The influence of leukotrienes C4 and D4 on the contractility of an inflamed porcine uterus.

The effect of leukotriene (LT) C4 (LTC4) and LTD4 on the contractility of an inflamed porcine uterus was investigated. On Day 3 of the estrous cycle (Day 0 of the study), either saline or Escherichia coli suspension was injected into each uterine horn. Although acute endometritis developed in all bacteria-inoculated gilts, a severe acute endometritis was noted more often on Day 8 than on Day 16. Myometrial and endometrial/myometrial strips were incubated with LTC4 or LTD4 alone, or together with a cysteinyl-LT receptor antagonist (BAY-u9773). Leukotriene C4 increased the contraction intensity in the saline- and bacteria-treated uteri on Day 8; however, its effect was lower in the myometrium of inflamed uteri. Contraction frequency was found to decrease in the saline-treated uteri as opposed to inflamed ones, in which it was elevated. On Day 16, contraction intensity increased in response to LTC4 in the saline-treated uteri but was reduced in the inflamed organs. The value of this parameter was lower in the inflamed uteri than that in the saline-treated ones. Leukotriene D4 (Days 8 and 16) augmented contractility in the saline-treated uteri, but despite increasing its intensity in the inflamed organs, it decreased contraction frequency. Leukotriene C4 or LTD4, added to BAY-u9773-pretreated saline- and bacteria-treated uteri on both days, decreased the contraction intensity. On Day 16 after treatment with BAY-u9773 and LTC4, contraction intensity in the endometrium/myometrium of the inflamed uteri was lower than that in the saline-treated organs. Data show that both LTC4 and LTD4 affect the contractility of inflamed porcine uteri, though LTC4 exerts a weaker contractile effect.

Cysteinyl leukotriene receptor (CysLT) antagonists decrease pentylenetetrazol-induced seizures and blood-brain barrier dysfunction.

Current evidence suggests that inflammation plays a role in the pathophysiology of seizures. In line with this view, selected pro-inflammatory arachidonic acid derivatives have been reported to facilitate seizures. Kainate-induced seizures are accompanied by leukotriene formation, and are reduced by inhibitors of LOX/COX pathway. Moreover, LTD4 receptor blockade and LTD4 synthesis inhibition suppress pentylenetetrazol (PTZ)-induced kindling and pilocarpine-induced recurrent seizures. Although there is convincing evidence supporting that blood-brain-barrier (BBB) dysfunction facilitates seizures, no study has investigated whether the anticonvulsant effect of montelukast is associated with its ability to maintain BBB integrity. In this study we investigated whether montelukast and other CysLT receptor antagonists decrease PTZ-induced seizures, as well as whether these antagonists preserve BBB during PTZ-induced seizures. Adult male albino Swiss mice were stereotaxically implanted with a cannula into the right lateral ventricle, and two electrodes were placed over the parietal cortex along with a ground lead positioned over the nasal sinus for electroencephalography (EEG) recording. The effects of montelukast (0.03 or 0.3 µmol/1 µL, i.c.v.), pranlukast (1 or 3 µmol/1 µL, i.c.v.), Bay u-9773 (0.3, 3 or 30 nmol/1 µL, i.c.v.), in the presence or absence of the agonist LTD4 (0.2, 2, 6 or 20 pmol/1 µL, i.c.v.), on PTZ (1.8 µmol/2 µL)-induced seizures and BBB permeability disruption were determined. The animals were injected with the antagonists, agonist or vehicle 30 min before PTZ, and monitored for additional 30 min for the appearance of seizures by electrographic and behavioral methods. BBB permeability was assessed by sodium fluorescein method and by confocal microscopy for CD45 and IgG immunoreactivity. Bay-u9973 (3 and 30 nmol), montelukast (0.03 and 0.3 µmol) and pranlukast (1 and 3 µmol), increased the latency to generalized seizures and decreased the mean amplitude of EEG recordings during seizures. LTD4 (0.2 and 2 pmol) reverted the anticonvulsant effect of montelukast (0.3 µmol). Montelukast (0.03 and 0.3 µmol) prevented PTZ-induced BBB disruption, an effect that was reversed by LTD4 at the dose of 6 pmol, but not at the doses 0.2 and 2 pmol. Moreover, the doses of LTD4 (0.2 and 2 pmol) that reverted the effect of montelukast on seizures did not alter montelukast-induced protection of BBB, dissociating BBB protection and anticonvulsant activity. Confocal microscopy analysis revealed that 1. PTZ increased the number of CD45+ and double-immunofluorescence staining for CD45 and IgG cells in the cerebral cortex, indicating BBB leakage with leukocyte infiltration; 2. while LTD4 (6 pmol) potentiated, montelukast decreased the effect of PTZ on leukocyte migration and BBB, assessed by double-immunofluorescence staining for CD45 and IgG cells in the cannulated hemisphere. Our data do not allow us ruling out that mechanisms unrelated and related to BBB protection may co-exist, resulting in decreased seizure susceptibility by montelukast. Notwithstanding, they suggest that CysLT1 receptors may be a suitable target for anticonvulsant development.

