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.     

Stimulus-specificity of the chemotactic deactivation of human neutrophilis 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 concentration 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.     

Direct demonstration of increased intracellular concentration of free calcium in rabbit and human neutrophils following stimulation by chemotactic factor

An increase in the level of intracellular free calcium concentration in rabbit and human neutrophils stimulated by chemotactic factors has been demonstrated directly using the calcium-sensitive fluorescent probe quin-2. Addition of f-Met-Leu-Phe (10(-9) M), C5a (3 x 10(-9) M) or leukotriene B4 (6 x 10(-8) M) to the neutrophils induces a rapid increase in the intracellular concentration of free calcium that reaches a maximum value 15 seconds following stimulation. At concentrations of f-Met-Leu-Phe less than 10(-8) M the enhancement is dose dependent with an ED50 of 8 x 10(-11) M and is significantly reduced in the presence of EGTA in the suspending medium.     

Evidence for mediation of acetyl glyceryl ether phosphorylcholine stimulation of adenosine 3′,5′-(cyclic)monophosphate levels in human polymorphonuclear leukocytes by leukotriene B4

Acetyl glyceryl ether phosphorylcholine induces human neutrophil aggregation. Incubation of neutrophils with either prostaglandin I₂, or the cyclic AMP-dependent phosphodiesterase inhibitor, RO 20-1724 before the addition of PAF-acether attenuates subsequent aggregation. Paradoxically, a small elevation in cyclic AMP is observed coincident with the initiation of PAF-acether-stimulated aggregation. The elevation in cyclic AMP in response to PAF-acether is amplified by RO 20-1724, and the magnitude of the response is dependent upon the concentration of PAF-acether. The elevation in cyclic AMP is not due to prostaglandins, because indomethacin actually enhances the elevation in cyclic AMP induced by PAF-acether. The involvement of the neutrophil 5-lipoxygenase, and subsequent leukotriene B₄ synthesis, is suggested by the observation that 5-lipoxygenase inhibitors limit both the elevation in cyclic AMP induced by PAF-acether, and the indomethacin enhancement. This indirect evidence is supported by the fact that leukotriene B₄ itself elevates neutrophil cyclic AMP levels in intact cells, and stimulates the adenylate cyclase in broken cell preparations. Although the elevation in cyclic AMP induced by either PAF-acether or leukotriene B₄ is coincident with the onset of neutrophil aggregation, it is not obligatory for aggregation. The adenylate cyclase inhibitor 2′,5′-dideoxyadenosine blocks the PAF-acether-stimulated increase in cyclic AMP, and actually enhances aggregation. It is suggested that the increase in cyclic AMP observed after the addition of PAF-acether is due to concomitant leukotriene B₄ synthesis, and is not obligatory for neutrophil aggregation, but is actually part of a feed-back regulatory system through which PAF-acether and leukotriene B₄ can limit their own activity in neutrophils.     

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.     

Mechanism of action of leukotriene B4: intracellular calcium redistribution in rabbit neutrophils

The possible involvement of membrane-bound calcium in the mechanism of action of leukotriene B4 was examined using the fluorescent chelate probe, chlortetracycline. Leukotriene B4 was found to cause a rapid release of membrane-bound calcium at physiologically relevant concentrations. This effect of leukotriene B4 is stereospecific and its magnitude is decreased upon the transformation of leukotriene B4 into its omega-hydroxy and omega-carboxy metabolites. The pool of calcium affected by leukotriene B4 appears to be the same as that released by other chemotactic factors such as formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). Similarly, preincubation with f-Met-Leu-Phe results in a decreased responsiveness of the cells to the addition of leukotriene B4. These results extend further the analogy between the mechanism of action of peptidic and lipid chemotactic factors, and emphasize the central role of the intracellular redistribution of calcium, as inferred and monitored by chlortetracycline fluorescence and steady-state isotopic flux studies, in neutrophil activation.     

