IL-33 Upregulates Cysteinyl Leukotriene Receptor Type 1 Expression in Human Peripheral Blood CD4+ T Lymphocytes.

IL-33 and cysteinyl leukotrienes (cysLTs) are key components of asthma pathogenesis, and both contribute to the initiation and maintenance of the type 2 inflammatory environment. However, little is known about the potential interactions between the two mediators. In this work, we aimed at studying the regulation of expression of the cysLT receptors CysLT1 and CysLT2 by IL-33 in human PBLs. Our results show that the IL-33/ST2L axis increases CysLT1 but not CysLT2 expression in a concentration- and time-dependent manner in PBLs. IL-33-induced CysLT1 upregulation was observed at the protein but not at the mRNA level and was accompanied by an increase in LTD4-induced calcium mobilization and migration of CD4+ T lymphocytes. We also show that purified naive CD4+ T lymphocytes expressed ST2L and responded to IL-33 in the absence of Ag or TCR stimulation, suggesting a mechanism independent of Ag presentation. These results contribute to expanding our knowledge in the field of IL-33 by proposing a new mode of action of the cytokine on T cells and by extending its role to the regulation of naive T cell trafficking, therefore reinforcing its interest as a potential therapeutic target for the treatment of asthma.

Expression of cysteinyl leukotriene receptor GPR17 in eosinophilic and non-eosinophilic chronic rhinosinusitis with nasal polyps.

BACKGROUND: The cysteinyl leukotrienes (cysLTs) are proinflammatory lipid mediators that act on the type 1 cysLT receptor (CysLT1R) in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). GPR17, a G protein-coupled orphan receptor with homology to the cysLT receptors, has been proposed as a damage sensor during inflammation. However, the expression and correlation of GPR17 and CysLT1R in eosinophilic CRSwNP (ECRS) and non-eosinophilic CRSwNP (non-ECRS) have not been well investigated. OBJECTIVE: To evaluate the expression of GPR17 and its correlation with CysLT1R in the 2 CRSwNP subsets. METHODS: Polyp tissues were collected from CRSwNP subjects (15 ECRS and 14 non-ECRS), and uncinate processes were collected from 12 CRSsNP subjects and 13 control subjects. The mRNA and protein levels of GPR17 and CysLT1R were examined using qRT-PCR, immunohistochemistry, and western blotting. Additionally, the correlation between GPR17 and CysLT1R at the mRNA and protein levels was evaluated. All assays were performed in a blinded manner. RESULTS: Polyp tissues exhibited significantly increased GPR17 expression relative to uncinate process tissues from CRSsNP patients, or healthy controls (P=0.0012 and P<0.0001, respectively). Compared with the non-ECRS subset, the ECRS subset showed significantly increased GPR17 expression. Moreover, the GPR17 expression was positively correlated with CysLT1R in nasal polyps. CONCLUSIONS: The increased expression of GPR17 in nasal polyps and the differential expression between eosinophilic and non-eosinophilic CRSwNP subsets suggest that these subsets may have distinct pathogenic mechanisms. The positive correlation between GPR17 and CysLT1R in polyp tissues might imply substantial regulatory mechanisms that must be elucidated.

Expression of CysLTR1 and 2 in Maturating Lymphocytes of Hyperplasic Tonsils Compared to Peripheral Cells in Children.