[Leukotriene D4 activates BV2 microglia in vitro].

OBJECTIVE: To investigate the effects of CysLT receptor agonist leukotriene D4(LTD4) and antagonists on activation of microglia BV2 cells. METHODS: The expression of CysLT1 and CysLT2 protein was determined by Western blotting and immunostaining in microglia BV2 cells. BV2 cells were pretreated with or without CysLT1 receptor selective antagonist montelukast, CysLT2 receptor selective antagonist HAMI 3379, or CysLT1/CysLT2 receptor dual antagonist BAY u9773 for 30 min, then the cells were treated with LTD4 for 24 h. Cell viability was detected by MTT reduction assay. Phagocytosis and mRNA expression of IL-6 were determined by fluorescent bead tracking and RT-PCR, respectively. RESULTS: In BV2 cells, LTD4 did not affect proliferation but significantly enhanced phagocytosis and increased IL-6 mRNA expression in a concentration-dependent manner. LTD4 at 100 nmol/L induced a 1.4-fold increase of phagocytic index and a 2-fold up-regulation of IL-6 mRNA expression (P<0.01). HAMI 3379 and BAY u9773 (100 nmol/L) further increased LTD4-induced phagocytosis; BAY u9773 and montelukast decreased LTD4-induced IL-6 mRNA expression, while HAMI 3379 had no effect on that. CONCLUSION: LTD4 activates BV2 cells in vitro and enhances IL-6 mRNA expression mediated by CysLT1 receptor, LTD4 induces phagocytosis which might be negatively regulated by CysLT2 receptor in BV2 cells.

A selective cysteinyl leukotriene receptor 2 antagonist blocks myocardial ischemia/reperfusion injury and vascular permeability in mice.

Cysteinyl leukotrienes (CysLTs) are potent inflammatory mediators that predominantly exert their effects by binding to cysteinyl leukotriene receptors of the G protein-coupled receptor family. CysLT receptor 2 (CysLT(2)R), expressed in endothelial cells of some vascular beds, has been implicated in a variety of cardiovascular functions. Endothelium-specific overexpression of human CysLT(2)R in transgenic mice (hEC-CysLT(2)R) greatly increases myocardial infarction damage. Investigation of this receptor, however, has been hindered by the lack of selective pharmacological antagonists. Here, we describe the characterization of 3-(((3-carboxycyclohexyl)amino)carbonyl)-4-(3-(4-(4-phenoxybutoxy)phenyl)-propoxy)benzoic acid (BayCysLT(2)) and explore the selective effects of this compound in attenuating myocardial ischemia/reperfusion damage and vascular leakage. Using a recently developed ß-galactosidase-ß-arrestin complementation assay for CysLT(2)R activity (Mol Pharmacol 79:270-278, 2011), we determined BayCysLT(2) to be ∼20-fold more potent than the nonselective dual CysLT receptor 1 (CysLT(1)R)/CysLT(2)R antagonist 4-(((1R,2E,4E,6Z,9Z)-1-((1S)-4-carboxy-1-hydroxybutyl)-2,4,6,9-pentadecatetraen-1-yl)thio)benzoic acid (Bay-u9773) (IC(50) 274 nM versus 4.6 µM, respectively). Intracellular calcium mobilization in response to cysteinyl leukotriene administration showed that BayCysLT(2) was >500-fold more selective for CysLT(2)R compared with CysLT(1)R. Intraperitoneal injection of BayCysLT(2) in mice significantly attenuated leukotriene D(4)-induced Evans blue dye leakage in the murine ear vasculature. BayCysLT(2) administration either before or after ischemia/reperfusion attenuated the aforementioned increased myocardial infarction damage in hEC-CysLT(2)R mice. Finally, decreased neutrophil infiltration and leukocyte adhesion molecule mRNA expression were observed in mice treated with antagonist compared with untreated controls. In conclusion, we present the characterization of a potent and selective antagonist for CysLT(2)R that is useful for discerning biological activities of this receptor.