Cellular regulatory role of leukotriene B4: its effects on cation homeostasis in rabbit neutrophils

We have found that exogenous leukotriene B4 modifies calcium homeostasis in rabbit neutrophils in a manner essentially analogous to that of the chemotactic peptide f-Met-Leu-Phe. Leukotriene B4 causes a rapid and dose-dependent increase in membrane permeability to calcium and a release of calcium from previously unexchangeable intracellular pool(s). The net result of these changes is to transiently elevate the intracellular level of exchangeable calcium. A stereoisomer of leukotriene B4 with greatly reduced secretory activity toward neutrophils (5S, 12S-di HETE) is essentially without effect on the rate of 45Ca uptake at concentrations equal to those that produce near maximal enhancement by leukotriene B4. Leukotriene B4, in addition to its effects on calcium metabolism, also increases the rate of 22Na influx into rabbit neutrophils. The relationships between the action of leukotriene B4 on calcium homeostasis and the neutrophil-directed activities of arachidonic acid and its lipoxygenase metabolites are discussed     

Altered leukotriene B4 metabolism in CYP4F18-deficient mice does not impact inflammation following renal ischemia

Inflammatory responses to infection and injury must be restrained and negatively regulated to minimize damage to host tissue. One proposed mechanism involves enzymatic inactivation of the pro-inflammatory mediator leukotriene B₄, but it is difficult to dissect the roles of various metabolic enzymes and pathways. A primary candidate for a regulatory pathway is omega oxidation of leukotriene B₄ in neutrophils, presumptively by CYP4F3A in humans and CYP4F18 in mice. This pathway generates ω, ω-1, and ω-2 hydroxylated products of leukotriene B₄, depending on species. We created mouse models targeting exons 8 and 9 of the Cyp4f18 allele that allows both conventional and conditional knockouts of Cyp4f18. Neutrophils from wild-type mice convert leukotriene B₄ to 19-hydroxy leukotriene B₄, and to a lesser extent 18-hydroxy leukotriene B₄, whereas these products were not detected in neutrophils from conventional Cyp4f18 knockouts. A mouse model of renal ischemia–reperfusion injury was used to investigate the consequences of loss of CYP4F18 in vivo. There were no significant changes in infiltration of neutrophils and other leukocytes into kidney tissue as determined by flow cytometry and immunohistochemistry, or renal injury as assessed by histological scoring and measurement of blood urea nitrogen. It is concluded that CYP4F18 is necessary for omega oxidation of leukotriene B₄ in neutrophils, and is not compensated by other CYP enzymes, but loss of this metabolic pathway is not sufficient to impact inflammation and injury following renal ischemia–reperfusion in mice.     

Stimulus-dependent changes in calcium metabolism in rabbit neutrophils

We have found that the changes in calcium metabolism in rabbit neutrophils produced by the chemotactic synthetic peptide f-Met-Leu-Phe are not sensitive to the calcium chelator EGTA. The present results demonstrate unambiguously that the previously described chemotactic factor induced changes in 45Ca fluxes in rabbit neutrophils do indeed reflect intracellular events. The pool of calcium mobilized by f-Met-Leu-Phe and increase in cell associated 45Ca upon stimulation are both insensitive to the presence of EGTA.     

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.     

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.     

Studies on the mechanism of formation of the 5S,12S-dihydroxy-6,8,10,14(E,Z,E,Z)-icosatetraenoic acid in leukocytes

Incubation of peripheral blood leukocytes with arachidonic acid (and ionophore A23187) led to the formation of leukotriene B₄, Δ⁶-trans-leukotriene B₄, Δ⁶-trans-12-epi-leukotriene B₄, 5-hydroxy-icosatetraenoic acid, 12-hydroxy-icosatetraenoic acid and of 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-icosatetraenoic acid (5S,12S-DiHETE). Incubation of leukocytes with leukotriene A₄ resulted in the formation of leukotriene B₄ and of its two Δ⁶-trans-isomers but not of the 5S,12S-DiHETE. ¹⁸O₂ labeling experiments have shown that the hydroxyl groups at C5 and C12 in the 5S,12S-DiHETE are derived from molecular oxygen. The tetraacetylenic analog of arachidonic acid was found to be a potent inhibitor of the formation of the 5S,12S-DiHETE whereas it potentiated the synthesis of the 5-hydroxy acid and of leukotriene B₄. Addition of the 12-hydroxy-icosatetraenoic acid to leukocytes, or of the 5-hydroxy-icosatetraenoic acid to a suspension of platelets caused the formation of the 5S,12S-DiHETE. It is concluded that the 5S,12S-DiHETE is not derived from leukotriene A₄ but is a product of the successive reactions of arachidonic acid with two lipoxygenases of different positional specificities.     

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.     

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.     

