Ethanol affects leukotriene generation and leukotriene-induced functional responses in human polymorphonuclear granulocytes

Since ethanol has been shown to inhibit the inflammatory response, we evaluated whether ethanol affected generation of leukotrienes in polymorphonuclear granulocytes (PMN) in vitro. Using the calcium ionophore A23187 as stimulus, the leukotriene B4 (LTB4) and leukotriene C4 (LTC4) generation were dose-dependently impaired by ethanol. No significant difference in the levels of the omega-oxidized metabolites was observed. However, the total LTB4 production (LTB4 plus omega-oxidized metabolites) was significantly decreased in the samples treated with ethanol. Furthermore, ethanol also modulated LTB4-induced functional responses. PMN aggregation, oxidative metabolism and elastase release were all inhibited in the presence of 1% ethanol (to 74 +/- 15%, 50 +/- 4% and 57 +/- 3% of controls, respectively). However, ethanol had no effect on intracellular calcium mobilization or on the change of the PMN membrane potential induced by either LTB4 or A23187. Thus, a possible mechanism for the reduced functional PMN responses in the presence of ethanol might be impaired generation of leukotrienes, but it is conceivable that ethanol impairs also other steps of the stimulus response coupling since the LTB4-induced functional responses were inhibited.     

Leukotriene A4 hydrolase and leukotriene C4 synthase activities in human chondrocytes: transcellular biosynthesis of Leukotrienes during granulocyte-chondrocyte interaction

OBJECTIVE: To investigate the cooperation of chondrocytes and polymorphonuclear cells (PMN) in the biosynthesis of leukotrienes (LT). METHODS: PMN, resting and interleukin-1beta-stimulated cultured human chondrocytes, and mixtures of both cell types were incubated with A23187 and/or 14C-arachidonic acid (14C-AA). To explore the presence of LTC4 synthase and LTA4 hydrolase, the chondrocytes were incubated with authentic LTA4. Eicosanoids were analyzed using high performance liquid chromatography techniques. RESULTS: Chondrocytes formed only prostaglandin E2 and minor amounts of 15-HETE and 11-HETE, the production of all of which was inhibited by 1 microM indomethacin. Incubation of PMN and chondrocytes produced more LTC4 from endogenous and exogenous AA, and more LTB4 from endogenous AA, than incubation of PMN alone, which was consistent with the presence of LTC4 synthase and LTA4 hydrolase activities in chondrocytes. Chondrocytes also slightly increased the level of PMN production of all 5-lipoxygenase (5-LO)-derived products from endogenous AA. CONCLUSION: Human chondrocytes form eicosanoids from AA only by the cyclooxygenase pathway. Chondrocytes cooperate in the transcellular biosynthesis of LT since they possess LTA4 hydrolase and LTC4 synthase activities and increase metabolism by the 5-LO pathway in PMN.     

LTB4 and LTC4 are absent in the cerebrospinal fluid of human immunodeficiency virus type 1-seropositive persons with toxoplasmic encephalitis: evidence for inhibition of 5-lipoxygenase by Toxoplasma gondii

Previous studies on macrophages have shown that Toxoplasma gondii alters the metabolism of arachidonic acid with subsequent inability to generate leukotrienes (LT)s. LTB4 and LTC4 were analyzed in cerebrospinal fluid of 3 groups of human immunodeficiency virus (HIV) type 1-seropositive patients: with toxoplasmic encephalitis (TE) (n=10), with herpes simplex encephalitis (n=5), and without encephalitis (n=10) and in HIV-1-seronegative controls without inflammatory diseases (n=30) by specific immunoassays and gas chromatography-mass spectrometry. In HIV-1-seropositive subjects with TE, LTB4 and LTC4 were below the detection limit (<5.0 pg/mL) and thus significantly decreased (P<.01) compared with HIV-1-seropositive patients with herpes simplex encephalitis (LTB4, 148.5+/-47.6 pg/mL; LTC4, 116.4+/-36.9 pg/mL) and in those without encephalitis (LTB4, 46.1+/-16.8 pg/mL; LTC4, 48.3+/-21.3 pg/mL), and in controls (LTB4, 43.6+/-21.2; LTC4, 45.2+/-18.9 pg/mL). These results point to an essential role of inhibition of 5-lipoxygenase with subsequent failure of LT release as an important mechanism for the survival of T. gondii in vivo.     