Effects of a cysteinyl leukotriene dual 1/2 receptor antagonist on antigen-induced airway hypersensitivity and airway inflammation in a guinea pig asthma model.

BACKGROUND: Little is known about the role of the cysteinyl leukotriene (cysLT) 2 receptor in the pathophysiology of asthma. The aim of this study is to investigate the effects of a cysLT1 receptor antagonist (montelukast) and a dual cysLT1/2 receptor antagonist (BAY-u9773) on airway hypersensitivity and airway inflammation induced by antigen challenge in ovalbumin (OVA)-sensitized guinea pigs. METHODS: Male Hartley guinea pigs sensitized with OVA were intraperitoneally administered 0.1, 1, or 10 mg/kg of montelukast or 0.1 mg/kg of BAY-u9773 and then challenged with inhaled OVA. Airway reactivity to acetylcholine, inflammatory cells in bronchoalveolar lavage (BAL) fluid, and eosinophil infiltration in airway walls after OVA challenge were evaluated. RESULTS: Pretreatment with 1 or 10 mg/kg, but not 0.1 mg/kg, of montelukast significantly suppressed airway hypersensitivity and eosinophil infiltration into the BAL fluid. Moreover, 0.1 mg/kg of BAY-u9773 significantly suppressed the development of these markers. The suppressive effects of BAY-u9773, although not significantly different, trended toward being greater than those of montelukast. Although all of the doses of montelukast tested and 0.1 mg/kg of BAY-u9773 significantly suppressed eosinophil infiltration in airway walls, the suppressive effect of BAY-u9773 was significantly greater than that of 0.1 mg/kg of montelukast. CONCLUSION: Signaling may contribute to the pathophysiology of asthma via the cysLT1/2 receptor.

Leukotriene-induced contraction is mediated by cysteinyl leukotriene receptor CysLT1 in guinea pig fundus but by CysLT1 and CysLT2 in antrum.

AIMS: Leukotriene D(4) (LTD(4)) causes contraction of the stomach through unclear receptors. The aim of the present study is to characterize the cysteinyl leukotriene receptor (CysLT) mediating leukotriene-induced muscle contraction in the stomach. MAIN METHODS: We measured contraction of gastric muscle strips isolated from the guinea pig fundus and antrum caused by cysteinyl leukotrienes, including LTC(4), LTD(4) and LTE(4), as well as the dihydroxy leukotriene LTB(4) in vitro. KEY FINDINGS: In both fundic and antral muscle strips, LTC(4) and LTD(4) caused marked whereas LTE(4) caused moderate, concentration-dependent contractions. In contrast, LTB(4) caused only small contraction. The relative potencies for cysteinyl leukotrienes to cause contraction in both fundus and antrum were LTC(4)=LTD(4)>LTE(4). The LTD(4)-induced contraction was not affected by tetrodotoxin or atropine, suggesting that the action is not neurally mediated. The LTD(4)-induced contraction in the fundus was almost abolished by the CysLT(1) selective antagonist montelukast. In contrast, the LTD(4)-induced contraction in the antrum was only partially inhibited by montelukast or the dual CysLT(1) and CysLT(2) antagonist BAY u9773. This antral contraction was almost abolished by the combination of montelukast and BAY u9773, indicating enhancement of inhibition. SIGNIFICANCE: The results of the present study demonstrate that cysteinyl leukotrienes LTC(4), LTD(4) and LTE(4) cause moderate to marked whereas the dihydroxy leukotriene LTB(4) causes small muscle contraction in the stomach in vitro. The leukotriene-induced contraction is mediated by CysLT(1) in fundus but by CysLT(1) and CysLT(2) in antrum.

CysLT2 receptor-mediated AQP4 up-regulation is involved in ischemic-like injury through activation of ERK and p38 MAPK in rat astrocytes.

