Enhanced generation of leukotriene B4 and superoxide radical from calcium ionophore (A23187) stimulated human neutrophils after priming with interferon-alpha

The present study was designed to investigate the effect of interferon-alpha ( INF-alpha) on the production of leukotrienes (LTs) and superoxide radicals when intact human neutrophils were stimulated with calcium ionophore (A23187). A reverse phase high performance liquid chromatography and UV spectroscopy were employed to detect and quantitate the released LTs; namely LTC4, LTB4 and its trans isomers, 6-trans LTB4 and 12-epi-6-trans LTB4. Preincubation of intact human neutrophils at 37 degrees C for 30 min with INF-alpha and stimulation with calcium ionophore A23187 for 1 min enhanced significantly the formation of LTB4. Preincubation of intact human neutrophils with INF-alpha and subsequent stimulation with calcium ionophore A23187 also enhanced significantly superoxide radical generation that reduced nitroblue tetrazolium into blue formazan. The in vivo effect of INF-alpha in rats demonstrated that the higher dose of INF-alpha that induced superoxide radical and LTB4 by A23187 stimulated intact human neutrophil in vitro, also induced a significant decrease in white blood cells and RBCs started at 4 h after i.p administration. The differential white blood cell count revealed that, the prime target for INF-alpha is the white blood cells of myeloid origin. These results might demonstrate the modulatory effects of INF-alpha on granulocyte functions.     

Inhibition of calcium ionophore-stimulated leukotriene generation from intact human neutrophils by captopril

Effects of captopril on the formation of leukotrienes (LTs) from stimulated intact human neutrophils were investigated. Neutrophils were stimulated with 1 microM calcium ionophore A23187 for the generation of LTs. A reverse phase high performance liquid chromatography technique and UV spectroscopy were used to detect and quantitate the released LTs namely, LTB4. LTC4. delta6-trans-LTB4 and delta6-trans-12-epi-LTB4. Preincubation of neutrophils with captopril significantly reduced LTB4 formation in a concentration-dependent manner, as compared to diluent-treated control cells. Since LTA4 is the substrate for both LTB4 and LTC4, thus in presence of captopril, shunting of LTA4 from synthesis of LTB4 was not directed to LTC4 formation. This finding was evidenced by the significant decrease of LTC4 production under the influence of high concentration of the drug. Formation of LTB4 stereoisomers, delta6-trans-LTB4 and delta6-trans-12-epi-LTB4 was not markedly altered by captopril. In subsequent experiments, when neutrophils were stimulated with A23187 after preincubation with exogenous arachidonic acid (75 microM), and treatment with captopril, similar findings were obtained for LTB4 and LTC4. Meanwhile, formation of the nonenzymatic hydrolysis products of LTA4 tended to rise reaching significant level in case of delta6-trans-LTB4, at the high concentration of captopril. These results demonstrate that captopril is an inhibitor of enzymatically generated LTs produced by intact human neutrophils, being more potent against LTB4. These effects of captopril on LTs are not mediated via an inhibition of arachidonic acid formation from membrane phospholipids. It could be suggested that captopril, at doses used clinically. could inhibit the generation of LTB4 without affecting LTC4. Consequently, these findings might account for possible antiinflammatory activity for captopril, and further suggest that some of the observed side effects of captopril might not be related to an overproduction of LTC4.     

Leukotriene B4 mediates p47phox phosphorylation and membrane translocation in polyunsaturated fatty acid-stimulated neutrophils

Polyunsaturated fatty acids (PUFAs) and leukotriene B(4) (LTB(4)) are involved in many inflammatory and physiological conditions. The role of arachidonic acid (AA) and linoleic acid (LA) in promoting the assembly of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits is well known, but the involvement of LTB(4) and other 5-lipoxygenase (5-LO) pathway metabolites of AA in hydrogen peroxide (H(2)O(2)) production by PUFA-stimulated polymorphonuclear leukocytes (PMNs) has not been investigated. We examined this question by determining H(2)O(2) production as well as phosphorylation and membrane translocation of the p47phox subunit of NADPH oxidase. Elicited peritoneal PMNs from rats and from 5-LO-deficient or wild-type mice were pretreated with or without inhibitors of LT biosynthesis and antagonists of the receptors for LTB(4) and cysteinyl LTs for 20 min before stimulation with AA (at 5 and 20 microM) or LA (at 20 microM). PUFAs elicited H(2)O(2) production in a dose-dependent manner, and pharmacologic or genetic inhibition of LT synthesis decreased H(2)O(2) production by approximately 40% when compared with untreated controls. LTB(4) was the moiety responsible for H(2)O(2) production, as revealed by studies using receptor antagonists and its exogenous addition. LTB(4) itself also promoted p47phox phosphorylation and translocation. These results identify a heretofore unrecognized role for activation of 5-LO and subsequent production of LTB(4) in stimulation of PMN NADPH oxidase activation by PUFAs.     