Characterization of the secretory activity of leukotriene B4 toward rabbit neutrophils

We have described in det ail the secretory activity of leukotriene B4 toward rabbit neutrophils. Leukotriene B4 rapidly and vigorously degranulates rabbit neutrophils. This activity is stereospecific, cytochalasin B-dependent, and is enhanced by extracellular calcium. Pretreatment with leukotriene B4 deactivates rabbit neutrophils, i.e., cells so treated do not respond to stimulation by an additional bolus of leukotriene B4. In addition, the secretory activity of leukotriene B4 is sharply dependent on the simultaneous presence of cytochalasin B. Rabbit neutrophils therefore exhibit the previously described desensitization to the effect of cytochalasin B. In these and other discussed respects the characteristics of the leukotriene B4-induced degranulation of rabbit neutrophils are strikingly similar to those of the chemotactic factors. These results support the hypothesis that leukotriene B4 mediates, at least in part, the secretory, and possibly other, activities of chemotactic factors.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A₄ hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A₄ to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B₄ was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and apparent K_m for leukotriene A₄ between 2 · 10^?5 and 3 · 10^?5 M. The purified leukotriene A₄ hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A₄ hydrolase from previously purified epoxide hydrolases.     

Chemotaxis of human neutrophils and eosinophils towards leukotriene B4 and its 20-w-oxidation products in vitro

Peripheral blood neutrophils and eosinophils from 70 patients and controls were studied for their in vitro chemotactic and chemokinetic responses towards synthetic leukotriene B₄ (LTB₄), 20-OH-LTB₄ and 20-COOH-LTB₄. All three factors induced chemotaxis and chemokinesis of cells. 20-OH-LTB₄ was always less and 20-COOH-LTB₄ even less active than the parent compound. Cells from patients with atopic eczema and T cell lymphoma moved less than cells from normal controls or from patients with psoriasis. In the presence of LTB₄, 20-OH-LTB₄ and buffer alone, more eosinophils than neutrophils moved to the lower side of the filter, while this did not occur with platelet activating factor as chemoattractant. Studies of neutrophil and eosinophil chemotaxis in the presence of LTB₄ should therefore always take into account a high variability of the quantitative response which is donor and disease dependent.     

Pertussis toxin inhibits the rise in the intracellular concentration of free calcium that is induced by chemotactic factors in rabbit neutrophils: possible role of the "G proteins" in calcium mobilization

The addition of pertussis toxin to rabbit neutrophils inhibits the rise in the intracellular concentration of free calcium induced by the chemotactic factors fMet-Leu-Phe and leukotriene B4. At high concentrations of fMet-Leu-Phe, the inhibitory effect of the toxin is more on the stimulus-induced increase in membrane permeability to calcium than on calcium mobilization from internal stores. These results suggest that the "G protein" system either directly or indirectly is involved in the regulation of the stimulus-induced changes in the calcium mobilization and/or gating systems.     

Leukotriene B4 biosynthesis by alveolar macrophages

Resting alveolar macrophages in culture synthesized small amount of leukotriene B₄. This synthesis was increased 2.5 fold following phagocytic stimulation by zymosan, and was increased 12.6 fold after stimulation with calcium and calcium ionophore A23187. The leukotriene B₄ synthesis could be completely inhibited by nordihydroguaiaretic acid (10^?5M). Phorbol myristate acetate, a membrane perturbant, has no effect on leukotriene B₄ production by macrophages.     

Platelet-activating factor as a stimulus of exocytosis in human neutrophils

The properties of platelet-activating factor (PAF-acether) 42–48ulus of exocytosis in human neutrophils have been re-investigated with particular attention to effects on cells that were not pretreated with cytochalasin B. Release of gelatinase, the most sensitive marker of exocytosis, was determined in addition to that of vitamin B-12-binding protein and β-glucuronidase. Superoxide production was assayed as a measure of the respiratory burst. The effects of PAF-acether were compared to those of leukotriene B₄ and N-formylmethionylleucylphenylalanine (fMet-Leu-Phe). Our results show that PAF-acether elicits marked secretion in untreated human neutrophils, and refute the prevalent view that cytochalasin B treatment is required for responsiveness. PAF-acether induced abundant release of gelatinase, increasing on average from 20% at 10 nM to 35% at 1 μM. This release was very rapid, i.e., almost complete after 2 min. fMet-Leu-Phe induced the same maximum response already at 0.1 μM, but release was considerably slower. Leukotriene B₄ was less potent with a maximum release of 20%. Exocytosis of gelatinase was always paralleled by liberation of smaller but significant amounts of vitamin B₁₂-binding protein from the specific granules. In contrast to their effect on exocytosis, PAF-acether and leukotriene B₄ were very weak stimuli of the respiratory burst when compared with fMet-Leu-Phe.