AIMS: We previously reported that cysteinyl leukotriene receptor 2 (CysLT(2)) mediates ischemic astrocyte injury, and leukotriene D(4)-activated CysLT(2) receptor up-regulates the water channel aquaporin 4 (AQP4). Here we investigated the mechanism underlying CysLT(2) receptor-mediated ischemic astrocyte injury induced by 4-h oxygen-glucose deprivation and 24-h recovery (OGD/R). MAIN METHODS: Primary cultures of rat astrocytes were treated by OGD/R to construct the cell injury model. AQP4 expression was inhibited by small interfering RNA (siRNA). The expressions of AQP4 and CysLTs receptors, and the MAPK signaling pathway were determined. KEY FINDINGS: OGD/R induced astrocyte injury, and increased expression of the CysLT(2) (but not CysLT(1)) receptor and AQP4. OGD/R-induced cell injury and AQP4 up-regulation were inhibited by a CysLT(2) receptor antagonist (Bay cysLT2) and a non-selective CysLT receptor antagonist (Bay u9773), but not by a CysLT(1) receptor antagonist (montelukast). Knockdown of AQP4 by siRNA attenuated OGD/R injury. Furthermore, OGD/R increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the cell injury and AQP4 up-regulation. SIGNIFICANCE: The CysLT(2) receptor mediates AQP4 up-regulation in astrocytes, and up-regulated AQP4 leads to OGD/R-induced injury, which results from activation of the ERK1/2 and p38 MAPK pathways.

Pranlukast prevents cysteinyl leukotriene-induced emesis in the least shrew (Cryptotis parva).

Many chemotherapeutic agents activate multiple signaling systems, including potentially emetogenic arachidonic acid metabolites. Of these messengers, the emetic role of the leukotriene family has been neglected. The aims of this study were to test the emetic potential of key leukotrienes (LTA(4), LTB(4), LTF(4), and the cysteinyl leukotrienes LTC(4), LTD(4) and LTE(4)), and to investigate whether the leukotriene CysLT(1) receptor antagonist pranlukast or mixed leukotriene CysLT(1/2) receptor antagonist Bay u9773 can prevent the LTC(4)-induced emesis. Least shrews were injected with varying doses of one of the six tested leukotrienes and vomiting parameters were measured for 30min. LTC(4) and LTD(4) were most efficacious, and significantly increased both the frequency and percentage of animals vomiting at doses from 0.1 and 0.05mg/kg, respectively. The other tested leukotrienes were either weakly emetic or ineffective at doses up to 4mg/kg. The relative emetogenic activities of the cysteinyl leukotrienes (LTC(4)=LTD(4)>LTE(4)) suggest that leukotriene CysLT(2) receptors have a key role in emesis. However, pranlukast dose-dependently, and at 10mg/kg completely, blocked LTC(4)-induced vomiting, implicating a leukotriene CysLT(1) receptor-mediated emetic effect. Bay u9773 dose-dependently reduced the percentage of animals vomiting, but did not significantly reduce vomiting frequency. Fos immunoreactivity, measured subsequent to LTC(4)-induced vomiting to define its putative anatomical substrates, was significantly increased in the enteric nervous system and medullary dorsal vagal complex following LTC(4) (P<0.05) versus vehicle injections. This study is the first to show that some leukotrienes induce emesis, possibly involving both central and peripheral leukotriene CysLT(1) and/or leukotriene CysLT(2) receptors.

IL-18 accelerates the cell apoptosis by up-regulating cysteinyl leukotriene 2 receptor expression in human umbilical vein endothelial cells at the early stage of administration.

The purpose of the present study is to identify whether interleukin (IL)-18 can modulate cysteinyl leukotriene 2 receptor (CysLT2R) expression in Human Umbilical Vein Endothelial Cells (HUVECs) and how it influences the cell death. According to the results from real-time reverse transcription PCR, confocal laser scanning microscopy, and western blotting, a dose-dependent augmentation of CysLT2R protein expression in HUVECs was triggered by IL-18 for the first 2 h followed by down-regulation within the next 22 h after IL-18 administration. The flow cytometry showed that non-selective CysLT1R and CysLT2R antagonist BAY-u9773 could attenuate the early stage apoptosis mediated by IL-18 whereas CysLT1R antagonist Montelukast couldn't. Also, pretreatment with BAY-u9773 suppressed calcium influx of HUVECs induced by IL-18 whereas Montelukast didn't work. The observation that progression of cell death aggravated by IL-18 could be attenuated by BAY-u9773 may offer a chance to develop a novel way to treat arteriosclerosis.