Cysteinyl-leukotriene receptors 1 and 2 (CysLTR1 and 2) are related to allergic inflammatory responses. Recent studies demonstrated their role in lymphocyte division and maturation in the bone marrow. Few data are available about CysLTRs function in lymphocyte maturation in tonsils. The objectives of this study are to compare CysLTRs expression in peripheral blood lymphocytes with expression in maturating lymphocytes of hyperplasic tonsil and to check the influence of respiratory allergies in this process. Leukocytes of peripheral blood (PL) and hyperplasic tonsils of children were immunostained for CysLTR1, CysLTR2, CD3 (T cells), and CD19 (B cells) and read in flow cytometer. Lymphocyte of tonsils were divided in differentiating small cells (SC) and mitotic large cells (LC); percentage of B and T cells expressing CysLTRs was determined, and comparison was done using ANOVA and Tukey's tests. Data were analyzed as a whole and categorizing patients according the presence of allergies. Sixty children were enrolled in this study. There was a large expression of CysLTR1 and 2 in CD3+ LC, and such expression decreased progressively in SC and PL. In B cells, the highest expression of CysLTR1 and 2 was found in PL while SC showed the lowest and LC showed the intermediate expression. This pattern kept unchanged in groups of allergic and non-allergic individuals. CysLTRs seem to be involved in lymphocyte maturation that occurs in tonsils, without influence of allergies. New studies aiming the clinic treatment of tonsil hyperplasia must be targeted to the development of drugs capable of blocking both CysLTR1 and 2.

Effect of Montelukast on Spinal Cord Ischemia- Reperfusion Injury.

AIM: Paraplegia due to ischemia-reperfusion (I/R) injury of the spinal cord is a devastating complication of thoracoabdominal aortic surgery. Cysteinyl leukotrienes are potent mediators of inflammation that are associated with I/R injury. The present study was designed to investigate the role of montelukast, a selective reversible CysLT1 receptor antagonist, on spinal cord I/R injury in an experimental model. MATERIAL AND METHODS: Twenty-one male Sprague-Dawley rats were randomly assigned to three groups (n=7 per group) as G1 (no aortic occlusion and montelukast administration), G2 (45 min. aortic occlusion; no montelukast administration) and G3 (45 min. aortic occlusion, 10 mg/kg montelukast administration). After neurologic evaluation using the Motor Deficit Index (MDI) score at the 48th hour of reperfusion, lumbar spinal cords were removed for histopathological evaluation and immunohistochemical staining for HSP70, interleukin-6 and myeloperoxidase (MPO). RESULTS: All rats in the G1 group had a normal neurological status and their MDI score was 0 (p < 0.05). The MDI score of G3 was significantly lower than G2 group (2.8 vs. 5.5; p < 0.05). Vacuolar congestion was found to be significantly lower in G1 than the other groups (p=0.0001). The interleukin-6 receptor level was found to be significantly lower in G3 group than the control group (p=0.013). There was no statistically significant difference found among the groups in terms of the degree of HSP70 and MPO staining. CONCLUSION: Increased generation of leukotrienes in postischemic organs play an important role in I/R injury. The findings of the current study demonstrated that montelukast improved motor recovery and decreased IL-6 levels in spinal cord I/R injury.

The cysteinyl leukotriene 2 receptor contributes to all-trans retinoic acid-induced differentiation of colon cancer cells.

BACKGROUND: Cysteinyl leukotrienes (CysLTs) are potent pro-inflammatory mediators that are increased in samples from patients with inflammatory bowel diseases (IBDs). Individuals with IBDs have enhanced susceptibility to colon carcinogenesis. In colorectal cancer, the balance between the pro-mitogenic cysteinyl leukotriene 1 receptor (CysLT(1)R) and the differentiation-promoting cysteinyl leukotriene 2 receptor (CysLT(2)R) is lost. Further, our previous data indicate that patients with high CysLT(1)R and low CysLT(2)R expression have a poor prognosis. In this study, we examined whether the balance between CysLT(1)R and CysLT(2)R could be restored by treatment with the cancer chemopreventive agent all-trans retinoic acid (ATRA). METHODS: To determine the effect of ATRA on CysLT(2)R promoter activation, mRNA level, and protein level, we performed luciferase gene reporter assays, real-time polymerase chain reactions, and Western blots in colon cancer cell lines under various conditions. RESULTS: ATRA treatment induces CysLT(2)R mRNA and protein expression without affecting CysLT(1)R levels. Experiments using siRNA and mutant cell lines indicate that the up-regulation is retinoic acid receptor (RAR) dependent. Interestingly, ATRA also up-regulates mRNA expression of leukotriene C4 synthase, the enzyme responsible for the production of the ligand for CysLT(2)R. Importantly, ATRA-induced differentiation of colorectal cancer cells as shown by increased expression of MUC-2 and production of alkaline phosphatase, both of which could be reduced by a CysLT(2)R-specific inhibitor. CONCLUSIONS: This study identifies a novel mechanism of action for ATRA in colorectal cancer cell differentiation and demonstrates that retinoids can have anti-tumorigenic effects through their action on the cysteinyl leukotriene pathway.

