Stimulus-specificity of the chemotactic deactivation of human neutrophils by lipoxygenase products of arachidonic acid

Preincubation of human neutrophils with chemotactic concentrations of 5(S)-hydroxy-eicosatetraenoic acid (5-HETE) or 5(S), 12(R)-dihydroxy-6,14 cis-8,10 trans-eicosatetraenoic acid (leukotriene B4) induces a state of preferential chemotactic unresponsiveness to the homologous factor, termed deactivation, and less suppression of the responses to other chemotactic stimuli. The ratio of the concentrations required for maximal chemotactic deactivation of neutrophils to that which stimulates chemotaxis optimally is greater for 5-HETE and leukotriene B4 than for peptide and protein factors. In contrast to other chemotactic factors, 5-hydroperoxy-eicosatetraenoic acid (5-OOHETE) induces neutrophil chemotactic deactivation that is independent of the nature of the subsequent stimulus and is more slowly reversible after elimination of the fluid-phase deactivating factor. The unique characteristics of the chemotactic deactivation of human neutrophils by 5-OOHETE may be attributable in part to its endogenous metabolism to potent deactivating factors or to covalent derivatization of subcellular structures of the neutrophils by the highly reactive 5-OOHETE.     

Deactivation of the effects of F-Met-Leu-Phe and leukotriene B4 on calcium mobilization in rabbit neutrophils

Preincubation of rabbit neutrophils with the synthetic chemotactic factor f-Met-Leu-Phe has been found to diminish the ability of these cells to mobilize calcium upon subsequent stimulation by f-Met-Leu-Phe or by leukotriene B₄. The preexposure of the neutrophils to leukotriene B₄ on the other hand results in a diminished subsequent response to itself but an unaltered response to f-Met-Leu-Phe. These results demonstrate that deactivation can be observed at the level of calcium mobilization, strengthen the postulated second messenger role of calcium in neutrophils and imply that neutrophil activation by chemotactic factors can bypass the arachidonic acid metabolic pathway.     

Selective feed-back inhibition of the 5-lipoxygenation of arachidonic acid in human T-lymphocytes

Purified human T-lymphocytes exhibit 5-lipoxygenase activity as demonstrated by the conversion of arachidonic acid to 5-hydroxy-eicosatetraenoic acid (5-HETE), 5(S),12(R)-di-hydroxy-eicosa-6,14 cis-8,10 trans-tetraenoic acid (leukotriene B₄), and 5,12-di-HETE isomers of leukotriene B₄ that lack a 6-cis double bond. The concentrations of leukotriene B₄, 5-HETE, 11-HETE and 15-HETE in suspensions of T-lymphocytes were increased significantly by concanavalin A and by the calcium ionophore A23187. Preincubation of T-lymphocytes with 15-HETE at μM concentrations, characteristic of suspensions of stimulated lymphocytes, inhibited selectively the increases in the levels of 5-HETE and leukotriene B₄, but not of 11-HETE and prostaglandin E₂.     

Formation of leukotrienes and hydroxy acids by human neutrophils and platelets exposed to monosodium urate

Monosodium urate (MSU) crystals stimulate the production of arachidonic acid metabolites by human neutrophils and platelets. Neutrophils exposed to MSU generated leukotriene B₄(LTB₄). 6- -LTB₄, 12- -6- -LTB₄, and 5S, 12S DHETE from endogenous sources of arachidonate. In addition to these metabolites both monohyroxyeicosatetraenoic acids (i.e., 5-HETE) and w-oxidation products (i.e., 20-COOH LTB₄) were formed by neutrophils exposed to MSU. Addition of exogenous arachidonic acid led to increased formation of each of these metabolites. When neutrophils were treated with colchicine (10 uM), LTB₄ but 5-HETE formation was impaired. (1-¹⁴C) Arachidonate-labeled platelets exposed to MSU released (1-¹⁴C)-arachidonate. (¹⁴C)-12 HETE, (¹⁴C)-HHT and (¹⁴C)-thromboxane B₂. Results indicate that MSU stimulates arachidonic acid metabolism in both human neutrophils and platelets. Moreover, they suggest not only that metabolites of arachidonate may be considered as possible candidates for mediators of inflammation in crystal-associated diseases, but that colchicine blocks the formation of LTB₄.     

