Cyclical binding,processing, and functional interactions of neutrophils with leukotriene B4

Leukotrene (LT) B₄ activates human polymorphonuclear neutrophils. (PMN) by binding to plasmalemmal receptors. It stimulates PMN to raise cytosolic calcium and degranulate. Both responses end within 15–30 sec. However, in < 15 sec, LTB₄-treated PMN lose the ability to respond further to LTB₄; decrease the affinity and number of high affinity receptors available for binding LTB₄ sequester LTB₄ in plasmalemma-associated sites that are inaccessible to a releasing buffei regi i men; and begin internalizing LTB₄. Over the next 90 min, the cells increasingly internalize LTB₄ and convert it to less potent metabolites; release the metabolites; recover LTB₄ binding sites; and become fully sensitive to LTB₄. Contrastingly, during the entire 90 min incubation with LTB₄. PMN retained the capacity to bind and respond normally to a second stimulus platelet-activating factor. We therefore suggest the following model. LTB₄ receptors, when ligand-bound, initiate function but rapidly lose this capacity as they lower their ligand binding affinity and sequester, internalize, or otherwise uncouple from transducing elements. These LTB₄ receptor changes contribute to terminating PMN responses and producing a stimulus-selective state of desensitization. During the desensitization period, PMN progressively process and metabolize LTB₄. This removes LTB₄ from the environment, thereby allowing PMN to recover functional receptors for and sensitivity to the ligand.     

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     

Synchronous free Ca2+ changes in individual neutrophils stimulated by leukotriene B4.

Calcium (Ca2+) signals were monitored in individual neutrophils using ratio imaging of fura-2. In contrast to N-formyl-L-leucyl-L-phenylalanine (f-met-leu-phe), which produced grossly asynchronous Ca2+ signals with delays in response (up to 60 s), leukotriene B4 (LTB4) provoked synchronous and immediate elevations in cytosolic free Ca2+. Some individual neutrophils which responded immediately to LTB4, subsequently displayed delayed Ca2+ signals in response to f-met-leu-phe. A sub-population of neutrophils failed to respond to both LTB4 and f-met-leu-phe. The asynchrony of the Ca2+ signalling to f-met-leu-phe is not, therefore, an obligatory property of signal transduction in neutrophils.     

The Arabidopsis sensor His-kinase, AHk4, can respond to cytokinins

His-to-Asp (His-->Asp) phosphorelay mechanisms are presumably involved in propagation of certain environmental stimuli, including phytohormones, in Arabidopsis thaliana. In addition to the previously characterized His-kinases, namely, the ETR1 family of ethylene receptors, CKI1 cytokinin-sensor, and ATHK1 osomo-sensor, this higher plant has three more His-kinases (named AHK2, AHK3, and AHK4). By employing the well-known His-->Asp phosphorelay systems in both the fission yeast and Escherichia coli, evidence is presented showing that the AHK4 His-kinase has an ability to serve as a cytokinin-responsive environmental sensor. Taking advantage of this AHK4-dependent His-->Asp phosphorelay system in E. coli, a phosphorelay interaction between the Arabidopsis His-kinase and histidine-containing phosphotransmitters (AHPs) was also demonstrated for the first time.     

Na+/H+ exchange modulates the production of leukotriene B4 by human neutrophils.

Human neutrophils produce various compounds of the 5-lipoxygenase pathway, including (5S)-hydroxyeicosatetraenoic acid, leukotriene B4, its 6-trans isomers and omega-oxidation metabolites of LTB4, when the cells are stimulated with the Ca2+ ionophore A23187. The elevation in the extracellular pH (pHo) facilitated the cytoplasmic alkalinization induced by the ionophore as determined fluorometrically using 2',7'-bis(carboxyethyl)carboxyfluorescein and enhanced the production of all the 5-lipoxygenase metabolites. The production decreased when the alkalinization was blocked by the decrease in the pHo, the removal of the extracellular Na+ or the addition of specific inhibitors of the Na+/H+ exchange, such as 5-(NN-hexamethylene)amiloride, 5-(N-methyl-N-isobutyl)amiloride and 5-(N-ethyl-N-isopropyl)amiloride. The alkalinization of the cytoplasm with methylamine completely restored the production suppressed by the removal of Na+ from the medium. These findings suggest that the change in the cytoplasmic pH (pHi) mediated by the Na+/H+ exchange regulates the production of the lipoxygenase metabolites. The site of the metabolism controlled by the pHi change seemed to be the 5-lipoxygenase, because the production of all the metabolites decreased in parallel and the release of [3H]arachidonic acid from the neutrophils in response to the ionophore was not affected by the pHi change. Furthermore, the production of the 5-lipoxygenase metabolites stimulated by A23187 with or without exogenous arachidonic acid showed a similar pHo-dependence and the production induced by N-formylmethionyl-leucylphenylalanine (chemotactic peptide) with exogenous arachidonic acid also decreased when the cytoplasmic alkalinization was inhibited.     