Leukotriene D4 stimulates the migration but not proliferation of endothelial cells mediated by the cysteinyl leukotriene cyslt(1) receptor via the extracellular signal-regulated kinase pathway.

The actions of cysteinyl leukotrienes (CysLTs) are mediated by activating CysLT receptors, CysLT(1), and CysLT(2). The CysLT(1) receptor mediates vascular responses to CysLTs; however, its effect on the proliferation and migration of endothelial cells is not clarified. To determine this effect, we observed proliferation and migration in EA.hy926 cells, a human endothelial cell line, and the involvement of activation of mitogen-activated protein kinases (MAPKs). We found that LTD(4) did not affect the proliferation, but significantly stimulated the migration of endothelial cells. LTD(4) also induced the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, but not those of p38 or JNK. The LTD(4)-induced migration and ERK1/2 phosphorylation were blocked by the CysLT(1)-receptor antagonist montelukast and the dual antagonist Bay u9773, but not by the CysLT(2)-receptor antagonist Bay cysLT2; the migration was also inhibited by the ERK1/2 inhibitor U0126. Our findings indicate that LTD(4) stimulates the CysLT(1) receptor-mediated migration of endothelial cells; this may be regulated by the ERK1/2 pathway.

Leukotriene pathways and in vitro adenotonsillar cell proliferation in children with obstructive sleep apnea.

INTRODUCTION: The abundant expression of leukotrienes (LTs) and their receptors in adenotonsillar tissues of children with obstructive sleep apnea (OSA) suggest that LT antagonists could be useful in treating OSA. METHODS: The effects of LTD4 and of LT receptor antagonists zileuton, montelukast, and BAY u9773 were examined on mixed cell cultures prepared from dissociated tonsils or adenoids harvested intraoperatively from children with polysomnographically diagnosed OSA. Proliferation was assessed by (3)[H]-thymidine incorporation, and inflammatory cytokine production (tumor necrosis factor [TNF]-alpha, interleukin [IL]-6, IL-8, IL-10, and IL-12) was assessed in supernatants using enzyme-linked immunosorbent assay. RESULTS: LTD4 elicited dose-dependent increases in adenotonsillar cell proliferation (p < 0.001; n = 12). All LT antagonists exhibited dose-dependent reductions in adenotonsillar cellular proliferation rates, with montelukast more than BAY u9773 more than zileuton (n = 14/group; p < 0.001). However, BAY u9773 showed partial agonist effects and increased cellular proliferation at higher concentrations (10(-4) mmol/L; p < 0.01; n = 12). LTD4 effects were partially blocked by montelukast and BAY u9773 but not by zileuton. All three antagonists reduced TNF-alpha, IL-6, and IL-12 concentrations, with selective changes in IL-8 and no effects on IL-10 levels. CONCLUSIONS: LT pathways mediate intrinsic proliferative and inflammatory signaling pathways in adenotonsillar tissues from children with OSA, and targeted pharmacologic disruption of these pathways may provide nonsurgical alternatives for prevention and treatment of this disease.

Cysteinyl leukotriene 2 receptor-mediated vascular permeability via transendothelial vesicle transport.

Cysteinyl leukotrienes (CysLTs) are potent mediators of inflammation synthesized by the concerted actions of 5-lipoxygenase (5-LO), 5-LO-activating protein (FLAP), leukotriene C(4) synthase, and additional downstream enzymes, starting with arachidonic acid substrate. CysLTs produced by macrophages, eosinophils, mast cells, and other inflammatory cells activate 3 different high-affinity CysLT receptors: CysLT(1)R, CysLT(2)R, and GPR 17. We sought to investigate vascular sites of CysLT(2)R expression and the role and mechanism of this receptor in mediating vascular permeability events. Vascular expression of CysLT(2)R was investigated by reporter gene expression in a novel CysLT(2)R deficient-LacZ mouse model. CysLT(2)R was expressed in small, but not large, vessels in mouse brain, bladder, skin, and cremaster muscle. Intravital, in addition to confocal and electron, microscopy investigations using FITC-labeled albumin in cremaster postcapillary venule preparations indicated rapid CysLT-mediated permeability, which was blocked by application of BAY-u9773, a dual CysLT(1)R/CysLT(2)R antagonist or by CysLT(2)R deficiency. Endothelial human CysLT(2)R overexpression in mice exacerbated vascular leakage even in the absence of exogenous ligand. The enhanced vascular permeability mediated by CysLT(2)R takes place via a transendothelial vesicle transport mechanism as opposed to a paracellular route and is controlled via Ca(2+) signaling. Our results reveal that CysLT(2)R can mediate inflammatory reactions in a vascular bed-specific manner by altering transendothelial vesicle transport-based vascular permeability.