Leukotrienes, LTC4 and LTB4, in bronchoalveolar lavage in bronchial asthma and other respiratory diseases

Leukotrienes (LTs) C4 and B4 are potent proinflammatory mediators with a wide variety of biologic activities, including smooth muscle contraction, mucus hypersecretion, and leukocyte activation, which may be of particular relevance to the pathology of asthma and other respiratory diseases. We measured the concentrations of LTC4 and LTB4 in bronchoalveolar lavage fluid from 16 atopic subjects with asthma (eight symptomatic and eight asymptomatic) and from 14 control subjects without asthma (six with hay fever and eight nonatopic). The amounts detected in symptomatic subjects with asthma were significantly higher than in control subjects (LTB4, 0.58 +/- 0.06 versus 0.36 +/- 0.05 pmol/ml, p less than 0.05; LTC4, 0.36 +/- 0.1 versus 0.12 +/- 0.02 pmol/ml, p less than 0.01). LTC4 and LTB4 were also measured in 17 patients: nine with interstitial lung disease of varying etiology (cryptogenic fibrosing alveolitis [CFA] or idiopathic pulmonary fibrosis), three with sarcoidosis, one with extrinsic allergic alveolitis, one with sulphonamide-induced pneumonia, and one patient with eosinophilic granuloma. The concentrations of LTB4 (but not LTC4) were significantly greater in patients with CFA compared with normal control subjects (0.69 +/- 0.3 versus 0.36 +/- 0.05 pmol/ml, p less than 0.01). There was a significant correlation (p less than 0.05) between the percentage of neutrophils and the concentration of LTB4 in the bronchoalveolar lavage fluid) of the group with interstitial lung disease as a whole. This study provides evidence for a role for LTs in the airways of subjects with day-to-day asthma and suggests that LTB4 may also be involved in the recruitment of granulocytes into the lung in patients with CFA.     

Effects of Leukotrienes on Neutrophil Migration, and on Production and Action of Lymphokines

The effect of the leukotrienes LTB4, LTC4 and LTD4 on the production of the lymphokine leukocyte migration inhibitory factor (LIF) from human peripheral blood mononuclear cells was tested. LTC4 and LTD4 failed to influence LIF production by cells stimulated by antigen or the polyclonal T cell activator, phytohemagglutinin (PHA). LTB4, at concentrations 10(-7) to 10(-6) M, showed significant inhibition of antigen-induced LIF production but was without effect when cells were stimulated by PHA. The cell levels of cyclic adenosine monophosphate (cAMP) were unaffected by 2.5 X 10(-7) M LTB4 in the absence or presence of theophylline. These results indicate that LTB4 acts on the early effector stage of the immune response without affecting the cellular cAMP content.     

Inhibition of leukotriene C4 and B4 generation by human eosinophils and neutrophils with the lipoxygenase pathway inhibitors U60257 and BW755C

Human eosinophils and neutrophils have the capacity to generate leukotriene C4 (LTC4) and leukotriene B4 (LTB4) respectively when stimulated by calcium ionophore A23187. Leukotriene production by mixtures of these cell types was measured by radioimmunoassay for LTC4 and LTB4, and the specificities of the assays determined by assessing cross-reactivities with a number of other arachidonic acid metabolites. The IC50S for LTC4 and LTB4 in their respective assays were 1.76 +/- 0.04 nmol and 3.00 +/- 0.08 nmol. Cross-reactivity for anti-LTC4 was shown by leukotriene D4 (LTD4) (70%) and leukotriene E4 (LTE4) (8%), when compared to LTC4, whereas in the radioimmunoassay for LTB4, only the 5(S), 12(R) 6-trans isomer of LTB4 showed appreciable interaction (12%). LTC4 production by eosinophil enriched cell fractions obtained from metrizamide gradients was inhibited in a dose-dependent fashion by the prostacyclin analogue, 6,9-deepoxy-6,9-phenylimino-delta 6,8-prostaglandin I, (U60257) and by 3-amino-1-(3-trifluoromethyl phenyl)-2-pyrazole (BW755C). The ID50 values for U60257 and BW755C were 2 X 10(-6) and 5 X 10(-6) M respectively. This demonstration of LTC4 production by human eosinophils, which are known to be important cells in clinical asthma, provides an in vitro model to assess 5-lipoxygenase inhibitors in human tissue.     