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.     

The endocannabinoid 2-arachidonoyl-glycerol activates human neutrophils: critical role of its hydrolysis and de novo leukotriene B4 biosynthesis

Although endocannabinoids are important players in nociception and obesity, their roles as immunomodulators remain elusive. The main endocannabinoids described to date, namely 2-arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA), induce an intriguing profile of pro- and anti-inflammatory effects. This could relate to cell-specific cannabinoid receptor expression and/or the action of endocannabinoid-derived metabolites. Importantly, 2-AG and AEA comprise a molecule of arachidonic acid (AA) in their structure and are hydrolyzed rapidly. We postulated the following: 1) the released AA from endocannabinoid hydrolysis would be metabolized into eicosanoids; and 2) these eicosanoids would mediate some of the effects of endocannabinoids. To confirm these hypotheses, experiments were performed in which freshly isolated human neutrophils were treated with endocannabinoids. Unlike AEA, 2-AG stimulated myeloperoxidase release, kinase activation, and calcium mobilization by neutrophils. Although 2-AG did not induce the migration of neutrophils, it induced the release of a migrating activity for neutrophils. 2-AG also rapidly (1 min) induced a robust biosynthesis of leukotrienes, similar to that observed with AA. The effects of 2-AG were not mimicked nor prevented by cannabinoid receptor agonists or antagonists, respectively. Finally, the blockade of either 2-AG hydrolysis, leukotriene (LT) B(4) biosynthesis, or LTB(4) receptor 1 activation prevented all the effects of 2-AG on neutrophil functions. In conclusion, we demonstrated that 2-AG potently activates human neutrophils. This is the consequence of 2-AG hydrolysis, de novo LTB(4) biosynthesis, and an autocrine activation loop involving LTB(4) receptor 1.     

Colchicine inhibits ionophore-induced formation of leukotriene B4 by human neutrophils: the role of microtubules

Neutrophils which ingest particles (serum-treated zymosan, monosodium urate crystals) or are exposed to calcium ionophore A23187 generate leukotriene B4 (LTB4). Earlier work has shown that cells exposed to colchicine before exposure to monosodium urate crystals produce less LTB4; the formation of 5-HETE is unaffected. To determine whether inhibition by colchicine of LTB4 generation was stimulus-specific and was mediated by microtubule integrity, the effects of colchicine (10 microM, 60 min) on the release of lipoxygenase products from neutrophils exposed to ionophore A23187 (10 microM, 5 min) were examined. In the presence of exogenous arachidonic acid (100 microM, 15 min), colchicine decreased LTB4 to 48% +/- 11.7 of control and 5-HETE to 60.5% +/- 5.7 of control (mean +/- SEM); 15-HETE was also decreased to 61% +/- 10.3 of control. In the absence of exogenous arachidonate, LTB4 was decreased to 22.2% +/- 11.7 of control and 5-HETE to 13% +/- 4.8 of control. Lumicolchicine did not significantly affect formation of 5-HETE or LTB4. However, vinblastine sulfate (20 microM, 60 min), another microtubule-disruptive agent, decreased the formation of both 5-lipoxygenase products. The effects of colchicine and vinblastine were not due to impairment of cell viability because the release of cytoplasmic lactic dehydrogenase was unaffected. Ultrastructural analysis of centriolar microtubules showed that decrements in microtubule numbers of colchicine- and vinblastine-treated cells paralleled decrements in 5-lipoxygenase products. These pharmacologic manipulations suggested that functional microtubules might be required for optimal lipoxygenase activity. Consequently, we prepared neutrophil-derived cytoplasts, devoid of an intact microtubule system. No significant decreases in the 5- or 15-lipoxygenase products were found when cytoplasts were exposed to colchicine in the presence of exogenous arachidonate and A23187. The data show that colchicine inhibits the formation of lipoxygenase products from neutrophils stimulated with A23187, most likely via its effect on microtubules, the integrity of which appears necessary for full expression of 5- and 15-lipoxygenases.     

