAF-acether is a mediator that is synthesized during cell activation in a process in which a phospholipase A2 and an acetyltransferase take part. These enzymes are finely regulated and accordingly PAF-acether release may be modulated. The authors have studied some of the transductory mechanisms which are triggered during cell stimulation and the effect of their pharmacological modulation on PAF-acether release. Theophylline, methylisobutylxanthine and dipyridamole, which block phosphodiesterase of cyclic nucleotides, induce a dose-dependent inhibition of PAF-acether release without affecting phagocytic uptake. Polyamines (dansylcadaverine, rimantadine and amantadine) reduced PAF-acether release and the phagocytic process in an order of potency similar to their ability to inhibit phospholipid methylation and the cholinephosphotransferase pathway. The calmodulin antagonist trifluoperazine induced a dose-dependent inhibition of PAF-acether release and acetyltransferase at concentrations from 10(-4) to 10(-5) M. Hence it appears that modulation of PAF-acether release can be obtained by different pharmacological blockades: phosphodiesterase of cyclic nucleotides, phospholipid metabolism and calcium-calmodulin.     

Tolfenamic acid inhibits leukotriene B4-induced chemotaxis of polymorphonuclear leukocytes in vitro

Inhibition of prostanoid synthesis is usually regarded as the mode of action of nonsteroidal antiinflammatory drugs (NSAIDs). In addition, some NSAIDs have been reported to have prostanoid-independent inhibitory effects on neutrophil functions. In the present study, we examined the effects of acetylsalicylic acid, diclofenac, indomethacin, ketoprofen, piroxicam and tolfenamic acid on leukotriene B₄ (LTB₄)-induced chemotaxis of human polymorphonuclear leukocytes (PMNs) in vitro. Tolfenamic acid inhibited LTB₄-induced chemotaxis (IC₅₀ 59M), whereas the other compounds were ineffective. Tolfenamic acid inhibited also FMLP-induced chemotaxis at the same concentration range (IC₅₀ 46M). About 25% reduction in the chemotactic response was achieved with therapeutic concentrations of tolfenamic acid. We suggest that the inhibition of PMN chemotaxis is an additional mechanism in the antiinflammatory action of tolfenamic acid and that this action is not ligand specific.     

Aggregating and prostanoid-releasing effects of platelet-activating factor and leukotrienes on human polymorphonuclear leukocytes and platelets

Aggregating and prostanoid-releasing properties of the inflammatory mediators, platelet-activating factor (PAF) and leukotrienes B4, C4 and D4 were studied in human polymorphonuclear leukocytes (PMNL) and in human platelet-rich plasma. Leukotriene B4 (LTB4) and PAF both induced a reversible aggregation of human PMNL with concomitant stimulation of PGE2 formation, whereas LTC4 had no effect on human PMNL. Arachidonic acid (AA) caused an irreversible aggregation of PMNL which was accompanied by formation of both PGE2 and TXB2. Inhibition of TXB2 synthesis by indomethacin or by OKY-1581, a thromboxane synthetase inhibitor, had no effect on the PMNL aggregation induced by LTB4, PAF or AA. Leukotrienes B4, C4 and D4 caused neither aggregation nor TXB2 release in human platelet-rich plasma. PAF, on the other hand, induced a dose-dependent, reversible platelet aggregation which was not accompanied by TXB2 formation nor inhibited by OKY-1581. The present study indicates that in addition to inducing PMNL aggregation, LTB4 is capable of releasing arachidonate metabolites from human PMNL but not from human platelets. Also the responses of PMNL and platelets to PAF seem to differ as the PAF-induced PMNL aggregation was accompanied by increased prostanoid formation whereas the PAF-induced reversible platelet aggregation was obviously independent from arachidonate metabolism.     

Effects of antirheumatic drugs on leukotriene B4 and prostanoid synthesis in human polymorphonuclear leukocytesin vitro

The effects ofd-penicillamine, sodium aurothiomalate, indomethacin, timegadine and tolfenamic acid on the lipoxygenase and cyclo-oxygenase pathways of arachidonic acid metabolism were studied in human polymorphonuclear leukocytes (PMNs)in vitro. In short-term incubations,d-penicillamine and aurothiomalate did not affect leukotriene B₄ (LTB₄), prostaglandin E₂ (PGE₂) or thromboxane B₂ (TXB₂) production. Each of the three non-steroidal anti-inflammatory drugs (NSAIDs) used were potent inhibitors of prostanoid synthesis. In higher concentrations they also reduced LTB₄ production; timegadine and tolfenamic acid were effective in concentrations comparable to those measured in plasma during drug therapy, whereas indomethacin was needed in ten times higher concentrations. The different effects of NSAIDs on 5-lipoxygenase activity may be of importance in their therapeutic actions as well as in the appearance of some side-effects, e.g. gastric irritation and aspirin-induced asthma.     