Increase in urinary leukotriene B4 glucuronide concentration in patients with aspirin-intolerant asthma after intravenous aspirin challenge

BACKGROUND: Aspirin challenge of aspirin-intolerant asthma (AIA) patients causes a significant increase in leukotriene E4 (LTE4) concentration in urine. However, knowledge on leukotriene B4 (LTB4) generation in patients with AIA is insufficient. Recent research has demonstrated that exogenously administered LTB4 is excreted as glucuronide into the urine in human healthy subjects. OBJECTIVE: The purpose of this study is to estimate urinary LTB4 glucuronide (LTBG) concentration in the clinically stable condition in healthy subjects and asthmatic patients and to investigate changes in urinary LTBG concentration in patients with AIA after aspirin challenge. METHODS: A provocation test was performed by intravenous aspirin challenge. After urine was hydrolysed by beta-glucuronidase, the fraction containing LTB4 was purified by high-performance liquid chromatography and LTB4 concentration was quantified by enzyme immunoassay. Urinary LTBG concentration was calculated as the difference between the concentration obtained with hydrolysis and that without hydrolysis. RESULTS: (1) After hydrolysis, the presence of urinary LTB4 was verified by gas chromatography-mass spectrometry-selected ion monitoring. (2) The urinary LTBG concentration was significantly higher in the asthmatic patients than in the healthy subjects (median, 5.37 pg/mg creatinine [range 1.2-13] vs. 3.32 pg/mg creatinine [range, 0.14-10.5], P = 0.0159). (3) The patients with AIA (n = 7), but not those with aspirin-tolerant asthma (n = 6), showed significant increases in LTBG and LTE4 excretions after aspirin challenge. (4) When the concentrations after aspirin challenge were analysed simultaneously, a significant linear correlation was observed between urinary LTBG concentration and urinary LTE4 concentration in patients with AIA (Spearman's rank correlation test, r = 0.817, P = 0.0003). CONCLUSION: LTBG is present in human urine, albeit at a concentration lower than urinary LTE4. In addition to a marked increase in cysteinyl-leukotriene production, aspirin challenge induced LTB4 production in AIA patients.     

Up-regulation of prostaglandin biosynthesis by leukotriene C4 in elicited mice peritoneal macrophages activated with lipopolysaccharide/interferon-{gamma}

Leukotrienes (LT) and prostaglandins (PG) are proinflammatory mediators generated by the conversion of arachidonic acid via 5-lipoxygenase (5-LO) and cyclooxygenase (COX) pathways. It has long been proposed that the inhibition of the 5-LO could enhance the COX pathway leading to an increased PG generation. We have found that in in vitro models of inflammation, such as mice-elicited peritoneal macrophages activated with lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma), the deletion of the gene encoding for 5-LO or the enzyme activity inhibition corresponded to a negative modulation of the COX pathway. Moreover, exogenously added LTC(4), but not LTD(4), LTE(4), and LTB(4), was able to increase PG production in stimulated cells from 5-LO wild-type and knockout mice. LTC(4) was not able to induce COX-2 expression by itself but rather potentiated the action of LPS/IFN-gamma through the extracellular signal-regulated kinase-1/2 activation, as demonstrated by the use of a specific mitogen-activated protein kinase (MAPK) kinase inhibitor. The LT-induced increase in PG generation, as well as MAPK activation, was dependent by a specific ligand-receptor interaction, as demonstrated by the use of a cys-LT1 receptor antagonist, although also a direct action of the antagonist used, on PG generation, cannot be excluded. Thus, the balance between COX and 5-LO metabolites could be of great importance in controlling macrophage functions and consequently, inflammation and tumor promotion.     