Extracellular signal-regulated kinases regulate leukotriene C4 generation, but not histamine release or IL-4 production from human basophils

Human basophils secrete histamine and leukotriene C4 (LTC4) in response to various stimuli, such as Ag and the bacterial product, FMLP. IgE-mediated stimulation also results in IL-4 secretion. However, the mechanisms of these three classes of secretion are unknown in human basophils. The activation of extracellular signal-regulated kinases (ERKs; ERK-1 and ERK-2) during IgE- and FMLP-mediated stimulation of human basophils was examined. Following FMLP stimulation, histamine release preceded phosphorylation of ERKs, whereas phosphorylation of cytosolic phospholipase A2 (cPLA2), and arachidonic acid (AA) and LTC4 release followed phosphorylation of ERKs. The phosphorylation of ERKs was transient, decreasing to baseline levels after 15 min. PD98059 (MEK inhibitor) inhibited the phosphorylation of ERKs and cPLA2 without inhibition of several other tyrosine phosphorylation events, including phosphorylation of p38 MAPK. PD98059 also inhibited LTC4 generation (IC50 = approximately 2 microM), but not histamine release. Stimulation with anti-IgE Ab resulted in the phosphorylation of ERKs, which was kinetically similar to both histamine and LTC4 release and decreased toward resting levels by 30 min. Similar to FMLP, PD98059 inhibited anti-IgE-mediated LTC4 release (IC50, approximately 2 microM), with only a modest effect on histamine release and IL-4 production at higher concentrations. Taken together, these results suggest that ERKs might selectively regulate the pathway leading to LTC4 generation by phosphorylating cPLA2, but not histamine release or IL-4 production, in human basophils.     

Optimization of assay conditions for leukotriene B4 synthesis by neutrophils or platelets isolated from peripheral blood of monogastric animals

Neutrophils are involved in inflammation through leukotriene (LT) production. The predominant proinflammatory leukotriene released from neutrophils is LTB4, which serves as a biological marker of inflammation. The purpose of this study was to optimize the conditions ex vivo for LTB4 production by neutrophils from horses and dogs, and platelets from chickens. Optimal production of LTB4 was characterized by incubation time (2.5, 5, 10, 15 or 20 min), temperature (25 or 37 degrees C), and calcium ionophore A23187 concentration (0.1, 1, 10 or 20 microM). Incubation longer than 2.5 min did not increase production of LTB4 in chickens or horses; in dogs, incubation for 2.5 and 10 min resulted in the highest concentrations of LTB4 (P相似文献   

Co-localization of leukotriene a4 hydrolase with 5-lipoxygenase in nuclei of alveolar macrophages and rat basophilic leukemia cells but not neutrophils.