Similarities in the mechanisms by which formyl-methionyl-leucyl-phenylalanine, arachidonic acid and leukotriene B4 increase calcium and sodium influxes in rabbit neutrophils

The chemotactic factors f-Met-Leu-Phe, arachidonic acid and leukotriene B₄ induce a rapid stimulation of both Ca²⁺ and Na⁺ influx in rabbit neutrophils. In the three cases the stimulation is rapid and the effects are not additive. Furthermore in all cases the stimulation of Na-influx but not of Ca-uptake is inhibited by the potassium-sparing diuretic amiloride. Preincubation with high concentrations of the chemotactic factor f-Met-Leu-Phe followed by washing of rabbit neutrophils reduces significantly the stimulation of calcium uptake induced by arachidonic acid, leukotriene B₄ and f-Met-Leu-Phe. These results strongly suggest that the exogenous addition of arachidonic acid or of leukotriene B₄ leads to the activation of the same permeation pathways as do better defined chemotactic factors.     

Unique aspects of the modulation of human neutrophil function by 12-L-hydroperoxy-5,8,10,14-eicosatetraenoic acid

12-L-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-OOHETE), a labile intermediate generated by the lipoxygenation of arachidonic acid in platelets, and 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-OHETE), the reduction product of 12-OOHETE, were examined for their effects on human neutrophil function . 12-OOHETE elicited a maximal neutrophil chemotactic response at 4 μg/ml, that exceeded by over 50% the maximal chemotactic response to 10–20 μg/ml of 12-OHETE. Similarly 12-OOHETE was more potent than 12-OHETE in evoking neutrophil chemokinetic responses and in enhancing the expression of C3b receptors on neutrophils. The concentration of guanosine 3′:5′ cyclic monophosphate (cGMP) in neutrophils was increased to the same plateau level by 5 ng/ml of 12-OOHETE and by 50 ng/ml of 12-OHETE. Elevations in the concentration of cGMP were maintained for 30 min or longer by a single dose of 12-OOHETE, but fell between 10 and 20 min after the introduction of 12-OHETE. The release of neutrophil lysosomal enzymes by the chemotactic fragments of C5 was augmented substantially by 12-OOHETE, while 12-OHETE had only a marginal effect. The non-chemotactic methyl ester of 12-OHETE failed to inhibit the chemotactic responses to 12-OOHETE at molar ratios that suppressed comparable responses to 12-OHETE by 42–86%. Thus 12-OOHETE is more potent than 12-OHETE in the stimulation of some human neutrophil functions and in the elevation of the cellular concentration of cGMP. Furthermore, 12-OOHETE may activate neutrophils by pathways not available to 12-OHETE.     

Mechanism involved in the mobilization of neutrophil calcium by 5-hydroxyeicosatetraenoate

5-Hydroxyeicosatetraenoate (5-HETE), like leukotriene B4 and platelet-activating factor, stimulated human polymorphonuclear neutrophils to mobilize intracellular calcium. The three compounds acted through mechanisms that were inhibited by pertussis toxin, cholera toxin, and PMA. Each agonist, furthermore, desensitized (or down-regulated) the neutrophil's calcium mobilization response to a second challenge with the same agonist. However, 5-HETE and leukotriene B4 had little or no activity in cross-desensitizing neutrophil responses to each other or to platelet-activating factor. Furthermore, 5-HETE interfered minimally or not at all with the binding of radiolabeled leukotriene B4 and platelet-activating factor to their respective receptors on neutrophils. Thus, 5-HETE mobilizes neutrophil calcium by a mechanism different from those used by leukotriene B4 and platelet-activating factor. This mechanism appears to involve specific 5-HETE receptors that couple to pertussis toxin-inhibitable, GTP-binding proteins.     