Aggravated inflammation and increased expression of cysteinyl leukotriene receptors in the brain after focal cerebral ischemia in AQP4-deficient mice.

OBJECTIVE: Aquaporin-4 (AQP4), the main water channel protein in the brain, plays a critical role in water homeostasis and brain edema. Here, we investigated its role in the inflammatory responses after focal cerebral ischemia. METHODS: In AQP4-knockout (KO) and wild-type mice, focal cerebral ischemia was induced by 30 min of middle cerebral arterial occlusion (MCAO). Ischemic neuronal injury and cellular inflammatory responses, as well as the expression and localization of cysteinyl leukotriene CysLT(2) and CysLT(1) receptors, were determined at 24 and 72 h after MCAO. RESULTS: AQP4-KO mice showed more neuronal loss, more severe microglial activation and neutrophil infiltration, but less astrocyte proliferation in the brain after MCAO than wild-type mice. In addition, the protein levels of both CysLT(1) and CysLT(2) receptors were up-regulated in the ischemic brain, and the up-regulation was more pronounced in AQP4-KO mice. The CysLT(1) and CysLT(2) receptors were primarily localized in neurons, microglia and neutrophils; those localized in microglia and neutrophils were enhanced in AQP4-KO mice. CONCLUSION: AQP4 may play an inhibitory role in postischemic inflammation.

CysLT1-R expression following allergen provocation in asthma and allergic rhinitis.

Cysteinyl leukotrienes (CysLTs) contribute to allergic and inflammatory diseases through CysLT(1)-R. We aimed to assess CysLT(1)-R mRNA expression in induced sputum of rhinitics with or without asthma before and following allergen challenges. Both groups underwent nasal and "low dose" lung allergen challenges. Asthmatics also underwent "standard" lung challenge. Sputum was obtained before and at different time-points following the challenges for CysLT(1)-R, 5-lipoxygenase (5-LO), and eotaxin mRNA assessments. At baseline, there was no difference in mediator levels between groups. An increase in CysLT(1)-R mRNA (p=0.04) and a trend towards an increase in 5-LO and eotaxin (p=0.06 for both) at 24 h post-nasal challenge were observed. Following "low dose" lung allergen challenge, there was a trend towards an increase in CysLT(1)-R (p=0.07). In conclusion, CysLT(1)-R gene expression changes can be detected in sputum following allergen challenges. No difference was observed between groups, suggesting that changes in CysLT(1)-R expression occur whether or not the subject has concurrent asthma.

Immunopharmacological role of the leukotriene receptor antagonists and inhibitors of leukotrienes generating enzymes in multiple sclerosis.

BACKGROUND AND AIM: Multiple sclerosis (MS) is a chronic inflammatory disease that involves central nervous system, and is generally associated with demyelination and axonal lesion. The effective factors for initiation of the inflammatory responses have not been known precisely so far. Leukotrienes (LTs) are inflammatory mediators with increased levels in the cerebrospinal fluid of MS patients and in experimental models of multiple sclerosis. Inhibition of LT receptors with specific antagonists can decrease inflammatory responses. In this review article we try to clarify the role of LT receptor antagonists and also inhibitors of enzymes which are involved in LTs generating pathway for treating multiple sclerosis as new targets for MS therapy. Moreover, we suggest that blockage of LT receptors by potent specific antagonists and/or agonists can be as a novel useful method in treatment of MS.

Pathogenic importance of cysteinyl leukotrienes in development of gastric lesions induced by ischemia/reperfusion in mice.