Endogenous arachidonic acid metabolism by calcium ionophore A23187-stimulated lamb lungs: effect of hypoxia

We have determined eicosanoid production from endogenous arachidonic acid by neonatal lamb lungs stimulated with calcium ionophore A23187 during normoxia and hypoxia. Lungs of lambs 19 to 25 d of age were isolated and perfused with cell-free Krebs' bicarbonate buffer at a flow rate of 15 to 20 ml/kg/min. After 30 min of equilibration in a recirculating system, A23187 was added to the perfusate in a 5-microM concentration and perfusion continued for 15 min more. Eicosanoids were measured in perfusate and lung homogenate supernatant. Cyclooxygenase metabolites prostaglandin (PG) E2, thromboxane A2, and PGI2, were measured by radioimmunoassay, and 5-lipoxygenase metabolites leukotrienes (LT) B4, C4, D4, and E4 by high performance liquid chromatography. During normoxia, all three cyclooxygenase metabolites were present in perfusate, but only PGI2 and thromboxane A2 were present in lung homogenate supernatant. Prostacyclin constituted 50% of all the cyclooxygenase products measured. LTC4 and LTD4 were detected in both perfusate and lung homogenate supernatant with little production of LTE4 and LTB4. During hypoxia, the profile of cyclooxygenase products was unchanged and prostacyclin production was not increased. However, the profile of leukotriene metabolites was altered. LTC4 synthesis was markedly reduced. The synthesis of LTE4 and LTB4 was increased 10-fold, with most of the leukotrienes being retained in lung tissue. We conclude that hypoxia significantly alters leukotriene metabolism of endogenous arachidonic acid by calcium ionophore-stimulated lungs. The increased production by stimulated lungs during hypoxia of LTE4, a substance that may increase lung capillary permeability, and that of LTB4, a powerful chemoattractant, may be important contributing factors to lung injury.     

(9-[4-acetyl-3-hydroxy-2-n-propylphenoxy) methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido [1,2-a] pyrimidin-4-one), AS-35, inhibits leukotriene synthesis

AS-35, (9-[4-acetyl-3-hydroxy-2-n-propylphenoxy) methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido[1, 2-a] pyrimidin-4-one), was developed as a leukotriene (LT) receptor antagonist, which also inhibited IgE-mediated release of leukotrienes (LTs). We have investigated the action of AS-35 on the enzyme activities which are involved in the synthesis of LTC(4) and LTB(4) (LT-synthesizing enzymes); cytosolic phospholipase A(2) (cPLA(2)), 5-lipoxygenase (5-LO), leukotriene (LT)C(4) synthase and LTA(4) hydrolase. AS-35 dose-dependently inhibited IgE- and A23187-stimulated production of LTC(4) by up to 71.5-84.8% and that of LTB(4) by 48.3-49.2% at 2. 5x10(-5) M. The assays for cPLA(2)(-), 5-LO-, LTC(4) synthase- and LTA(4) hydrolase-activities revealed that the inhibition is attributable to suppression of cPLA(2), 5-LO and LTC(4) synthase but not LTA(4) hydrolase. We have also studied the action of AS-35 on the release of beta-hexosaminidase (beta-HEX) as a marker of preformed mediators. AS-35 had only weak inhibitory action on the release of beta-HEX. The results indicate that anti-allergic action of AS-35 is predominantly attributable to its inhibition of LT synthesis by suppressing three consecutive enzymes for LTC(4) synthesis.     

Genistein, a selective protein tyrosine kinase inhibitor, inhibits interleukin-2 and leukotriene B4 production from human mononuclear cells.

In this study, genistein, a selective protein tyrosine kinase (PTK) inhibitor, inhibited peripheral blood mononuclear cell (PBMC) proliferation and interleukin-2 production from cultures that were stimulated with phytohemagglutinin (PHA), phorbol 12-myristate 13-acetate (PMA) plus A23187, or PHA plus PMA, and genistein effectively blocked the PHA plus IL-2-induced PBMC proliferation. Further, we also found that genistein inhibited LTB4 production from A23187-stimulated cultures whereas H-7, a PKC inhibitor, had no effect on LTB4 production. Our results suggest that PTK may be necessary for the synthesis of LTB4.     