Late appearance of phospholipid platelet-activating factor and leukotriene B4 in human skin after repeated antigen challenge

Inflammatory mediators were assessed in supernatants of chamber fluids from eight ragweed- or grass-sensitive subjects during antigen-induced cutaneous inflammatory responses. Platelet activating factor (PAF) accumulated at concentrations of 1 pm to 90 mumol/L in six of eight subjects beginning at 3 hours and continuing for 9 hours after antigen challenge. Leukotriene B4 (LTB4) was detectable at cutaneous sites of antigen challenge in five of five subjects throughout the 9-hour period at levels from 1 to 36 nmol, a range of 38% to 80% of which were omega-oxidation metabolites. Histamine levels peaked in the first hour at 106 +/- 18 ng/ml and decreased to a plateau of 11 to 13 ng/ml at 3 to 9 hours after antigen challenge. No PAF and only very low levels of LTB4 (0.1 to 1.3 nmol) and of histamine (less than 2 ng/ml) were detected at buffer-control sites during the 9 hours of study. Continuous antigen exposure thus results in the persistent release of histamine and LTB4 and the late appearance of PAF, all of which may contribute to the chronicity of allergic disorders and may have a bearing on the IgE-mediated, late-phase cutaneous response.     

Metabolism of leukotriene B4 by activated human polymorphonuclear granulocytes

Human polymorphonuclear granulocytes (PMNs) synthesize leukotriene B4 (LTB4) as a response of cell activation. Inactivation of the potent inflammatory mediator proceeds via omega-oxidation, resulting in the formation of 20-hydroxy- and 20-carboxy-LTB4. The main metabolite after stimulation with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) is 20-carboxy-LTB4, and after stimulation with the calcium ionophore A23187 is 20-hydroxy-LTB4. Differences in the LTB4 inactivation pathway were also observed when the catabolism of exogenously added LTB4 was analysed. In contrast to resting cells or cells preactivated with FMLP, prestimulation with the ionophore or with phorbol esters resulted in the inhibition of 20-carboxy-LTB4-generation. This decrease correlated with the reduction in specific [3H] LTB4-receptor expression. Studies with the non-penetrating diazonium salt of sulphanilic acid, which is known to interact with ectoenzymes, revealed that LTB4 is metabolized via receptor-mediated uptake. Our data suggest that the reduction in the amount of LTB4-receptor sites inhibits the conversion of 20-OH-LTB4 into 20-COOH-LTB4.     

Leukotriene B4 modulates phospholipid methylation and chemotaxis in human polymorphonuclear leukocytes

Formation of phosphatidylcholine from phosphatidylethanolamine via the S-adenosylmethionine (AdoMet) pathway has been shown to be required for signal transduction of receptor-ligand interactions in a variety of cells. These interactions result in the remodeling of phospholipid pools and phospholipase activation. To extend these observations and to explore the role of the phosphatidylcholine synthesis pathway in transduction of the leukotriene B4 (LTB4) receptor-ligand response, we examined phospholipid methylation in human polymorphonuclear leukocytes (PMN) following stimulation by LTB4, a potent chemotactic agent that is a metabolite of arachidonic acid. At early time points (approximately 3-10 min), formation of methylated phospholipids was enhanced following LTB4 stimulation. The LTB4 analogs 6-trans LTB4 as well as LTB4 epimers induced less methylation compared with LTB4, and the potencies of these analogs in inducing methylation correlated with their diminished ability to induce chemotaxis. Furthermore, the ability of these agonists to induce methylation also correlated with the binding affinity of these agents to the LTB4 receptors on these cells. Synthesis of phosphatidylcholine by the choline transferase pathway was not affected by LTB4. Inhibition of the AdoMet reaction with 3- deazaadenosine, L-homocysteine homolactone, or erythro-9-[2-hydroxy-3-nonyl] adenine (EHNA) abrogated LTB4-induced phospholipid methylation and the chemotactic response. The potencies of these inhibitors in blocking phospholipid methylation also correlated with their ability to abrogate the LTB4-induced chemotactic response. These data suggest that phospholipid methylation and phospholipase activation play an important role in transduction of the LTB4 receptor-ligand interaction in PMN, which results in chemotaxis.     