Formation of leukotrienes and other hydroxy acids during platelet-neutrophil interactions in vitro

Interactions of human platelets with neutrophils were studied in suspensions of [³H]arachidonate-labeled platelets and unlabeled neutrophils stimulated with ionophore A23187. Several radioactive arachidonate metabolites, not produced by platelets alone, were detected, including [³H]-labeled leukotriene B₄ (LTB₄), dihydroxyeicosatetraenoic acid (DHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). When [³H]12-HETE, a platelet product, was added to stimulated neutrophils, DHETE was formed. Similarly, when [³H]5-HETE, a neutrophil product, was added to stimulated platelets, DHETE was the major product. These results suggest that upon stimulation: 1) platelet-derived arachidonate may serve as precursor for the neutrophil-derived eicosanoids LTB₄ and 5-HETE, and 2) that platelet-derived 12-HETE can be converted to DHETE by human neutrophils. The present investigation documents cell-cell interactions via the lipoxygenase pathway, which may be important in hemostasis, thrombosis and inflammation.     

Unique aspects of the modulation of human neutrophil function by 12-L-hydroperoxy-5,8,10,14-eicosatetraenoic acid

12-L-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-OOHETE), a labile intermediate generated by the lipoxygenation of arachidonic acid in platelets, and 12-L-hydroxy-5,8,10.14-eicosatetraenoic acid (12-OHETE), the reduction product of 12-OOHETE, were examined for their effects on human neutrophil function in vitro. 12-OOHETE elicited a maximal neutrophil chemotactic response at 4 microgram/ml, that exceeded by over 50% the maximal chemotactic response to 10-20 microgram/ml of 12-OHETE. Similarly 12-OOHETE was more potent than 12-OHETE in evoking neutrophil chemokinetic responses and in enhancing the expression of C3b receptors on neutrophils. The concentration of guanosine 3':5' cyclic monophosphate (cGMP) in neutrophils was increased to the same plateau level by 5 ng/ml of 12-OOHETE and by 50 ng/ml of 12-OHETE. Elevations in the concentration of cGMP were maintained for 30 min or longer by a single dose of 12-OOHETE, but fell between 10 and 20 min after the introduction of 12-OHETE. The release of neutrophil lysosomal enzymes by the chemotactic fragments of C5 was augmented substantially by 12-OOHETE, while 12-OHETE had only a marginal effect. The non-chemotactic methyl ester of 12-OHETE failed to inhibit the chemotactic responses to 12-OOHETE at molar ratios that suppressed comparable response to 12-OHETE by 42-86%. Thus 12-OOHETE is more potent than 12-OHETE in the stimulation of some human neutrophil functions and in the elevation of the cellular concentration of cGMP. Furthermore, 12-OOHETE may activate neutrophils by pathways not available to 12-OHETE.     

Effect of 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin 1(1) (U-60,257), an inhibitor of leukotriene synthesis, on human neutrophil function

A23187, a calcium ionophore, stimulated a time-dependent generation of 5(S), 12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid (leukotriene B₄), production of superoxide anion (O₂^?) and release of granule-associated β-glucuronidase and lysozyme by human neutrophils. Leukotriene B₄ also elicited the selective release of granule enzymes from cytochalasin B-treated neutrophils. U-60,257, a recently identified inhibitor of leukotriene (LT) C₄ and D₄ synthesis, caused a dose-related (1–10 μM) suppression of LTB₄ production by A23187-activated neutrophils. Degranulation and O₂^? generation by neutrophils exposed to A23187 and the chemotactic oligopeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), were also inhibited with U-60,257.     

15-Hydroxyeicosatetraenoic Acid Inhibits Superoxide Anion Generation by Human Neutrophils: Relationship to Lipoxin Production

Human neutrophils can aggregate, degranulate, and release mediators of inflammation including oxygen radicals and lipoxygenase (LO)-derived products of arachidonic acid. The regulation of 5– and 15-lipoxy-genases appears to be important since their products (e.g. leukotrienes and lipoxins) display unique spectra of bioactions. Addition of 15-HETE. a product of the 15-LO, to neutrophils in suspension dramatically shifted the LO products generated and led to a dose-dependent increase in lipoxins, while the production of leukotriene B₄ and its μ-oxidation products (i.e. 20-COOH-LTB₄ and 20-OH-LTB₄) was inhibited. Exogenous 15-HETE also dose-dependently inhibited the generation of superoxide anions induced by either the chemotactic peptide f-met-leu-phe or the divalent cation ionophore A23187. Neither lipoxin A, nor lipoxin B₄ (10^?8?10^?6M) inhibited O₂^?_? generation induced by either f-met-leu-phe or A23187. These results indicate that in addition to serving as a substrate for lipoxin generation, 15-HETE also inhibits superoxide anion generation by human neutrophils. Together they provide further evidence to suggest that products of the 15-lipoxygenase may serve a regulatory role at inflammatory loci.     