Neutrophil-mediated changes in vascular permeability are inhibited by topical application of aspirin-triggered 15-epi-lipoxin A4 and novel lipoxin B4 stable analogues

Neutrophil (PMN) activation is critical in inflammation and reperfusion injury, suggesting that PMN-directed therapies may be of clinical use. Here, leukotriene B4 (LTB4)-induced PMN influx in ear skin was equivalent between 5-lipoxygenase knockout and wild-type mice. To explore actions of lipoxin (LX) in PMN-mediated tissue injury, we prepared several novel LX stable analogues, including analogues of LXA4 and aspirin-triggered 15-epi-LXA4 as well as LXB4, and examined their impact in PMN infiltration and vascular permeability. Each applied topically to mouse ears inhibited dramatically PMN-mediated increases in vascular permeability (IC50 range of 13-26 nmol) with a rank order of 15(R/S)-methyl-LXA4 > 16-para-fluoro-phenoxy-LXA4 approximately 5(S)-methyl-LXB4 >/= 16-phenoxy-LXA4 > 5(R)-methyl-LXB4. These LX mimetics were as potent as an LTB4 receptor antagonist, yet results from microphysiometry with mouse leukocytes indicated that they do not act as LTB4 receptor level antagonists. In addition, within 24 h of delivery, > 90% were cleared from ear biopsies. Neither IL-8, FMLP, C5a, LTD4, nor platelet-activating factor act topically to promote PMN influx. When applied with LTB4, PGE2 enhanced sharply both infiltration and vascular permeability, which were inhibited by a fluorinated stable analogue of aspirin-triggered LX. These results indicate that mimetics of LXs and aspirin-triggered 15-epi-LXA4 are topically active in this model and are potent inhibitors of both PMN infiltration and PMN-mediated vascular injury.     

Suppression of leukotriene formation in RBL-2H3 cells that overexpressed phospholipid hydroperoxide glutathione peroxidase

The overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) by RBL-2H3 cells was used as the basis for an investigation of the effects of PHGPx on the formation of leukotrienes. The rates of production of leukotriene C4 (LTC4) and leukotriene B4 (LTB4) in cells that overexpressed PHGPx were 8 times lower than those in a control line of cells. The reduction in rates of production of leukotrienes apparently resulted from the increase in the PHGPx activity since control rates of formation of leukotrienes could be achieved in PHGPx-overexpressing cells upon inhibition of PHGPx activity by diethyl malate. The conversion of radioactively labeled arachidonic acid to intermediates in the lipoxygenase pathway, such as 5-hydroxyeicosatetraenoic acid (5-HETE), LTC4, and LTB4, was strongly inhibited in PHGPx-overexpressing cells that had been prelabeled with [14C]arachidonic acid. PHGPx apparently inactivated the 5-lipoxygenase that catalyzed the conversion of arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) since 5-HPETE is a common precursor of 5-HETE, LTC4, and LTB4. The rates of formation of LTC4 and LTB4 in PHGPx-overexpressing cells returned to control rates upon the addition of a small amount of 12-HPETE. Flow cytometric analysis revealed that the rapid burst of formation of lipid hydroperoxides induced by A23187 was suppressed in PHGPx-overexpressing cells as compared with the control lines of cells. Subcellular fractionation analysis showed that the amount of PHGPx associated with nuclear fractions from PHGPx-overexpressing cells was 3.5 times higher than that from the control line of cells. These results indicate that PHGPx might be involved in inactivation of 5-lipoxygenase via reductions in levels of the fatty acid hydroperoxides that are required for the full activation of 5-lipoxygenase. Thus, in addition to its role as an antioxidant enzyme, PHGPx appears to have a novel function as a modulator of the production of leukotrienes.     