The synthesis of leukotriene B(4) from arachidonic acid requires the sequential action of two enzymes: 5-lipoxygenase and leukotriene A(4) hydrolase. 5-Lipoxygenase is known to be present in the cytoplasm of some leukocytes and able to accumulate in the nucleoplasm of others. In this study, we asked if leukotriene A(4) hydrolase co-localizes with 5-lipoxygenase in different types of leukocytes. Examination of rat basophilic leukemia cells by both immunocytochemistry and immunofluorescence revealed that leukotriene A(4) hydrolase, like 5-lipoxygenase, was most abundant in the nucleus, with only minor occurrences in the cytoplasm. The finding of abundant leukotriene A(4) hydrolase in the soluble nuclear fraction was substantiated by two different cell fractionation techniques. Leukotriene A(4) hydrolase was also found to accumulate together with 5-lipoxygenase in the nucleus of alveolar macrophages. This result was obtained using both in situ and ex vivo techniques. In contrast to these results, peripheral blood neutrophils contained both leukotriene A(4) hydrolase and 5-lipoxygenase exclusively in the cytoplasm. After adherence of neutrophils, 5-lipoxygenase was rapidly imported into the nucleus, whereas leukotriene A(4) hydrolase remained cytosolic. Similarly, 5-lipoxygenase was localized in the nucleus of neutrophils recruited into inflamed appendix tissue, whereas leukotriene A(4) hydrolase remained cytosolic. These results demonstrate for the first time that leukotriene A(4) hydrolase can be accumulated in the nucleus, where it co-localizes with 5-lipoxygenase. As with 5-lipoxygenase, the subcellular distribution of leukotriene A(4) hydrolase is cell-specific and dynamic, but differences in the mechanisms regulating nuclear import must exist. The degree to which these two enzymes are co-localized may influence their metabolic coupling in the conversion of arachidonic acid to leukotriene B(4).     

Intracellular retention of the 5-lipoxygenase pathway product, leukotriene B4, by human neutrophils activated with unopsonized zymosan

The cellular and extracellular distribution of leukotriene B4 (LTB4) generated in human neutrophilic polymorphonuclear leukocytes (PMN) stimulated with unopsonized zymosan has been compared with that generated in PMN activated by the calcium ionophore. The amounts of extracellular and intracellular LTB4 were quantitated by radioimmunoassay. The authenticity of the immunoreactive LTB4 was confirmed by the elution of a single immunoreactive peak after reverse phase-high performance liquid chromatography (RP-HPLC) at the retention time of synthetic LTB4, by the identical elution time of a peak of radiolabeled product derived from [3H]arachidonic acid-labeled PMN with the immunoreactive product, and by the comparable chemotactic activity on a weight basis of immunoreactive LTB4 and synthetic LTB4 standard. Under optimal conditions of stimulation by unopsonized zymosan, more than 78% of the generated immunoreactive LTB4 remained intracellular, whereas with optimal activation by the ionophore, less than 8.6% of immunoreactive LTB4 was retained. Resolution by RP-HPLC of the products from the supernatants and cell extracts of [3H]arachidonic acid-labeled PMN stimulated with unopsonized zymosan and those stimulated with calcium ionophore allowed identification and measurement of 5-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-LTB4, LTB4, and omega oxidation products of LTB4 by radioactivity. With zymosan stimulation of PMN, 5-HETE and the 6-trans-LTB4 diastereoisomers were not released, LTB4 was partially released, and the omega oxidation products of LTB4 were preferentially extracellular in distribution. In contrast, with ionophore stimulation, only 5-HETE had any duration of intracellular residence being equally distributed intra- and extracellularly throughout the 30-min period of observation; 6-trans-LTB4, LTB4, and the omega oxidation products of LTB4 were retained at less than 19%. The respective distributions of 5-HETE after zymosan and ionophore stimulation were not altered by the introduction of albumin to the reaction mixtures to prevent reacylation, or by hydrolysis of the cell extract to uncover any product that had been reacylated. The finding that stimulation of PMN with unopsonized zymosan results in the cellular retention of 5-lipoxygenase products suggests that release of these metabolites may be an event that is regulated separately from their generation.     

B2 but not B1 cells can contribute to CD4+ T-cell-mediated clearance of rotavirus in SCID mice