Inhibitory effect of amlexanox (AA-673) on the immunological and non-immunological release of histamine or leukotrienes

The effect of amlexanox on the non-immunological or immunological release of histamine or leukotrienes (LTs) from passively sensitized human lung fragments and atopic human leukocytes was investigated and compared with those of AA-861, tranilast, azelastine and disodium cromoglycate. 1) Amlexanox at concentrations of 10(-7)-10(-4) M showed an inhibition of histamine, LTB4, LTC4, LTD4 and LTE4 release from passively sensitized human lung fragments in a concentration-dependent fashion. A selective and competitive inhibitor of the 5-lipoxygenase activity, AA-861 modestly affected the histamine release and potently suppressed the any LT release at 10(-7) and 10(-6) M. Antiallergic drugs, tranilast and disodium cromoglycate also suppressed these chemical mediator release, but the inhibition potency was somewhat weaker than that of amlexanox. 2) Ca ionophore A23187-induced release of LTB4 and LTC4 from atopic human leukocytes was slightly enhanced up to 10(-6) M of amlexanox. However, 10(-4) M of the drug strongly diminished both of LT release. From these results, it is suggested that amlexanox is a clinically effective drug for atopic diseases, especially allergic asthma and rhinitis.     

Clinical evaluation of urinary leukotriene E4 levels in children with atopic dermatitis]

To evaluate the significance of peptide leukotrienes in children with atopic dermatitis, we measured urinary LTE4 levels which are thought to reflect in vivo production of peptide LTs. Urine was collected in the early morning. There was no significant difference in urinary LTE4 levels among atopic children with different severities. On the other hand, urinary LTE4 levels were significantly elevated in the patients who had severe nocturnal itches compared with the patients who had mild nocturnal itches and normal controls. These results suggested that increased production of peptide LTs may be relevant to nocturnal itch of atopic dermatitis.     

Analysis of exhaled leukotrienes in nonasthmatic adult patients with seasonal allergic rhinitis

BACKGROUND: Leukotrienes (LTs) are increased in exhaled breath condensate (EBC) in patients with asthma. So far no data have been reported about LT levels in nonasthmatic patients with seasonal allergic rhinitis (SAR). The aim of the study was to find out whether the LT levels in EBC were increased in the nonasthmatic adult patients with SAR both during and after the pollen season in comparison with healthy controls and to assess the changes of the LT levels after the pollen season. METHODS: Twenty-nine nonasthmatic adult patients with SAR underwent measurement of exhaled LTs in the EBC during and after the pollen season. Leukotrienes B(4), C(4), D(4) and E(4) were analysed by a specific and sensitive gas chromatography/mass spectrometry (GC/MS) assay and compared with 50 healthy nonsmoking controls. Spirometry, skin prick tests and nonspecific IgE were evaluated. RESULTS: Leukotrienes concentrations (B(4), E(4) but not D(4)) were significantly increased in and after the pollen season in patients with SAR in comparison with healthy controls. In most of the samples, LT C(4) was undetectable. The values of all exhaled LTs were significantly decreased after the pollen season compared with the seasonal baseline: LTB(4) (P = 0.023), LTD(4) (P = 0.020), LTE(4) (P = 0.047). CONCLUSIONS: Levels of exhaled LTB(4) and LTE(4) were higher in SAR patients than in healthy controls and decreased after the pollen season as compared with levels in season. The SAR patients with the highest in season LT levels had also the post-season levels elevated and this may be an early marker of inflammatory process in the lower airways despite the absence of clinical symptoms of asthma.     

Urinary leukotriene E4 in Henoch–Schonlein purpura

BACKGROUND: Children with Henoch-Schonlein purpura (HSP) occasionally have allergic disease. We have previously shown that pranlukast hydrate was effective for purpura in HSP. Pranlukast hydrate is a leukotriene (LT) receptor antagonist; therefore, it is likely that LTs take part in the cause of HSP. Urinary leukotriene E4 (LTE4u) levels are a useful index of whole-body cysteinyl LT production in vivo. In this study, LTE4u was examined in children with HSP. OBJECTIVE: The purpose of this study was to examine the relation between the level of LTE4u and the cause of HSP. METHODS: Eighteen HSP children (six boys and 12 girls) and six healthy children were enrolled. RESULTS: LTE4u levels in patients with HSP were significantly higher (P< 0.05) at the onset than those in healthy children. Four weeks therapy with pranlukast hydrate lowers LTE4u levels in patients with HSP (P< 0.05). There were no differences in LTE4u between the group of HSP patients with purpura nephritis and the group of HSP patients without purpura nephritis. CONCLUSION: These results indicate that csyteinyl LTs may play a role in the pathophysiology of purpura in HSP.     