Inhibition of mucociliary clearance of the eustachian tube by leukotrienes C4 and D4

The effect of leukotrienes C4 (LTC4) and D4 (LTD4) and prostaglandin E2 (PGE2) on mucociliary clearance of the eustachian tube was investigated in vitro and in vivo. Normal ciliated epithelium was obtained from the eustachian tube of guinea pigs and incubated separately with LTC4, LTD4, and PGE2 at concentrations of 10(-8) mol/L and 10(-6) mol/L. Ciliary activity was measured photoelectrically. Leukotriene D4 progressively inhibited ciliary activity, while PGE2 promoted it. Leukotriene C4 also induced ciliary inhibition. One milliliter each of 10(-5) mol/L LTC4, LTD4, and PGE2 was directly injected into the tympanic bullae of chinchillas under anesthesia. The middle ears were examined by otomicroscopy, tympanometry, and auditory brain stem response over time. Clearance of middle ear effusion was delayed by LTC4 and LTD4, as compared with PGE2 and the control. These findings indicate that LTC4 and LTD4 inhibit mucociliary clearance of the eustachian tube.     

Cloricromene inhibits leukotriene formation by human polymorphonuclear leucocytes by suppressing arachidonate release from membrane phospholipids

Cloricromene, an antithrombotic agent known to inhibit the release of arachidonic acid (AA) in stimulated human platelets, was tested for its effects on arachidonate release and metabolism in human polymorphonuclear leucocytes (PMNs). Cloricromene dose-dependently suppressed the release of leukotriene B4 (LTB4), as assessed by radioimmunoassay, from both isolated PMNs and human whole blood stimulated with the calcium ionophore A23187 or with serum-treated zymosan (STZ). The inhibitory effect was higher when the concentration of the stimulating agent was weaker. Cloricromene also inhibited dose-dependently the liberation of LTB4, LTC4, LTD4 and 5-hydroxy-6,8,11,14-eicosatraenoic acid as assessed by HPLC in the supernantant of A23187-stimulated PMNs. Finally, the drug was able to suppress the release of [3H]AA from purified human PMNs prelabeled with the radioactive fatty acid and stimulated with either A23187 or with STZ. The A23187-induced decrease in the radioactivity of phosphatidylinositol, the phospholipid class mainly involved in AA release in stimulated PMNs, was also inhibited by cloricromene. Cloricromene suppresses leukotriene formation in human PMNs by reducing AA release from membrane phospholipids, possibly through interference with phospholipase A2 activation; this activity may contribute to the leucocyte-inhibitory effects reported previously for cloricromene.     

Decreased leukotriene C4 synthesis accompanies adherence-dependent nuclear import of 5-lipoxygenase in human blood eosinophils

The enzyme 5-lipoxygenase (5-LO) catalyzes the synthesis of leukotrienes (LTs) from arachidonic acid (AA). Adherence or recruitment of polymorphonuclear neutrophils (PMN) induces nuclear import of 5-LO from the cytosol, which is associated with enhanced LTB4 synthesis upon subsequent cell stimulation. In this study, we asked whether adherence of human eosinophils (EOS) causes a similar redistribution of 5-LO and an increase in LTC4 synthesis. Purified blood EOS examined either in suspension or after adherence to fibronectin for 5 min contained only cytosolic 5-LO. Cell stimulation resulted in activation of 5-LO, as evidenced by its translocation to membranes and LTC4 synthesis. As with PMN, adherence of EOS to fibronectin for 120 min caused nuclear import of 5-LO. Unexpectedly, however, adherence also caused a time-dependent decrease in LTC4 synthesis: EOS adhered for 120 min produced 90% less LTC4 than did cells adhered for 5 min. Adherence did not diminish the release of [3H]AA from prelabeled EOS or reduce the synthesis of the prostanoids thromboxane and PGE2. Also, inhibition of LTC4 production caused by adherence could not be overcome by the addition of exogenous AA. Adherence increased, rather than decreased, LTC4 synthase activity. However, the stimulation of adherent EOS failed to induce translocation of 5-LO from the nucleoplasm to the nuclear envelope. This resistance to activation of the nuclear pool of 5-LO with diminished LT production represents a novel mode of regulation of the enzyme, distinct from the paradigm of up-regulated LT synthesis associated with intranuclear localization of 5-LO observed in PMN and other cell types.     