The conversion of leukotriene C4 to isomers of leukotriene B4 by human eosinophil peroxidase

The smooth muscle contractile and vasoactive mediator leukotriene C₄ (5(S)-hydroxy-6(R)-sulfido-glutathionyl-eicosatetraenoic acid; LTC₄) is converted by phorbol ester-stimulated human eosinophils to two isomers of leukotriene B₄, 5(S),12(R)-6,8,10 trans-14 cis-eicosatetraenoic acid (5(S),12(R)-“all-trans”-LTB₄) and 5(S),12(S)-“all-trans”-LTB₄, which are leukocyte chemotactic factors lacking the humoral functions of LTC₄. Optimal conversion of LTC₄ to the “all-trans” isomers of LTB₄ by intact eosinophils and soluble eosinophil peroxidase requires both H₂O₂ and halide ions. Oxidative metabolism of leukotrienes may represent an important regulatory function of eosinophils in hypersensitivity reactions.     

Stereospecificity of leukotriene B4 and structure-function relationships for chemotaxis of human neutrophils

The chemotactic activity of leukotriene B4 (5S, 12R Dihydroxy 6, 14 cis, 8, 10 trans eicosatetraenoic acid) (LTB4) was examined by using a sensitive Boyden-chamber assay. The activity of LTB4 was compared to other biosynthetic stereoisomers: 5S, 12R Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (6-trans LTB4); 5S, 12S Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (12-epi-6-trans LTB4), 5S, 12S DiHETE; the metabolic product 20-Hydroxy LTB4 (20-OH LTB4); methylated LTB4 (Methyl-LTB4), and the related monoHETE 5-HETE and 12-HETE. The compounds were purified by several steps of reverse phase and straight phase HPLC. The LTB4 exhibits measurable chemotactic activity at 10(-9) M with maximal activity at 10(-7) M and an ED50 of 10(-8) M. The LTB4 isomers and monoHETE were less chemotactic than previously reported. The monoHETE (5-HETE and 12-HETE), the isomer 12-epi-6-trans LTB4, and 5S, 12S DiHETE fail to attract neutrophils at levels between 10(-6) and 10(-5) M. If these compounds are chemotactic, then activity is at least four orders of magnitude less than that of LTB4. The isomer 6-trans LTB4 at 10(-6) to 10(-5) M induced chemotaxis with an extrapolated ED50 value of 10(-5) M, indicating that a trans for cis change in configuration at position 6 reduces the chemotactic activity of LTB4 by 1000-fold. Conversely, the metabolic product 20-OH LTB4 is at least as active as the native compound LTB4. Methylation of the carboxyl group of LTB4 reduces its chemotactic activity by two orders of magnitude. These results indicate a high degree of stereospecificity for the LTB4 receptor with strict dependence on hydroxyl group, and triene configuration and considerable dependence on the carboxyl group. Modification at the aliphatic omega end of the LTB4 molecule has a minimal effect on function, suggesting that the hydrophobicity of this portion of the molecule is not important for optimal activity. Furthermore, we propose that metabolic products of LTB4 may be of greater importance than LTB4 as physiologic inflammatory mediators in vivo.     

Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.     

The identification of hydroxy fatty acids in psoriatic skin

Psoriasis is a common chronic inflammatory and proliferative skin disease characterised by epidermal neutrophil infiltration which may be induced by chemotactic substances in the involved epidermis. Superficial psoriatic scale was shown to contain biologically active amounts of leukotriene B₄ and monohydroxy-eicosatetraenoic acid (HETE)- like material as determined by assay for chemokinetic activity in high performance liquid chromatography (HPLC) fractions of scale extracts. Extracts of scale and chamber fluid from abraded lesional and uninvolved psoriatic skin were purified by HPLC and appropriate fractions were analysed by gas chromatography - mass spectrometry (GC-MS). The following monohydroxy metabolites of arachidonic, linoleic and 11,14-eicosadienoic acids were identified : 15-HETE, 12-HETE, 11-HETE, 9-HETE, 8-HETE, 5-HETE, 13-hydroxy-octadecadienoic acid (13-HODD), 9-HODD and 15-hydroxy-eicosadienoic acid (15-HEDE). The results suggested that 12-HETE, 13-HODD and 9-HODD are the most abundant monohydroxy fatty acids in the psoriatic skin extracts described above. Assays of 13-HODD, 9-HODD and 15-HEDE for chemokinetic activity were negative with concentrations up to 10^?4M. The biological significance of these three compounds in not known, but some of the hydroxylated metabolites of arachidonic acid may, by virtue of their chemotactic properties, be relevant to the pathogenesis of the psoriatic neutrophil infiltrate.     