We examined the role of cysteinyl leukotrienes (CysLTs) in the gastric ulcerogenic response to ischemia/reperfusion (I/R) in mice. Experiments were performed in male C57BL/6J mice after 18-h fasting. Under urethane anesthesia, the celiac artery was clamped for 30 min, and then reperfusion was achieved by removing the clamp. The stomach was examined for lesions 60 min thereafter. The severity of I/R-induced gastric damage was reduced by prior administration of pranlukast [CysLT receptor type 1 (CysLT(1)R) antagonist] as well as 1-[[5'-(3''-methoxy-4''-ethoxycarbonyl-oxyphenyl)-2',4'-pentadienoyl]aminoethyl]-4-diphenylmethoxypiperidine [TMK688; 5-lipoxygenase (5-LOX) inhibitor]. On the contrary, these lesions were markedly worsened by pretreatment with indomethacin, and this response was abrogated by the coadministration of TMK688 or pranlukast. The gene expression of CysLT(1)R but not 5-LOX was up-regulated in the stomach after I/R, but both expressions were increased under I/R in the presence of indomethacin. I/R slightly increased the mucosal CysLT content of the stomach, yet this increase was markedly enhanced when the animals were pretreated with indomethacin. The increased CysLT biosynthetic response to indomethacin during I/R was attenuated by TMK688. Indomethacin alone caused a slight increase of CysLT(1)R expression and markedly up-regulated 5-LOX expression in the stomach. We concluded that I/R up-regulated the expression of CysLT(1)R in the stomach; CysLTs play a role in the pathogenesis of I/R-induced gastric damage through the activation of CysLT(1)R; and the aggravation by indomethacin of these lesions may be brought about by the increase of CysLT production and the up-regulation of 5-LOX expression, in addition to the decreased prostaglandin production.

Variable expression of cysteinyl leukotriene type I receptor splice variants in asthmatic females with different promoter haplotypes.

BACKGROUND: Cysteinyl leukotrienes are potent inflammatory mediators implicated in the pathogenesis of asthma. Human cysteinyl leukotriene receptor 1 (CYSLTR1) gene contains five exons that are variably spliced. Within its promoter few polymorphisms were described. To date, there has been no evidence about the expression of different splice variants of CysLT1 in asthma and their association with CYSLTR1 promoter polymorphisms.The goal of our study was to investigate CysLT1 alternative transcripts expression in asthmatic patients with different CYSLTR1 promoter haplotypes.The study groups consisted of 44 patients with asthma, diagnosed according to GINA 2008 criteria and 18 healthy subjects. Genomic DNA and total RNA was extracted from peripheral blood mononuclear cells. Real-time PCR was performed with specific primers for transcript I [GenBank:DQ131799] and II [GenBank:DQ131800]. Fragments of the CYSLTR1 promoter were amplified by PCR and sequenced directly to identify four single nucleotide polymorphisms: C/T [SNP:rs321029], A/C [SNP:rs2637204], A/G [SNP:rs2806489] and C/T [SNP:rs7066737]. RESULTS: The expression of CysLT1 transcript I and II in asthma did not differ from its expression in healthy control group. However, in major alleles homozygotic CAAC/CAAC women with asthma we found significantly higher expression of transcript I as compared to heterozygous CAAC/TCGC women in that loci. CysLT1 transcript I expression tended to negative correlation with episodes of acute respiratory infection in our asthmatic population. Moreover, expression of CysLT1 transcript II in CAAC/CAAC homozygotic women with asthma was significantly lower than in CAAC/CAAC healthy control females. CONCLUSIONS: Genetic variants of CYSLTR1 promoter might be associated with gender specific expression of CysLT1 alternative transcripts in patients with asthma. CysLT1 splice variants expression might also correlate with the susceptibility to infection in asthmatic population.

Participation of leukotriene C(4) in the regulation of suicidal erythrocyte death.