5-Lipoxygenase reaction products modulate alveolar macrophage phagocytosis of Klebsiella pneumoniae

The leukotrienes are potent lipid mediators of inflammation formed by the 5-lipoxygenase-catalyzed oxidation of arachidonic acid. Although the effects of leukotrienes on neutrophil chemotaxis and activation have been established, their role in modulating innate host defense mechanisms is poorly understood. In a previous study (M. Bailie, T. Standiford, L. Laichalk, M. Coffey, R. Strieter, and M. Peters-Golden, J. Immunol. 157:5221-5224, 1996), we used 5-lipoxygenase knockout mice to establish a critical role for endogenous leukotrienes in pulmonary clearance and alveolar macrophage phagocytosis of Klebsiella pneumoniae. In the present study, we investigated the role of specific endogenous leukotrienes in phagocytosis of K. pneumoniae and explored the possibility that exogenous leukotrienes could restore phagocytosis in alveolar macrophages with endogenous leukotriene synthesis inhibition and enhance this process in leukotriene-competent cells. Rat alveolar macrophages produced leukotriene B4 (LTB4), LTC4, and 5-hydoxyeicosatetraenoic acid (5-HETE) during the process of phagocytosis, and the inhibition of endogenous leukotriene synthesis with zileuton and MK-886 dramatically attenuated phagocytosis. We also observed a reduction in phagocytosis when we treated alveolar macrophages with antagonists to the plasma membrane receptors for either LTB4, cysteinyl-leukotrienes, or both. In leukotriene-competent cells, LTC4 augmented phagocytosis to the greatest extent, followed by 5-HETE and LTB4. These 5-lipoxygenase reaction products demonstrated similar relative abilities to reconstitute phagocytosis in zileuton-treated rat alveolar macrophages and in alveolar macrophages from 5-lipoxygenase knockout mice. We conclude that endogenous synthesis of all major 5-lipoxygenase reaction products plays an essential role in phagocytosis. The restorative and pharmacologic effects of LTC4, LTB4, and 5-HETE may provide a basis for their exogenous administration as an adjunctive treatment for patients with gram-negative bacterial pneumonia.     

Leukotriene C4-synthesis deficiency: a new inborn error of metabolism linked to a fatal developmental syndrome

BACKGROUND: Cysteinyl leukotrienes (LTC4, LTD4, LTE4) are potent lipid mediators derived from arachidonic acid in the 5-lipoxygenase pathway that exert profound biological effects. We investigated synthesis and metabolism of leukotrienes in an infant who presented with muscular hypotonia, psychomotor retardation, failure to thrive, and microcephaly. The course of the disease was rapidly progressive and the infant died aged 6 months. METHODS: Cysteinyl leukotrienes and LTB4 were analysed in cerebrospinal fluid, plasma, urine, and stimulated monocytes by EIA. We measured [3H]-LTC4 formation from [3H]-LTA4 in monocytes and platelets by radio-high-pressure liquid chromatography. FINDINGS: Concentrations of LTC4 and its metabolites were below the detection limit in the cerebrospinal fluid, plasma and urine. LTC4 could not be generated in stimulated monocytes, whereas LTB4 synthesis was increased. [3H]-LTC4 could not be made from [3H]-LTA4 in the patient's monocytes or platelets. INTERPRETATION: In this patient, inability to synthesise LTC4 suggests a deficiency of LTC4 synthase. This defect is a new inborn error of human eicosanoid metabolism and may be associated with the clinical disorder. Leukotriene analysis should be done in all patients with neurological symptoms who are candidates for metabolic diseases.     