15(S)-HETE modulates LTB(4) production and neutrophil chemotaxis in chronic bronchitis

We evaluated the levels of15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE] and the expression of15-lipoxygenase (15-LO) mRNA in induced sputum obtained from 10 controland 15 chronic bronchitis subjects. 15(S)-HETE was evaluated by reversephase high-performance liquid chromatography separationfollowed by specific RIA. 15-LO mRNA expression was determined byprimed in situ labeling. The levels of both soluble and cell-associated 15(S)-HETE resulted significantly higher in chronic bronchitis than incontrol subjects. The percentage of cells expressing 15-LO mRNA wassignificantly higher in chronic bronchitis than in control subjects(P < 0.01). Double staining for specific cell typemarkers and 15-LO mRNA showed macrophages and neutrophils positive for 15-LO, whereas similar staining of peripheral blood neutrophils did notshow evidence for 15-LO expression, suggesting that expression of 15-LOin neutrophils takes place on migration into the airways. Because15(S)-HETE inversely correlated with the percentage of neutrophils insputum of chronic bronchitis subjects, we studied the effect of15(S)-HETE on leukotriene B₄ (LTB₄) productionin vitro and evaluated the concentration of LTB₄ in inducedsputum and the contribution of LTB₄ to the chemotacticactivity of induced sputum samples ex vivo. The results obtainedindicate that macrophages and neutrophils present within the airways ofchronic bronchitis subjects express 15-LO mRNA; increased basal levelsof 15(S)-HETE may contribute to modulate, through the inhibition of5-lipoxygenase metabolites production, neutrophil infiltration andairway inflammation associated with chronic bronchitis.

     

Requirement of free arachidonic acid for leukotriene B4 biosynthesis by 12-hydroperoxyeicosatetraenoic acid-stimulated neutrophils

Stimulation of human neutrophils with 12-hydroperoxyeicosatetraenoic acid (12-HPETE) led to formation of 5S, 12S-dihydroxyeicosatetraenoic acid (DiHETE), but leukotriene B4 (LTB4) or 5-hydroxyeicosatetraenoic acid (5-HETE) was not detectable by reversed-phase high-performance liquid chromatography analysis. N-formylmethionylleucylphenylalanine (FMLP) induced the additional synthesis of small amounts of LTB4 in 12-HPETE-stimulated neutrophils. The addition of arachidonic acid greatly increased the synthesis of LTB4 and 5-HETE by neutrophils incubated with 12-HPETE. In experiments using [1-14C]arachidonate-labeled neutrophils, little radioactivity was released by 12-HPETE alone or by 12-HPETE plus FMLP, while several radiolabeled compounds, including LTB4 and 5-HETE, were released by A23187. These findings demonstrate that LTB4 biosynthesis by 12-HPETE-stimulated neutrophils requires free arachidonic acid which may be endogenous or exogenous.     

Human Red Cells Enhance the Formation of 5-Lipoxygenase-Derived Products by Neutrophils

Upon activation, human neutrophils generate 5-lipoxygenase products which are involved in inflammation as well as other physiological and pathophysiological processes. We have examined the influence of red cells on the generation of lipoxygenase-derived products by neutrophils utilizing high pressure liquid chromato-graphy system which permitted quantitation of SHETE, leukotriene B₄ (and its isomers) and the omega oxidation products of leukotriene B₄ (20-hydroxyleukotriene B₄, 20-carboxyleukotriene B₄) within the same sample. Co-incubation of red cells with neutrophils (50:1, red cells:neutrophils) resulted in a 722 percent increase in 5-hydroxyeicosatetraenoic acid production and a slight increase in leukotriene B₄ and its omega oxidation products which were not accompanied by increases in 15-hydroxyeicosatetraenoic acid production. The role of the sulfhydryl status of the red cell and its ability to scavenge hydrogen peroxide were assessed in relationship to the interaction of red cells on the neutrophil-derived lipoxygenase products. Together, these findings indicate that red cells can regulate the levels of lipid-derived mediators produced by neutrophils. Moreover, they suggest that red cell-neutrophil interactions may be of importance in inflammatory reactions.     