Eryptosis, the suicidal death of erythrocytes, is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by increase in cytosolic Ca(2+) concentration upon energy depletion. The present study explored the involvement of leukotrienes. Western blotting was employed to detect the cysteinyl-leukotriene receptor cysLT1, competitive immune assay to determine leukotriene release from erythrocytes, Fluo3 fluorescence to estimate cytosolic Ca(2+) concentration, forward scatter to analyse cell volume and annexin V-binding to disclose phosphatidylserine exposure. As a result, erythrocytes expressed the leukotriene receptor CysLT1. Glucose depletion (24 hours) significantly increased the formation of the cysteinyl-leukotrienes C(4)/D(4)/E(4). Leukotriene C(4) (10 nM) increased Ca(2+) entry, decreased forward scatter, activated caspases 3 and 8, and stimulated annexin V-binding. Glucose depletion similarly increased annexin V-binding, an effect significantly blunted in the presence of the leukotriene receptor antagonist cinalukast (1 microM) or the 5-lipoxygenase inhibitor BW B70C (1 microM). In conclusion, upon energy depletion erythrocytes form leukotrienes, which in turn activate cation channels, leading to Ca(2+) entry, cell shrinkage and cell membrane scrambling. Cysteinyl-leukotrienes thus participate in the signaling of eryptosis during energy depletion.

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.

Evaluation of cysteinyl leukotriene-induced contraction of human cultured bronchial smooth muscle cells.

BACKGROUND: Cysteinyl leukotrienes (cysLTs) are major mediators involved in bronchial asthma, particularly bronchial constriction. However, a contractile response of human bronchial smooth muscle cells (hBSMCs) to cysLTs has not been well characterized at cellular level. METHODS: A contraction assay using collagen gel embedded with cultured hBSMCs was established to analyze contractile responses at cellular level. Contractile responses to several constrictors including cysLTs were evaluated in 6-day cultured gels with varying fetal bovine serum (FBS) concentrations. RESULTS: Removal of FBS from the culture for the designated time periods resulted in increased contractile responses. CysLT-induced contraction of the gel became more pronounced and reproducible. CysLT(1)R expression on the hBSMCs was significantly increased by the removal of FBS. CONCLUSION: Contractile responses of hBSMCs to cysLTs can be evaluated at cellular level for the first time. This experimental system may be useful for the in vitro evaluation and future development of cysLTs antagonists.

Effects of KP-496, a novel dual antagonist for cysteinyl leukotriene receptor 1 and thromboxane A2 receptor, on Sephadex-induced airway inflammation in rats.

Bronchial asthma is characterized by chronic airway inflammation. Eosinophils are involved in airway inflammation and play crucial roles in asthma. There is accumulating evidence to suggest contributions of cysteinyl leukotrienes (cysLTs) and thromboxane (TX) A(2) to the recruitment of eosinophils into lung in asthmatics. KP-496 is a novel dual antagonist for CysLT receptor type 1 and TXA(2) receptors. The aim of this study was to evaluate the anti-inflammatory effects of KP-496 on Sephadex-induced airway inflammation. Sephadex suspension was intratracheally injected into rats. Amounts of regulated on activation, normal T cell expressed and secreted (RANTES) and eotaxin, and numbers of infiltrating cells in bronchoalveolar lavage fluid were measured 24 and 48 h after Sephadex injection, respectively. KP-496 (30, 100 microg/head) was intratracheally administered to rats 1 h before and 7 h after Sephadex injection. KP-496 and prednisolone (10 mg/kg, per os) exhibited significant inhibitory effects on infiltration of total cells and eosinophils into lung. Production of RANTES was significantly inhibited by KP-496 and prednisolone. Production of eotaxin was significantly inhibited by prednisolone. KP-496 also inhibited the production of eotaxin, though this effect was not significant. These results demonstrate that KP-496 exhibited the anti-inflammatory effects by inhibiting infiltration of inflammatory cells and productions of RANTES and eotaxin.

Relationship between cysteinyl-leukotriene-1 receptor and human transitional cell carcinoma in bladder.