Cysteinyl leucotriene receptor type 1 mediates contraction in human and guinea-pig oesophagus.

Leucotriene D(4) (LTD(4)) causes contraction of the guinea-pig and cat oesophagus. Effects of cysteinyl leucotrienes in the human oesophagus were unknown. To investigate and compare the cysteinyl leucotriene effects in the human oesophagus with those in the guinea-pig oesophagus, we measured contraction of muscularis mucosae strips isolated from the human and guinea-pig oesophagus caused by cysteinyl leucotrienes, LTC(4), LTD(4) and LTE(4), as well as the dihydroxy leucotriene, LTB(4). Effects of leucotrienes in human were similar to those in guinea-pig oesophagus. LTC(4) and LTD(4) caused moderate, whereas LTE(4) caused mild, concentration-dependent contraction. LTE(4) was a partial agonist. In contrast, LTB(4) did not cause any contraction. The relative potencies for cysteinyl leucotrienes to cause contraction were LTD(4) = LTC(4) > LTE(4). The LTD(4)-induced contraction was moderately inhibited by two selective CysLT(1) receptor antagonists, montelukast and zafirlukast, in both human and guinea-pig oesophagus. In addition, the LTD(4)-induced contraction was not and only slightly inhibited by BAY u9773, the CysLT(1) and CysLT(2) receptor antagonist, in the human and guinea-pig oesophageal muscularis mucosae respectively. These indicate the existence of the CysLT(1) mediating oesophageal contraction in both human and guinea-pig oesophagus. The LTD(4)-induced contraction was not affected by tetrodotoxin, atropine or capsaicin, suggesting a direct effect. These results demonstrate that cysteinyl leucotrienes but not the dihydroxy leucotriene cause contraction in the human and guinea-pig oesophagus. CysLT(1) mediates contraction in both human and guinea-pig oesophagus.

Guinea pig cysteinyl leukotriene receptor 2 (gpCysLT2) mediates cell proliferation and intracellular calcium mobilization by LTC4 and LTD4.

We cloned and pharmacologically characterized the guinea pig cysteinyl leukotriene (CysLT) 2 receptor (gpCysLT2). gpCysLT2 consists of 317 amino acids with 75.3%, 75.2%, 73.3% identity to those of humans, mice and rats, respectively. The gpCysLT2 gene is highly expressed in the lung, moderately in eosinophils, skin, spleen, stomach, colon, and modestly in the small intestine. CysLTs accelerated the proliferation of gpCysLT2-expressing HEK293. Leukotriene C4 (LTC4) and Leukotriene D4 (LTD4) enhanced the cell proliferation higher than Bay-u9773, a CysLT2 selective partial agonist and a nonselective antagonist for CysLT receptors. Bay-u9773 did not antagonize the cell proliferation by LTC4 and LTD4. Despite the equipotency of the mitogenic effect among these chemicals, calcium mobilization (CM) levels were variable (LTC4> LTD4>> Bay-u9773), and Bay-u9773 antagonized the CM by LTC4. Moreover, the Gi/o inhibitor pertussis toxin perfectly inhibited agonist-induced cell proliferation. These results reveal that cell proliferation via CysLT2 signaling was mediated by Gi/o signaling but independent of calcium mobilization.

Agonist- and T(H)2 cytokine-induced up-regulation of cysteinyl leukotriene receptor messenger RNA in human monocytes.