Leukotrienes in renal transplant rejection in rats. Distinct roles for leukotriene B4 and peptidoleukotrienes in the pathogenesis of allograft injury

To investigate the role of leukotrienes in renal allograft rejection, we studied the effects of specific leukotriene inhibitors in a rat kidney transplant model. The enhanced renal production of leukotrienes observed in allograft recipients was reduced in a dose-dependent manner by the specific 5-lipoxygenase inhibitor MK886. This suppression of leukotriene production caused a substantial improvement in renal function. Inhibition of 5-lipoxygenase also reduced the severity of vascular inflammation and endothelial injury in allografts, and profoundly inhibited expression of donor MHC class II Ag on kidney cells. Survival of renal allograft recipients was prolonged from 10 +/- 1 days in controls to 16 +/- 1 days in animals that received a 6-day course of MK886 (p < 0.05). To investigate the relative roles of LTB4 compared to peptidoleukotrienes in these processes, we treated a separate group of animals with the specific peptidoleukotriene receptor antagonist SKF106203. This agent inhibits the interaction of peptidoleukotrienes with their receptor(s) but does not affect the biologic actions of LTB4. In these studies, SKF106203 caused a modest improvement in renal allograft function that was of lesser magnitude than that seen with the 5-lipoxygenase inhibitor. SKF106203 also reduced vascular inflammation in allografts, but had no effect on expression of MHC class II Ag. We conclude that leukotrienes play a key role in the pathogenesis of renal allograft rejection. Furthermore, the detrimental effects of leukotrienes in rejection are mediated by distinct actions of LTB4 and peptidoleukotrienes.     

The effects of leukotrienes C4 and D4 on ciliary activity of human paranasal sinus mucosa in vitro

The effects of leukotrienes C4 and D4 on ciliary activity of human paranasal sinus mucosa were investigated in vitro. Normal mucosa was surgically obtained from human paranasal sinuses and incubated in the form of tissue culture. Ciliated cells were magnified under an inverted microscope, and ciliary activity was photoelectrically measured. LTD4 progressively inhibited ciliary activity, and showed a more potent effect on ciliary activity compared to LTC4. The concentrations of LTC4 and LTD4 in the incubation medium were determined by radioimmunoassay when the mucosa was incubated with 10(-8) M LTC4. The concentration of LTD4 gradually increased and after 90 min reached the maximum of 0.71 x 10(-8) M, while that of LTC4 was reduced to about 10% of its initial concentration within 60 min. These results suggested the possible conversion of LTC4 to LTD4 on the mucosa, and that LTC4 can inhibit ciliary activity by means of LTD4.     

Eicosanoid synthesis by warm- and cold-acclimated American bullfrog (Rana catesbeiana) brain

Previous studies in bullfrogs have demonstrated the presence of leukotriene (LT)C4 binding sites in the brain. However, synthesis of eicosanoids by brain tissue has not been examined. Because prostaglandin (PG) synthesis differs in warm- and cold-acclimated bullfrog lung tissue, this study compared the synthesis of prostaglandins and leukotrienes in brains from warm-(22 degrees C) and cold-acclimated (5 degrees C) animals. Initial experiments determined that leukotriene and prostaglandin production rates were greatest during the initial 30 min time period. Therefore, tissues were incubated in Munsick's solution and gassed with 95% O2, 5% CO2 for 30 min. Media were analyzed by radioimmunoassay for LTC4, LTB4, PGE2, PGF2 alpha, TXB2, and 6-keto PGF1 alpha. In warm-acclimated bullfrog brains, production was as follows: LTC4 > PGE2 > 6-keto PGF1 alpha, thromboxane (TX)B2, LTB4, and PGF2 alpha. Brain tissues from cold-acclimated animals incubated at 22 degrees C produced significantly greater quantities of PGE2 and 6-keto PGF1 alpha than did brains from warm-acclimated animals. Stimulation of TXB2 levels was observed when the animal was stunned with a blow to the head prior to decapitation. Indomethacin, a cyclooxygenase inhibitor, decreased prostaglandin but not leukotriene synthesis. Epinephrine (4 x 10(-8) M), the amphibian sympathetic postganglionic neurotransmitter, stimulated leukotriene synthesis by brains from warm-acclimated bullfrogs, and the effect was blocked with the 5-lipoxygenase inhibitor MK-886 (5 x 10(-5) M). These results clearly indicate that the bullfrog brain synthesized both leukotrienes and prostaglandins. Further studies are necessary to determine their function in the amphibian central nervous system.     