Involvement of LTB4 in zymosan-induced joint nociception in mice: participation of neutrophils and PGE2

Leukotriene B4 (LTB4) mediates different inflammatory events such as neutrophil migration and pain. The present study addressed the mechanisms of LTB4-mediated joint inflammation-induced hypernociception. It was observed that zymosan-induced articular hypernociception and neutrophil migration were reduced dose-dependently by the pretreatment with MK886 (1-9 mg/kg; LT synthesis inhibitor) as well as in 5-lypoxygenase-deficient mice (5LO(-/-)) or by the selective antagonist of the LTB(4) receptor (CP105696; 3 mg/kg). Histological analysis showed reduced zymosan-induced articular inflammatory damage in 5LO(-/-) mice. The hypernociceptive role of LTB4 was confirmed further by the demonstration that joint injection of LTB4 induces a dose (8.3, 25, and 75 ng)-dependent articular hypernociception. Furthermore, zymosan induced an increase in joint LTB4 production. Investigating the mechanism underlying LTB4 mediation of zymosan-induced hypernociception, LTB4-induced hypernociception was reduced by indomethacin (5 mg/kg), MK886 (3 mg/kg), celecoxib (10 mg/kg), antineutrophil antibody (100 mug, two doses), and fucoidan (20 mg/kg) treatments as well as in 5LO(-/-) mice. The production of LTB4 induced by zymosan in the joint was reduced by the pretreatment with fucoidan or antineutrophil antibody as well as the production of PGE2 induced by LTB4. Therefore, besides reinforcing the role of endogenous LTB4 as an important mediator of inflamed joint hypernociception, these results also suggested that the mechanism of LTB4-induced articular hypernociception depends on prostanoid and neutrophil recruitment. Furthermore, the results also demonstrated clearly that LTB4-induced hypernociception depends on the additional release of endogenous LTs. Concluding, targeting LTB4 synthesis/action might constitute useful therapeutic approaches to inhibit articular inflammatory hypernociception.     

Leukotriene generation and metabolism in isolated human lung macrophages.

We studied the generation and metabolism of leukotrienes (LTs) in human lung macrophages obtained from lung tissue of patients with central bronchial carcinoma. By counterflow centrifugation macrophages were enriched with a purity of more than 95-100%. A time and dose dependent generation of LTB4 and LTC4 was determined by specific radioimmunoassays after stimulation with the Ca-ionophore and anti-IgE. The amount of LTB4 exceeded the amount of LTC4. The concentrations of leukotrienes in the macrophage fraction amounted to 4.3 +/- 2.2 ng LTB4 and 0.6 +/- 0.05 ng LTC4/1 x 10(7) cells after 5 min of incubation with the Ca-ionophore. The LTB4 levels decreased to 3.0 +/- 0.6 ng after 60 min indicating the metabolism of the generated LTB4 by human lung macrophages. This was confirmed by incubation of the cells with exogenously added [3H]LTB4. LTB4 was converted into unpolar products as was identified by thin-layer chromatography and high-performance liquid chromatography; a comparison with the fibroblast cell line L929 which is known to convert LTB4 into the dihydro-LTB4 metabolite (5,12-dihydroxyeicosatrienoic acid) indicates that human lung macrophages use the same pathway of metabolization. Biological inactivation as determined by chemotaxis and cross-reaction with the LTB4 antiserum correlates with the degree of LTB4 metabolism. Moreover, the macrophages convert LTC4 into LTD4 and LTE4 by the enzymatic activity of the gamma-glutamyltranspeptidase and dipeptidase. Our data emphasize that the human alveolar macrophage not only produces arachidonic acid metabolites but modulates the local inflammatory potential by its metabolizing capacity for leukotrienes.     

Human bronchial epithelial cells express an active and inducible biosynthetic pathway for leukotrienes B4 and C4