Decreased release of leukotriene B4 from monocytes and polymorphonuclear leukocytes in patients with systemic lupus erythematosus

The leukotriene B4 (LTB4) releasing capacities of monocytes and polymorphonuclear leukocytes (PMN) were studied using a specific radioimmunoassay in 28 patients with systemic lupus erythematosus (SLE) and 9 normal controls. LTB4 release from both monocytes (7.3 +/- 3.3 ng) and PMN (6.6 +/- 3.4 ng) in SLE patients was decreased compared with that (15.2 +/- 4.3 ng for monocytes, 20.7 +/- 4.5 ng for PMN) in normal controls. There were no differences in the releasing capacities of monocytes and PMN between patients with active and inactive disease. LTB4 suppressed lymphocyte blastogenesis by PHA-P and induced suppressor cells. The significance of the decreased release of LTB4 from monocytes and PMN in SLE patients was discussed.     

Feline polymorphonuclear leukocytes respond chemotactically to leukotriene B4 and activated serum but not to F-met-leu-phe

The chemotactic response of feline polymorphonuclear leukocytes (PMNs) to three types of chemoattractants was studied. Feline PMNs responded to leukotriene B₄ as well as to agarose-activated autologous and homologous serum. However, no response was obtained to N-formylmethionylleucylphenylalanine (FMLP), and four similar peptides that activate the FMLP receptor (N-formylnorleucylleucylphenylalanine, N-formylmethionylphenylalanine, methionylleucylphenylalanine, and pepstatin.) Thus, feline PMNs are similar to equine, porcine, bovine and canine PMNs which also do not respond chemotactically to these peptides.     

Histamine release from human leukocytes by platelet-activating factor

Platelet-activating factor (PAF) was found to induce histamine release from human basophils in mixed leukocytes in the presence of cytochalasin B. The reaction was rapid and dependent on temperature. The release was lower at a high concentration of PAF and was inhibited by EDTA, suggesting a noncytolytic mechanism. CV-3988, a PAF antagonist, inhibited the reaction dose-dependently. PAF acted as an IgE-independent stimulus, but PAF-induced histamine release from leukocytes of allergic asthmatics, who had elevated serum IgE levels, was significantly higher than that from leukocytes of controls. These results suggest that PAF-induced histamine release is useful for studying the role of PAF in the pathogenesis of allergic disorders including bronchial asthma.     

Regulation and kinetics of platelet-activating factor and leukotriene C4 synthesis by activated human basophils

BACKGROUND: Allergic disease is the result of an interplay of many different cell types, including basophils and mast cells, in combination with various inflammatory lipid mediators, such as platelet-activating factor (PAF) and leukotrienes (LT). LTC4 synthesis by human basophils has been studied quite extensively. However, not much is known about the synthesis of PAF by human basophils. OBJECTIVE: In this study, we have made a comprehensive comparison between the kinetics of PAF and LTC4 synthesis, in highly purified basophils, activated with different stimuli or with combinations of stimuli. METHODS : Synthesis of PAF and LTC4 by human basophils was determined with commercially available assay kits. The basophils were activated with C5a, fMLP, PMA, allergen or anti-IgE, in the absence and presence of IL-3 and/or in combination with elevation of cytosolic free Ca2+ by the sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. RESULTS: Most stimuli were found to induce both PAF and LTC4 synthesis. PAF synthesis and LTC4 release were enhanced by preincubation of the basophils with IL-3 or by elevation of cytosolic free Ca2+ by thapsigargin. Incubation of human basophils with IL-3 alone or thapsigargin alone did not result in detectable synthesis of PAF and LTC4, whereas the combination of the two resulted in high amounts of PAF and LTC4 synthesis. Depending on the stimulus used, LTC4 release was 5-100-fold higher than PAF synthesis. In addition, PAF, but not LTC4, was transiently detected, probably due to PAF degradation. LTC4 and PAF synthesis was strongly blocked by inhibitors of cytosolic phospholipase A2, indicating that this enzyme is involved in PAF and LTC4 synthesis by activated human basophils. CONCLUSION: This study provides a first comprehensive comparison of PAF and LTC4 synthesis in highly purified human basophils, stimulated with a variety of stimuli.