Studies utilizing various immunodeficient mouse models of rotavirus (RV) infection demonstrated significant roles of RV-specific secretory immunoglobulin A (IgA), CD4+ T cells, and CD8+ T cells in the clearance of RV and protection from secondary infection. Secretion of small but detectable amounts of IgA in RV-infected alphabeta T-cell receptor knockout mice (11) and distinctive anatomical localization and physiology of B1 cells suggested that B1 cells might be capable of producing RV-specific intestinal IgA in a T-cell-independent fashion and, therefore, be responsible for ablation of RV shedding. We investigated the role of B1 cells in the resolution of primary RV infection using a SCID mouse model. We found that the adoptive transfer of unseparated peritoneal exudate cells ablates RV shedding and leads to the production of high levels of RV-specific intestinal IgA. In contrast, purified B1 cells do not ablate RV shedding and do not induce a T-cell-independent or T-cell-dependent, RV-specific IgA response but do secrete large amounts of polyclonal (total) intestinal IgA. Cotransfer of mixtures of purified B1 cells and B1-cell-depleted peritoneal exudate cells differing in IgA allotypic markers also demonstrated that B2 cells (B1-cell-depleted peritoneal exudate cells) and not B1 cells produced RV-specific IgA. To our knowledge, this is the first observation that B1 cells are unable to cooperate with CD4+ T cells and produce virus-specific intestinal IgA antibody. We also observed that transferred CD4+ T cells alone are capable of resolving RV shedding, although no IgA is secreted. These data suggest that RV-specific IgA may not be obligatory for RV clearance but may protect from reinfection and that effector CD4+ T cells alone can mediate the resolution of primary RV infection. Reconstitution of RV-infected SCID mice with B1 cells results in the outgrowth of contaminating, donor CD4+ T cells that are unable to clear RV, possibly because their oligoclonal specificities may be ineffective against RV antigens.     

Human natural cytotoxic cell activity: Enhancement by leukotrienes (LT) A4, B4 and D4, but not by stereoisomers of LTB4 or HETES

Natural cytotoxic activity of human lymphocytes was significantly enhanced by incubation with LTA₄, LTB₄ or LTD₄. In contrast, stereoisomers of LTB₄ as well as 5-HETE and 12-HETE were not active. These findings suggest that leukotrienes may play an important role in host defense mechanisms.     

Phospholipase D activation by platelet-activating factor, leukotriene B4, and formyl-methionyl-leucyl-phenylalanine in rabbit neutrophils. Phospholipase D activation is involved in enzyme release.

Lipid chemoattractants, such as platelet-activating factor and leukotriene B4, as well as the peptide chemoattractant FMLP, were found to stimulate [3H]phosphatidic acid ([3H]PA) formation in 1-O-[3H]octadecyl-lyso platelet-activating factor-labeled rabbit neutrophils. The stimulation of [3H]PA formation appears to result from the activation of phospholipase D (PLD), because in the presence of ethanol, chemoattractant stimulation produced [3H]phosphatidylethanol, the characteristic compound produced by PLD at the expense of [3H]PA formation. The PLD activation by all chemoattractants tested was primed by cytochalasin B and revealed a similar time dependence. However, lipid chemoattractants were less potent as compared with FMLP, and the maximal stimulation by the former was lower than that by the latter. From these results, it is concluded that the mechanism of PLD activation by lipid chemoattractants is similar to, but different from, that by FMLP. Cytochalasin B stimulated degranulation and [3H]PA formation in agonist-stimulated neutrophils, and their stimulations were well correlated. Ethanol inhibited both agonist-stimulated [3H]PA formation and degranulation in a concentration-dependent manner, but the inhibition in degranulation was much less than that in [3H]PA formation. These results suggest that PLD activation is involved in degranulation, but another signaling pathway may also be required for full stimulation of degranulation. When the radiolabeled neutrophils were stimulated by chemoattractants for 5 min, 1,2-[3H]diglyceride was found to accumulate. The accumulation was inhibited by either ethanol or the phosphatidate phosphohydrolase inhibitor propranolol, which indicates that PA produced by PLD can be converted to 1,2-diglyceride by phosphatidate phosphohydrolase. Under these conditions, propranolol did not inhibit degranulation stimulated by chemoattractants. These results indicate that PA produced by PLD is more important than its metabolite diglyceride for the degranulation of rabbit neutrophils.     