BACKGROUND: Human bronchial epithelial cells synthesize cyclooxygenase and 15-lipoxygenase products, but the 5-lipoxygenase (5-LO) pathway that generates the leukotriene (LT) family of bronchoconstrictor and pro-inflammatory mediators is thought to be restricted to leucocytes. OBJECTIVE: We hypothesized that human bronchial epithelial cells (HBECs) express a complete and active 5-LO pathway for the synthesis of LTB4 and LTC4, either constitutively or after stimulation. METHODS: Flow cytometry, RT-PCR, Western blotting, enzyme immunoassays and reverse-phase high-performance liquid chromatography were used to investigate constitutive and stimulated expression of 5-LO pathway enzymes and the synthesis of LTs B4 and C4 in primary HBECs and in the 16-HBE 14o- cell line. RESULTS: Constitutive mRNA and protein expression for 5-LO, 5-LO-activating protein (FLAP), LTA4 hydrolase and LTC4 synthase were demonstrated in primary HBECs and in the 16-HBE 14o- cell line. In 16-HBE 14o- cells, treatment with calcium ionophore A23187, bradykinin or LPS up-regulated the expression of these enzymes. The up-regulation of 5-LO was blocked by the anti-inflammatory glucocorticoid dexamethasone. Human bronchial epithelial cells were shown to generate bioactive LTs, with primary HBECs generating 11-fold more LTC4 and five-fold more LTB4 than 16-HBE 14o- cells. LT production was enhanced by ionophore treatment and blocked by the FLAP inhibitor MK-886. CONCLUSIONS: Expression of an active and inducible 5-LO pathway in HBEC suggests that damaged or inflamed bronchial epithelium may synthesize LTs that contribute directly to bronchoconstriction and leucocytosis in airway inflammation.     

Independent regulation of leukotriene B4-elicited polymorphonuclear leukocyte exocytosis and superoxide generation by a serum factor

Serum from a patient with inactive systemic lupus erythematosus (SLE) and ibuprofen-induced transient neutropenia was used as a probe to define further the control of human polymorphonuclear leukocyte (PMN) exocytosis and superoxide (O2-) generation. Thirty-minute preincubation of normal PMNs with 10-50% v/v of this serum, followed by washing, produced a specific dose-related suppression of leukotriene B4 (LTB4)-elicited beta-glucuronidase and lysozyme release of up to 45% and 30% respectively. If cells were not washed, the inhibition increased to 60% and 40%. Superoxide production stimulated by LTB4 was unaffected. The serum had no effect on formyl-met-leu-phe (FMLP) or phorbol myristate acetate-stimulated O2- or exocytosis. O2- and beta-glucuronidase release elicited by zymosan-treated serum (ZTS) were both decreased by 15%, but there was no increased inhibition seen if cells were not washed, or if the time of preincubation was increased from 7 to 30 min. In contradistinction, the serum inhibition of LTB4 exocytosis did show time dependence. Serum obtained when the patient was not leukopenic and sera from 6 normal controls, 2 patients with inactive SLE, 1 patient with SLE and chronic leukopenia, and 2 controls taking ibuprofen did not influence any PMN function. The serum inhibition of ZTS-induced functions was qualitatively similar to that observed when PMNs were preincubated and desensitized with ZTS in vitro. Selective inhibition of LTB4 exocytosis was not seen when PMNs were desensitized with LTB4 in vitro. These observations indicate that LTB4-elicited O2- and exocytosis can be independently and specifically regulated. The cellular site at which this serum factor acts is not clear, but the current studies strongly suggest that this inhibition is not due to in vitro deactivation by LTB4 activity.     

Leukotriene B4 synthesis and neutrophil chemotaxis in chronic granulocytic leukaemia.

A sensitive gas chromatography-mass spectrometric method was used to measure the generation in whole blood of leukotriene B4 (LTB4), a potent stimulator of neutrophil chemotaxis, in eight patients with chronic granulocytic leukaemia (CGL) and 12 healthy controls. LTB4 was detectable in unstimulated samples from all the patients (mean 194 (70 SEM) pg/ml), and the capacity for LTB4 production after stimulation with calcium ionophore (A23187) was similar in patients (32.1 (11) ng/10(6) leucocytes) and controls (38.1 (4) ng/10(6) leucocytes). In response to stimuli which induce neutrophil activation, LTB4 production was significantly greater in the patients than in controls: 35.6 (13) v 13.0 (3) ng/ml, p less than 0.05 (f-met-leu-phe); and 42.4 (16) v 14.7 (4) ng/ml, p less than 0.02 (opsonised zymosan). Anti-IgE stimulated considerably more LTB4 production in patients with CGL than in controls (3.86 (1.6) v 0.83 (0.43) ng/ml; p less than 0.005) and this correlated significantly (p less than 0.05) with the basophil count. Neutrophil chemotaxis to LTB4, however, was significantly impaired in the patients with CGL even at the highest concentration of LTB4 (10(-5) M). Chemotaxis to f-met-leu-phe, phagocytosis, and bacterial killing were normal. Thus although LTB4 synthesis is normal or even enhanced in patients with CGL, specific defects in LTB4-mediated responses may contribute to neutrophil dysfunction in this disease.