OBJECTIVES: To investigate the leukotriene (LT) D(4) (LTD(4)) receptor (cysteinyl-LT(1) receptor [CysLT(1)R]) expression in transitional cell carcinoma (TCC) of the bladder, as well as the effects of the CysLT(1)R antagonist on cell proliferation in TCC cell lines. The metabolism of arachidonic acid by either cyclooxygenase or lipoxygenase is thought to play an important role in carcinogenesis. LTD(4) is a pro-inflammatory mediator derived from arachidonic acid through various enzymatic steps, and 5-lipoxygenase is an important factor in generating LTD(4). METHODS: CysLT(1)R expression in TCC tissue and normal bladder tissue was examined. CysLT(1)R expression was detected using immunohistochemistry. The effects of the CysLT(1)R antagonist on TCC cell growth were examined by 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyltetrazolium bromide assay and reverse transcriptase-polymerase chain reaction. Flow cytometry was used to determine whether the CysLT(1)R antagonist induced apoptosis. RESULTS: Initially, only slight CysLT(1)R expression was detected in normal bladder tissues and marked CysLT(1)R expression was detected in the TCC tissues. CysLT(1)R expression was greater in high-grade cancer than in low-grade cancer. Furthermore, CysLT(1)R expression was also greater in advanced-stage cancer than in early-stage cancer. Finally, the CysLT(1)R antagonist caused marked inhibition of TCC cells by inducing early apoptosis. CONCLUSIONS: CysLT(1)R was induced in TCC. The results suggest that the CysLT(1)R antagonist might mediate potent antiproliferative effects on TCC cells. Thus, the target of the CysLT(1)R is potentially a new therapy in the treatment of TCC.

Ação dos leucotrienos na fagocitose via receptores Fc`GAMA´ e manose em macrófagos alveolares e mecanismos moleculares envolvidos / Importance of leukotrienes en phagocytosis of Fc`GAMA´ and mannose receptor in alveolar macrophages and molecular mecanismes involved

Os leucotrienos aumentam a fagocitose e a atividade microbicida contra uma série de patógenos. Em macrófagos alveolares, LTB`IND.4´ e LTD`IND.4´ aumentam a fagocitose via Fc`GAMA´R de modo dependente de PKC. Entretanto, o papel das isoformas específicas da PKC, das MAPK, e da Pi3K neste processo, ainda não é conhecido. Além disso, pouco se sabe sobre a importância dos leucotrienos na fagocitose via outros receptores. Os objetivos deste trabalho são: a) ampliar o conhecimento sobre as vias de sinalização ativadas pelos leucotrienos durante a fagocitose de hemácias opsonizadas por IgG; b) avaliar o efeito dos leucotrienos na fagocitose de Candida albicans, por macrófagos alveolares e as vias de sinalização intracelular envolvidas. Observou-se que os leucotrienos endogenamente produzidos ou adicionados aos macrófagos alveolares, aumentam a fagocitose via Fc`GAMA´R e para isso utilizam distintas vias de sinalização intracelular. A ação do LTB`IND.4´ envolveu predominantemente a via ERK1/2 e PKC`ALFA´ e com menor intensidade da PKC`DELTA´...

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.

Expression of enzymes and receptors of the leukotriene pathway in human neuroblastoma promotes tumor survival and provides a target for therapy.

The metabolism of arachidonic acid by the cyclooxygenase (COX) or lipoxygenase (LO) pathways generates eicosanoids that have been implicated in the pathogenesis of a variety of human diseases, including cancer. In this study, we examined the expression and significance of components within the 5-LO pathway in human neuroblastoma, an embryonal tumor of the sympathetic nervous system. High expression of 5-LO, 5-LO-activating protein (FLAP), leukotriene A(4) hydrolase, leukotriene C(4) synthase, and leukotriene receptors was detected in a majority of primary neuroblastoma tumors and all cell lines investigated. Expression of 5-LO and FLAP was evident in tumor cells but not in nonmalignant adrenal medulla where neuroblastomas typically arise. Moreover, neuroblastoma cells produce leukotrienes, and stimulation of neuroblastoma cells with leukotrienes increased neuroblastoma cell viability. Inhibitors of 5-LO (AA-861), FLAP (MK-886), or the leukotriene receptor antagonist montelukast inhibited neuroblastoma cell growth by induction of G(1)-cell cycle arrest and apoptosis. Similarly, specific 5-LO and leukotriene receptor silencing by small interfering RNA decreased neuroblastoma cell growth. These findings provide new insights into the pathobiology of neuroblastoma, and the use of leukotriene pathway inhibitors as a novel adjuvant therapy for children with neuroblastoma warrants further consideration.