Leukotriene B4 activates the NADPH oxidase in eosinophils by a pertussis toxin-sensitive mechanism that is largely independent of arachidonic acid mobilization

Experiments were designed to investigate whether leukotriene (LTB4) receptors can couple directly to phospholipase A2 (PLA2) in guinea pig eosinophils and the role of endogenous arachidonic acid (AA) in LTB4-induced activation of the NADPH oxidase. LTB4 (EC50 approximately 16 nM) and AA (EC50 approximately 6 microM) generated hydrogen peroxide (H2O2) in a concentration-dependent manner and at an equivalent maximum rate (5-6 nmol/min/10(6) cells). LTB4 stimulated PLA2 over a similar concentration range that activated the NADPH oxidase, although kinetic studies revealed that the release of [3H]AA (t1/2 approximately 2 s) preceded H2O2 generation (t1/2 > 30 s). Pretreatment of eosinophils with pertussis toxin abolished the increase in inositol(1,4,5)trisphosphate mass, [Ca2+]c, [3H]AA release, and H2O2 generation evoked by LTB4. Qualitatively identical results were obtained in eosinophils in which phospholipase C (PLC) was desensitized by 4beta-phorbol 12,13-dibutyrate with the exception that [3H]AA release was largely unaffected. Additional studies performed with the protein kinase C inhibitor, Ro 31-8220, and under conditions in which Ca2+ mobilization was abolished, provided further evidence that LTB4 released [3H]AA independently of signal molecules derived from the hydrolysis of phosphatidylinositol(4,5)bisphosphate by PLC. Pretreatment of eosinophils with the PLA2 inhibitor, mepacrine, abolished LTB4-induced [3H]AA release at a concentration that inhibited H2O2 by only 36%. Collectively, the results of this study indicate that agonism of LTB4 receptors on guinea pig eosinophils mobilizes AA by a mechanism that does not involve the activation of PLC. In addition, although LTB4 effectively stimulated PLA2, a central role for AA in the activation of the NADPH oxidase was excluded.     

5-lipoxygenase inhibitors in asthma therapy

Leukotrienes are biological active lipids produced from arachidonic acid in cell membrane by the enzyme 5-lipoxygenase. During anaphylaxis, large amounts of leukotrienes are generated in the lungs, causing bronchoconstriction. Leukotrienes exacerbate asthma in different ways including cellular infiltration, chemotaxis aggregation, and degranulation (LTB4), as well as airway constriction, vasopermeability, mucous hypersecretion, mucosal edema and reduced mucociliary clearance (LTC4, LTD4 and LTE4). It is expected that 5-lipoxygenase inhibitors are effective in bronchial asthma by blocking leukotriene synthesis, and its indication is chronic therapy to prevent symptoms of bronchospasm. Zileuton is the 5-lipoxygenase inhibitor most advanced in clinical development. As bronchial asthma is thought to be provoked by a variety of causes, therapeutic drug should be selected according to the state of the disease.     

Toxoplasma gondii alters eicosanoid release by human mononuclear phagocytes: role of leukotrienes in interferon gamma-induced antitoxoplasma activity

Toxoplasma gondii tachyzoites markedly alter the profile of eicosanoids released by human mononuclear phagocytes. Freshly isolated, 2-h adherent human monocytes release both cyclooxygenase (e.g., thromboxane [TX] B2, prostaglandin [PG] E2) and 5-lipoxygenase (e.g., leukotriene [LT] B4, LTC4) products of arachidonic acid metabolism after stimulation by the calcium ionophore A23187 or ingestion of opsonized zymosan particles or heat-killed T. gondii. However, after incubation with viable T. gondii, normal and chronic granulomatous disease monocytes release only the cyclooxygenase products TXB2 and PGE2 and fail to form LTB4, LTC4, or other 5-lipoxygenase products. Monocytes maintained in culture for 5 d lose this capacity to release TXB2 and PGE2 after incubation with T. gondii. T. gondii significantly inhibit calcium ionophore A23187-induced LTB4 release by monocyte-derived macrophages; heat-killed organisms do not affect this calcium ionophore A23187-induced release of LTB4. T. gondii-induced inhibition of LTB4 release by calcium ionophore A23187-stimulated monocyte-derived macrophage is reversed by interferon (IFN)-gamma treatment of the monolayers. LTB4 induced extensive damage to the cellular membranes and cytoplasmic contents of the organisms as observed by transmission electron microscopy. Exogenous LTB4 (10(-6) M) induced intracellular killing of ingested T. gondii by non-IFN-gamma-treated monocyte-derived macrophages. IFN-gamma-induced antitoxoplasma activity in monocyte-derived macrophages was inhibited by the selective 5-lipoxygenase inhibitor zileuton but not by the cyclooxygenase inhibitor indomethacin. These findings suggest a novel role for 5-lipoxygenase arachidonic acid products in human macrophage IFN-gamma-induced antitoxoplasma activity.     