Products of 5-lipoxygenase and myeloperoxidase activities are increased in young male cigarette smokers

Abstract

The significance of 5-lipoxygenase and myeloperoxidase activities has not been extensively studied among young male smokers. Leukotriene B₄, 20-hydroxy-leukotriene B₄, 20-carboxy-leukotriene B₄ and 3-chlorotyrosine were measured in plasma and urinary samples of young male smokers at 8 hours following cigarette abstinence and an hour after cigarette smoking. Leukotriene B₄ and 3-chlorotyrosine were determined in neutrophils isolated from these individuals. The levels of these markers were compared with those of age-matched controls. In vitro studies were performed to evaluate the production of leukotriene B₄ and 3-chlorotyrosine from human neutrophils following exposure to nicotine and cotinine. Thirty male smokers (mean age, 27.4 years) and 28 male non-smokers (mean age, 28.7 years) were studied. Plasma levels of leukotriene B₄, 20-carboxy-leukotriene B₄ and 3-chlorotyrosine were higher in smokers than in non-smokers; leukotriene B₄ and 20-carboxy-leukotriene B₄ levels increased further an hour after cigarette smoking. Peripheral neutrophils isolated from smokers showed greater expressions of myeloperoxidase and 5-lipoxygenase activities compared with non-smokers, while plasma leukotriene B₄ and 3-chlorotyrosine were correlated significantly with high-sensitivity C-reactive protein and plasma nicotine concentrations. Exposure of human neutrophils to nicotine and cotinine resulted in a higher production of leukotriene B₄ and 3-chlorotyrosine. To conclude, leukotriene B₄ and 3-chlorotyrosine levels are increased in young male cigarette smokers. These results suggest that cigarette smoking aggravates neutrophil-mediated inflammation by modulating the activities of myeloperoxidase and 5-lipoxygenase pathways.     

Interaction of human neutrophils with airway epithelial cells: Reduction of leukotriene B4 generation by epithelial cell derived prostaglandin E2

Airway epithelial cells (AEC) play an active role in the regulation of inflammatory airway disease. In the present study we analyzed the interaction of AEC with polymorphonuclear leukocytes (PMN) in coincubation with respect to their arachidonic acid (AA) metabolism using reversed phase-HPLC and post-HPLC-ELISA. Primary cultures of porcine AEC released predominantly PGE₂, PGF_2a, and 15-hydroxyeicosatetraenoic acid (15-HETE), whereas the major human PMN-derived AA metabolite was the chemotactic factor leukotriene B₄ (LTB₄). In AEC-PMN cocultures stimulated with the calcium ionophore A23187, PMN-related 5-lipoxygenase products were decreased by 45%. This reduction in LTB₄ formation in the presence of AEC was mainly due to PGE₂ generated by the epithelial cells, whereas 15-HETE made a minor contribution. Most of the effect was inhibited by AEC pretreatment with acetylsalicylic acid and restored by addition of equivalent amounts of exogenous PGE₂. LTB₄ degradation was not enhanced in PMN-AEC coincubations. Moreover, reduction of LTB₄ formation in this system did not require an intimate cell-to-cell contact as shown by studies involving filter membranes for PMN-AEC separation. Superoxide anion concentrations were also decreased in PMN-AEC coincubations; this effect, however, was unrelated to PGE₂ for quantitative reasons and was probably due to 2 is the major mediator in the coincubation of porcine AEC and human PMN that downregulates neutrophil responses by activating receptors on the neutrophil. A minor contributor in this course of PMN-AEC interaction may be the 15-HETE transcellular pathway. Overall, airway epithelium appears to play an antiinflammatory role by damping the proinflammatory potential of neutrophils. J. Cell. Physiol. 175:268–275, 1998. © 1998 Wiley-Liss, Inc.