Corticosteroids as inhibitors of cysteinyl leukotriene metabolic and signaling pathways.

BACKGROUND: Corticosteroids (CCSs) do not influence secretion of cysteinyl leukotrienes (CysLTs) that occurs on cellular activation during allergic reactions nor do they modulate bronchospastic responses to inhalation challenges with leukotrienes (LTs). OBJECTIVES: We speculated that CCSs might modulate pathways responsible for CysLT production and diminish the ability of cellular activation to cause their release. Similarly, CCSs could reduce expression of CysLT receptor 1 (CysLTR1) and CysLT receptor 2 (CysLT2R) and modulate their responsiveness. METHODS: We investigated influences of fluticasone on expression of mRNA for LTC(4) synthase (LTC(4)S), CysLT1R, and CysLT2R within T lymphocytes, monocytes, and eosinophils by means of quantitative PCR. Effects on receptor protein expression were evaluated by means of flow cytometry. RESULTS: Circulating immune cells (T cells, monocytes, and eosinophils) express low levels of LTC(4)S mRNA, and this was not influenced by CCSs. However, IL-4 induced transcripts in T lymphocytes, and this was prevented by fluticasone. Paradoxically, CCSs synergized with IL-4 to increase LTC(4)S expression in monocytes. Although not influencing basal or IL-4-stimulated CysLT1R expression, fluticasone inhibited basal CysLT2R transcript expression on monocytes and IL-4-induced expression in all 3 cell types. CONCLUSIONS: In addition to not blocking the acute release of CysLTs on cellular activation, CCSs do not diminish the capacity of cells to synthesize these compounds. CCSs do not diminish CysLT1R expression, consistent with their lack of influence on bronchospasm, which is mediated through this receptor.

Ligation of Toll-like receptor 3 differentially regulates M2 and M3 muscarinic receptor expression and function in human airway smooth muscle cells.

BACKGROUND: Viral infection causes asthma exacerbations and airway hyperreactivity. Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA) of viral or synthetic origin in a fashion different from protein kinase R (PKR). The aim of this study was to examine the expression and function of TLR3 in human airway smooth muscle (ASM) cells. METHODS: Expression of TLR3 and muscarinic receptor (MR), histamine receptor (HR), and cysteinyl leukotriene receptor (CysLTR) subtypes was analyzed by quantitative real-time PCR, flow cytometry, or Western blotting. It was assessed whether ASM cells respond to polyinosinic-polycytidylic acid (poly I:C), a synthetic analog of dsRNA, with alterations in M2R, M3R, H1R, and CysLT1R expression. The function of these subtypes was evaluated by cholinergic regulation of forskolin-stimulated cyclic AMP accumulation or by mobilization of intracellular calcium upon stimulation. RESULTS: ASM cells expressed TLR3 and PKR, and intracellular TLR3 expression was demonstrated. Poly I:C caused decreased M2R and increased M3R expression, without affecting H1R and CysLT1R expression. Poly I:C-treated cells showed decreased cholinergic inhibition of forskolin-stimulated cyclic AMP accumulation and enhanced calcium flux in response to acetylcholine, but not to histamine and LTD4. These modulating effects of poly I:C were reversed by chloroquine, but not by 2-aminopurine. CONCLUSIONS: The data indicate that poly I:C internalized by ASM cells differentially regulates M2R and M3R expression and function by interacting with TLR3 rather than with PKR, suggesting that these changes may contribute to airway hyperreactivity.