Effect of trans-resveratrol, a natural polyphenolic compound, on human polymorphonuclear leukocyte function

1. Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD). 2. Epidemiological and laboratory evidence suggests that red wine, by virtue of its polyphenolic constituents, may be more effective than other alcoholic beverages in reducing the risk of CHD mortality. 3 The aim of the present study was to investigate the effects of trans-resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenol present in most red wines, on functional and biochemical responses of PMN, upon in vitro activation. 4. trans-Resveratrol exerted a strong inhibitory effect on reactive oxygen species produced by PMN stimulated with 1 microM formyl methionyl leucyl phenylalamine (fMLP) (IC50 1.3+/-0.13 microM, mean+/-s.e.mean), as evaluated by luminol-amplified chemiluminescence. 5. trans-Resveratrol prevented the release of elastase and beta-glucuronidase by PMN stimulated with the receptor agonists fMLP (1 microM, IC50 18.4+/-1.8 and 31+/-1.8 microM), and C5a (0.1 microM, IC50 41.6+/-3.5 and 42+/-8.3 microM), and also inhibited elastase and beta-glucuronidase secretion (IC50 37.7+/-7 and 25.4+/-2.2 microM) and production of 5-lipoxygenase metabolites leukotriene B4 (LTB4), 6-trans-LTB4 and 12-trans-epi-LTB4 (IC50 48+/-7 microM) by PMN stimulated with the calcium ionophore A23187 (5 microM). 6. trans-Resveratrol significantly reduced the expression and activation of the beta2 integrin MAC-1 on PMN surface following stimulation, as revealed by FACS analysis of the binding of an anti-MAC-1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb, recognizing an activation-dependent epitope on MAC-1. Consistently, PMN homotypic aggregation and formation of mixed cell-conjugates between PMN and thrombin-stimulated fixed platelets in a dynamic system were also prevented by transresveratrol. 7. These results, indicating that trans-resveratrol interferes with the release of inflammatory mediators by activated PMN and down-regulates adhesion-dependent thrombogenic PMN functions, may provide some biological plausibility to the protective effect of red wine consumption against CHD.     

Survival and water-balance characteristics of unfed adult Amblyomma cajennense (Acari: Ixodidae)

The specific type of phospholipase A2 (PLA2) involved in formation of leukotriene B4 (LTB4) and platelet activating factor (PAF) in inflammatory cells has been controversial. In a recent report we characterized activation of the 'cytosolic' form of PLA2 (cPLA2) in human neutrophils (PMN) permeabilized with Staphylococcus aureus alpha-toxin under conditions where the secretory form of PLA2 (sPLA2) was inactive. In the current study, generation of both LTB4 and PAF in porated PMN are demonstrated. PMN, prelabeled with [3H]arachidonic acid (3H-AA, to assess AA release and LTB4 production) or with 1-O-[9',10'-3H]hexadecyl-2-lyso-glycero-3-phosphocholine (3H-lyso-PAF, for determination of lyso-PAF and PAF formation), were permeabilized with alpha-toxin in a 'cytoplasmic' buffer supplemented with acetyl CoA. Maximum production of both PAF and LTB4 required addition of 500 nM Ca2+, G-protein activation induced with 10 microM GTP gamma S, and stimulation with the chemotactic peptide, N-formyl-Met-Leu-Phe (FMLP, 1 microM); LTB4 production was confirmed by radioimmunoassay. Removal of acetyl CoA from the system had little effect on LTB4 generation but blocked PAF production with a concomitant increase in lyso-PAF formation LTB4 and PAF production occurred in parallel over time and at differing ATP and Ca2+ concentrations. Further work demonstrated that: (i) maximum production of both inflammatory mediators required a hydrolyzable form of ATP; (ii) blocking phosphorylation with staurosporin inhibited production of both; (iii) the reducing agent, dithiotreitol, had little affect on LTB4 formation but slightly enhanced PAF generation. This study clearly shows that cPLA2 activation can provide precursors for both LTB4 and PAF, that maximum PAF and LTB4 formation occur under conditions that induced optimal cPLA2 activation, that a close coupling between LTB4 and PAF formation exists, and that, after substrate generation, no additional requirements are necessary for LTB4 and PAF generation in the permeabilized PMN system.     