Transepithelial migration of neutrophils in response to leukotriene B4 is mediated by a reactive oxygen species-extracellular signal-regulated kinase-linked cascade

The epithelial cells that form a barrier lining the lung airway are key regulators of neutrophil trafficking into the airway lumen in a variety of lung inflammatory diseases. Although the lipid mediator leukotriene B(4) (LTB(4)) is known to be a principal chemoattractant for recruiting neutrophils to inflamed sites across the airway epithelium, the precise signaling mechanism involved remains largely unknown. In the present study, therefore, we investigated the signaling pathway through which LTB(4) induces transepithelial migration of neutrophils. We found that LTB(4) induces concentration-dependent transmigration of DMSO-differentiated HL-60 neutrophils and human polymorphonuclear neutrophils across A549 human lung epithelium. This effect was mediated via specific LTB(4) receptors and was inhibited by pretreating the cells with N-acetylcysteine (NAC), an oxygen free radical scavenger, with diphenylene iodonium (DPI), an inhibitor of NADPH oxidase-like flavoproteins, or with PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. Consistent with those findings, LTB(4)-induced ERK phosphorylation was completely blocked by pretreating cells with NAC or DPI. Taken together, our observations suggest LTB(4) signaling to transepithelial migration is mediated via generation of reactive oxygen species, which leads to downstream activation of ERK. The physiological relevance of this signaling pathway was demonstrated in BALB/c mice, in which intratracheal instillation of LTB(4) led to acute recruitment of neutrophils into the airway across the lung epithelium. Notably, the response to LTB(4) was blocked by NAC, DPI, PD98059, or CP105696, a specific LTB(4) receptor antagonist.     

IL-4, but not IL-5, can act synergistically with B cell activating factor (BCAF) to induce proliferation of resting B cells

B cell activating factor (BCAF) was initially identified in the supernatant of the murine T helper cell clone 52-3 (52-3 SN) because of its ability to promote activation and proliferation of resting B cells in the absence of any other costimulus. In this paper, we show that 52-3 T helper cells also secrete IL-4 and IL-5 and we have analyzed the influence of these two lymphokines on B cell proliferation induced by BCAF-containing 52-3 SN. Using the neutralizing anti-IL-4 monoclonal antibody 11B11, we observed partial inhibition of B cell proliferation. 52-3 SN free of IL-4 prepared using an immunoabsorbent column was still able to induce significant B cell proliferation. Although recombinant IL-4 alone does not induce B cell proliferation, it increased the proliferation induced by IL-4-free 52-3 SN. Kinetic studies showed that IL-4 is required at the start of B cell cultures in order to exert optimal synergistic effects. In contrast, anti-IL-5 monoclonal antibody NC17 did not affect the B cell proliferative activity of 52-3 SN whether or not IL-4 was present. When 52-3 SN was tested on dextran-sulfate-activated B cells, IL-5 and BCAF activities were detected but only the IL-5 activity was neutralized by monoclonal antibody NC17. These results demonstrate that (i) BCAF-containing SN can induce proliferation of resting B cells independently of IL-4 and IL-5, and (ii) IL-4, but not IL-5, can act synergistically with BCAF to induce B cell proliferation.     

Release of adenosine from human neutrophils stimulated by platelet activating factor, leukotriene B(4) and opsonized zymosan

Isolated human polymorphonuclear leukocytes (PMNL) stimulated by platelet activating factor (PAF), leukotriene B(4) (LTB(4)) or opsonized zymosan (OZ) released adenosine measured by thermospray high performance liquid chromatography mass spectrometry in the cell-free supernatants. Stimulation by PAF or LTB(4) resulted in a bellshaped concentration-effect curve; 5 x 10(-7) M PAF, 10(-8) M LTB(4) and 500 mug ml(-1) OZ induced peak adenosine release, thus cytotoxic concentrations did not elevate adenosine level in the supernatants. Therefore adenosine release was characteristic of viable cells. As calculated from concentration-effect curves, the rank order of potency for adenosine release was PAF > LTB > OZ. These resuits suggest that adenosine, when bound specifically to membrane receptor sites, may initiate signal transduction, and, in co-operation with other inflammatory mediators, may modulate phagocyte function, e.g. production of chemoluminescence (CL).     