BACKGROUND: The cysteinyl leukotrienes (CysLTs) are lipid mediators that have been implicated in the pathogenesis of allergic diseases, and their actions are mediated via specific receptors named CysLT1 receptor (CysLT1R) and CysLT2 receptor (CysLT2R). Little information is known about the role of T(H)2 cytokines in the regulation of both CysLT1R and CysLT2R expression. OBJECTIVE: To investigate the possible modulation of both CysLT1R and CysLT2R messenger RNA (mRNA) expression, we have developed a real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay based on the TaqMan fluorescence method to quantify CysLT1R and CysLT2R mRNA in human monocytes. METHODS: Human monocytes were stimulated with leukotriene D4 or interleukin (IL) 4 or IL-13, and the levels of CysLT1R and CysLT2R mRNA were measured by the quantitative RT-PCR. RESULTS: CysLT1R and CysLT2R mRNA was increased after stimulation with leukotriene D4. CysLT1R mRNA was augmented 150-fold after treatment with IL-4; however, no significant increase was observed in CysLT2R mRNA level. IL-13 could induce a biphasic augmentation of CysLT1R mRNA level. In contrast to IL-4, IL-13 enhanced CysLT2R mRNA level, with a maximal effect at 2 hours of incubation. CONCLUSIONS: CysLT1R and CysLT2R expression can be regulated by CysLT itself and T(H)2 cytokines at the transcriptional level.

Cysteinyl leukotrienes synergize with growth factors to induce proliferation of human bronchial fibroblasts.

BACKGROUND: Cysteinyl leukotrienes (cys-LTs) are potent asthma-related mediators that function through their G protein-coupled receptors, cys-LT receptor type 1 (CysLT1R) and cys-LT receptor type 2 (CysLT2R). OBJECTIVE: Because many G protein-coupled receptors transactivate the epidermal growth factor receptor (EGFR) through metalloprotease-mediated ligand shedding, we investigated the effects of cys-LTs on signal transduction and proliferation of bronchial fibroblasts. METHODS: Human bronchial fibroblasts were grown from biopsy specimens of healthy subjects. Mitogenesis was assessed on the basis of tritiated methylthymidine incorporation. RESULTS: Leukotriene (LT) D(4) alone did not increase mitogenesis but dose-dependently increased thymidine incorporation and cell proliferation in the presence of epidermal growth factor (EGF). The enhancement was not prevented by CysLT1R antagonists (MK-571 and montelukast) or by a dual antagonist (BAY u9773), which is consistent with the lack of detectable mRNA for CysLT1R and CysLT2R in bronchial fibroblasts. LTD(4) did not cause EGFR transphosphorylation nor was the synergism blocked by the metalloprotease inhibitor GM6001. The EGFR-selective kinase inhibitor AG1478 suppressed the synergy between LTD(4) and EGF but had no effect on synergistic interactions of LTD(4) with other receptor tyrosine kinase growth factors. The effect of LTD(4) involved a pertussis toxin-sensitive and protein kinase C-mediated intracellular pathway, leading to sustained growth factor-dependent phosphorylation of extracellular signal-regulated kinase 1/2 and protein kinase B (PKB/Akt). CONCLUSION: Cys-LTs do not transactivate EGFR but have a broader capability to synergize with receptor tyrosine kinase pathways. CLINICAL IMPLICATIONS: This study implies a critical role of cys-LTs in airway fibrosis in asthma and other chronic airway diseases, which might not be blocked by therapy with current LT receptor antagonists.

Distinct roles of CysLT1 and CysLT2 receptors in oxygen glucose deprivation-induced PC12 cell death.

Cysteinyl leukotrienes are involved in ischemic brain injury, and their receptors (CysLT(1) and CysLT(2)) have been cloned. To clarify which subtype mediates the ischemic neuronal injury, we performed permanent transfection to increase CysLT(1) and CysLT(2) receptor expressions in PC12 cells. Oxygen glucose deprivation (OGD)-induced cell death was detected by Hoechst 33258 and propidium iodide fluorescent staining as well as by flow cytometry. OGD induced late phase apoptosis mainly and necrosis minimally. Over-expression of CysLT(1) receptor decreased and over-expression of CysLT(2) receptor increased OGD-induced cell death. An agonist LTD(4) (10(-7)M) also induced apoptosis, especially in CysLT(2) receptor over-expressing cells. A selective CysLT(1) receptor antagonist montelukast did not affect OGD-induced apoptosis; while non-selective CysLT receptor antagonist Bay u9773 inhibited OGD-induced apoptosis, especially in CysLT(2) receptor over-expressing cells. Thus, CysLT(1) and CysLT(2) receptors play distinct roles in OGD-induced PC12 cell death; CysLT(1) attenuates while CysLT(2) facilitates the cell death.

Membrane IgE binds and activates Fc epsilon RI in an antigen-independent manner.