Increased production of eicosanoids, TXA2, PGI2 and LTC4 in experimental spinal cord injuries

Arachidonate metabolites have many kinds of bioactivities. Thromboxane A2 (TXA2) stimulates platelet aggregation and vasoconstriction, whereas prostaglandin I2 (PGI2) antagonises its activities. Thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) are determined in biological materials. Production of TXB2, 6-keto-PGF1 alpha and leukotriene C4 (LTC4), which have potent vascular permeability, was measured by radioimmunoassay in experimental spinal cord injured animals. TXB2 level in the rat spinal cord reached a peak concentration of 133.6 +/- 3.8 pmol/g cord, and 6-keto-PGF1 alpha increased to 26.2 +/- 11.7 pmol/g cord 5 minutes after the injury. There was good correlation between TXB2 production and vascular damage as monitored by fluorescein uptake. When OKY-046 ((E)-3-[4-(1-imidazolylmethyl) phenyl]-2-propenoic acid), which selectively inhibits TXA2 synthetase activity, was administered 10 minutes before injury, the increase in TXB2 production was inhibited by more than 80%, but the degree of vascular damage was reduced by only 40%. In the guinea pig spinal cord, LTC4 levels reached a peak concentration of 2.2 +/- 0.4 pmol/g cord 10 minutes after compression, while that of TXB2 reached 146.8 +/- 6.2 pmol/g cord. The increased production of TXB2 was correlated with the degree of compression injury while that of LTC4 production did not. These findings suggest that vasoactive eicosanoids, TXA2, PGI2 and LTC4, play important roles in secondary damage following spinal cord injury, although their roles may be different among species of animals.     

Impact of arachidonic versus eicosapentaenoic acid on exotonin-induced lung vascular leakage: relation to 4-series versus 5-series leukotriene generation

Escherichia coli hemolysin (HlyA) is a proteinaceous pore-forming exotoxin that is implicated as a significant pathogenicity factor in extraintestinal E. coli infections including sepsis. In perfused rabbit lungs, subcytolytic concentrations of the toxin evoke thromboxane-mediated vasoconstriction and prostanoid-independent protracted vascular permeability increase (11). In the present study, the influence of submicromolar concentrations of free arachidonic acid (AA) and eicosapentaenoic acid (EPA) on the HlyA-induced leakage response was investigated. HlyA at concentration from 0.02 to 0.06 hemolytic units/ml provoked a dose-dependent, severalfold increase in the capillary filtration coefficient (Kfc), accompanied by the release of leukotriene(LT)B4, LTC4, and LTE4 into the recirculating buffer fluid. Simultaneous application of 100 nmol/L AA markedly augmented the HlyA-elicited leakage response, concomitant with an amplification of LTB4 release and a change in the kinetics of cysteinyl-LT generation. In contrast, 50 to 200 nmol/L EPA suppressed in a dose-dependent manner the HlyA-induced increase in Kfc values. This was accompanied by a blockage of 4-series LT generation and a dose-dependent appearance of LTB5, LTC5, and LTE5. In addition, EPA fully antagonized the AA-induced amplification of the HlyA-provoked Kfc increase, again accompanied by a shift from 4-series to 5-series LT generation. We conclude that the vascular leakage provoked by HlyA in rabbit lungs is differentially influenced by free AA versus free EPA, related to the generation of 4- versus 5-series leukotrienes. The composition of lipid emulsions used for parenteral nutrition may thus influence inflammatory capillary leakage.