Modulation of the heterogeneous membrane potential response of neutrophils to N-formyl-methionyl-leucyl-phenylalanine (FMLP) by leukotriene B4: evidence for cell recruitment

Individual human neutrophils (PMN) isolated by Hypaque-Ficoll gradient sedimentation, dextran sedimentation, or buffy coat preparation were assessed for the effects of leukotriene B4 (5S,12R dihydroxy 6,14-cis-8, 10 trans eicosatetraenoic acid (LTB4)-pretreatment on N-formylmethionyl-leucyl-phenylalanine (FMLP)-mediated membrane potential or oxidative responses by using flow cytometry and a lipophilic probe of membrane potential (di-pentyl-oxacarbocyanine, di-O-C(5)3), or the nitroblue tetrazolium dye (NBT) reduction test, respectively. Although exposure to LTB4 (10(-7) M) had no effect on the membrane potential of resting PMN and little effect on oxidant production, pretreating PMN with LTB4 followed by FMLP (10(-6) M) demonstrated a significant enhancement in the proportion of depolarizing PMN over that seen with FMLP alone (p = 0.0014, N = 9). This recruitment of previously unresponsive cells by LTB4 was dose and time dependent, with the maximal relative increase in the proportion of depolarizing cells occurring at LTB4 concentrations of 10(-8) to 10(-7) M and within 1 min of LTB4 addition. The recruitment effect persisted despite vigorous washing of the cells. LTB4 also increased the proportion of NBT-positive PMN in response to FMLP. Although LTB4 alone did not depolarize PMN it did induce a light scatter shift indicative of cell activation. 3H-FMLP binding studied at 0 degree C comparing buffer and LTB4-treated PMN indicated no significant change in the number or affinity of FMLP binding. The data provide evidence for the recruitment of a greater proportion of cells into a FMLP-responsive state as a mechanism for the enhanced functional response of PMN pretreated with LTB4, as well as for a dissociation of the membrane potential and light scattering responses of cells to this pro-inflammatory LT. The mechanism of recruitment remains unclear, but it most likely involves the modulation of a post-FMLP binding step.     

Biosynthesis of 20,20,20-trifluoroleukotriene B4 from 20,20,20-trifluoroarachidonic acid: a metabolically stable analog of leukotriene B4 and its application to a study of stimulation of leukotriene B4 synthesis by immunoglobulin G1,2

Leukotriene B4 is rapidly metabolized through omega-oxidation, preventing its detection when it is produced under certain biological conditions. To investigate leukotriene B4 production in various physiological conditions, analogs of arachidonic acid which are converted to metabolically stable analogs of leukotriene B4 would be useful. We have synthesized 20,20,20-trifluoroarachidonic acid by the cis-selective Wittig reaction of the C12-C20 fragment with phosphonium salt. 20,20,20-trifluoroarachidonic acid was transformed into 20,20,20-trifluoroleukotriene B4 when incubated with human neutrophils in the presence of the calcium ionophore A23187. The product was identified by uv absorption spectrophotometry, gas chromatography-mass spectrometry, and coelution on high-performance liquid chromatography with 20,20,20-trifluoroleukotriene B4, which was enantioselectively synthesized by the reaction of the fluorine-containing C11-C20 fragment with the C1-C10 phosphonate. The fluorinated leukotriene B4 demonstrated as much chemotactic activity on human neutrophils as natural leukotriene B4 and was metabolically stable when incubated with human neutrophils, probably by blocking omega-oxidation. Also, enzymes catalyzing the transformation of arachidonic acid (AA) into leukotriene B4 did not discriminate the fluorinated precursors from the natural, nonfluorinated AA, thus 20-F3-AA is a valid analog of AA to be used in the study of AA metabolism. When 50 microM of the fluorinated acid was incubated with neutrophils stimulated with heat-aggregated human immunoglobulin G, a significant amount of fluorinated leukotriene B4 (4.3 ng/10(6) cells/40 min, at most) was formed in a dose-dependent manner while little leukotriene B4 was detected with incubation with 50 microM arachidonic acid, probably due to omega-oxidation of the product, leukotriene B4. 20,20,20-Trifluoroarachidonic acid appears to be a useful tool for studying the capacity of leukotriene B4 synthesis in various biological systems while long-lasting 20,20,20-trifluoroleukotriene B4 would serve as an excellent analog of leukotriene B4 in pharmacological studies to understand functions of leukotrienes B4.