Interaction of secretory IgE with FcepsilonRI is the prerequisite for allergen-driven cellular responses, fundamental events in immediate and chronic allergic manifestations. Previous studies reported the binding of soluble FcepsilonRIalpha to membrane IgE exposed on B cells. In this study, the functional interaction between human membrane IgE and human FcepsilonRI is presented. Four different IgE versions were expressed in mouse B cell lines, namely: a truncation at the Cepsilon2-Cepsilon3 junction of membrane IgE isoform long, membrane IgE isoform long (without Igalpha/Igbeta BCR accessory proteins), and both epsilonBCRs (containing membrane IgE isoforms short and long). All membrane IgE versions activated a rat basophilic leukemia cell line transfected with human FcepsilonRI, as detected by measuring the release of both preformed and newly synthesized mediators. The interaction led also to Ca(2+) responses in the basophil cell line, while membrane IgE-FcepsilonRI complexes were detected by immunoprecipitation. FcepsilonRI activation by membrane IgE occurs in an Ag-independent manner. Noteworthily, human peripheral blood basophils and monocytes also were activated upon contact with cells bearing membrane IgE. In humans, the presence of FcepsilonRI in several cellular entities suggests a possible membrane IgE-FcepsilonRI-driven cell-cell dialogue, with likely implications for IgE homeostasis in physiology and pathology.

Cysteinyl-leukotrienes receptor activation in brain inflammatory reactions and cerebral edema formation: a role for transcellular biosynthesis of cysteinyl-leukotrienes.

We studied the effect of intravascular activation of human neutrophils on the synthesis of cysteinyl leukotrienes (cysLT) and the formation of cerebral edema in guinea-pig brains. Challenge with the chemotactic formylated tripeptide fMLP (0.1 microM) of neutrophil-perfused brain in vitro resulted in blood-brain barrier disruption associated with a significant increase of cysLT. Both events were completely prevented by neutrophil pretreatment with a specific 5-lipoxygenase (5-LO) inhibitor. Perfusion with the 5-LO metabolite leukotriene B4 (10 nM), together with neutrophils treated with the 5-LO inhibitor, did not restore the alteration in permeability observed upon perfusion with untreated and activated neutrophils. The dual cysLT1-cysLT2 receptor antagonist BAYu9773 was more potent and more effective than a selective cysLT1 antagonist in preventing the brain permeability alteration induced by neutrophil activation. RT-PCR showed significant expression of cysLT2 receptor mRNA in human umbilical vein endothelial cells. Intravital microscopy in mice showed that inhibition of leukotriene synthesis significantly reduced firm adhesion of neutrophils to cerebral vessels without affecting rolling. These data support the hypothesis that neutrophil and endothelial cells cooperate toward the local synthesis of cysLT within the brain vasculature and, acting via the cysLT2 receptor on endothelial cells, may represent a contributing pathogenic mechanism in the development of cerebral inflammation and edema.

A cysteinyl leukotriene 2 receptor variant is associated with atopy in the population of Tristan da Cunha.

The clinical heterogeneity of asthma suggests that the contribution of genetic variability in candidate gene loci to well-defined phenotypes, such as atopy, may be examined to identify appropriate genetic risk factors for asthma. The gene encoding the cysteinyl leukotriene 2 (CysLT2) receptor has been implicated in atopy since it is localized to a region of chromosome 13q14 that has been linked to atopy in several populations and the cysteinyl leukotrienes are known to activate eosinophils and mast cells in atopy. Accordingly, we analysed the contribution of CysLT2 receptor gene variation to atopy in the inhabitants of Tristan da Cunha, a population characterized by both a founder effect and a 47% prevalence of atopy. Single-stranded conformational polymorphism analysis revealed four variants. Among these, the M201V [corrected] variant was activated with four-fold less potency by leukotriene D4 (LTD4) in a calcium flux assay. The CysLT2 receptor partial agonist, BAY u9773, also showed four-fold lower potency on the M201V [corrected] variant. The M201V [corrected] mutation is located within the extracellular region of the fifth transmembrane spanning domain of CysLT2 receptor, a position that may alter ligand binding and effector signalling. The novel M201V [corrected] CysLT2 receptor variant was associated with atopy (21%) on Tristan da Cunha compared with those who were non-atopic (7%) (Fisher's exact test, P=0.0016) in a manner that was independent of asthma (two-way ANOVA, P=0.0015). This represents the first association of a coding mutation in the CysLT2 receptor gene, located on chromosome 13q14, with the atopic phenotype found in the Tristan da Cunha population.