CysLT1 Expression Research Articles

Montelukast Inhibits Lung Cancer Cell Migration by Suppressing Cysteinyl Leukotriene Receptor 1 Expression In vitro.

Lung cancer is a major threat to public health and remains difficult to treat. Repositioning of existing drugs has emerged as a therapeutic strategy in lung cancer. Clinically, low-dose montelukast has been used to treat asthma. We evaluated the potential of using montelukast to treat lung cancer. Migration was detected using wound-healing and Transwell assays, the expression of CysLT1 using western blotting, and subcellular localization of CysLT1 using immunofluorescence. CRISPR/Cas9 technology was used to further investigate the function of CysLT1. Subcellular localization staining showed that the CysLT1 distribution varied in murine and human lung cancer cell lines. Furthermore, montelukast suppressed CysLT1 expression in lung cancer cells. The treated cells also showed weaker migration ability compared with control cells. Knockout of CysLT1 using CRISPR/Cas9 editing in A549 cells further impaired the cell migration ability. Montelukast inhibits the migration of lung cancer cells by suppressing CysLT1 expression, demonstrating the potential of using CysLT1 as a therapeutic target in lung cancer.

Baicalin Ameliorates Radiation-Induced Lung Injury by Inhibiting the CysLTs/CysLT1 Signaling Pathway.

Objective Radiation-induced lung injury (RILI) is a common complication of radiotherapy for thoracic tumors. This study investigated the alleviating effect of baicalin (BA) on RILI and its possible mechanism. Methods RILI model was established by chest irradiation (IR) of C57BL/6 mice for 16 weeks. Different concentrations of BA were administered, and dexamethasone (DXM) was used as a positive control. Then, the lung pathological changes were observed by HE and Masson staining. The levels of TGF-β, TNF-α, IL-1β, IL-6, CysLT, LTC4, and LTE4 were measured by ELISA. The CysLT1 expression was detected by qPCR, immunohistochemistry, and western blot. Type II AEC cells were pretreated with LTD-4 to establish the RILI cell model and intervened with different concentrations of BA. Then, the collagen I protein level was measured by ELISA. The CysLT1 and α-SMA expression were detected by qPCR, immunofluorescence, and western blot. Results BA could effectively improve lung histopathological changes and pulmonary fibrosis. In vivo, BA could inhibit the levels of TGF-β, TNF-α, IL-1β, and IL-6 and reduce the levels of CysLT, LTC4, and LTE4. In vitro, different concentrations of LTD4 could reduce the viability of type II AEC cells, which could be reversed by the administration of different concentrations of BA. In addition, BA could reduce CysLT1 mRNA, as well as CysLT1 and α-SMA protein levels in vitro and in vivo. Conclusion BA attenuated lung inflammation and pulmonary fibrosis by inhibiting the CysLTs/CysLT1 pathway, thereby protecting against RILI.

High Cysteinyl Leukotriene Receptor 1 Expression Correlates with Poor Survival of Uveal Melanoma Patients and Cognate Antagonist Drugs Modulate the Growth, Cancer Secretome, and Metabolism of Uveal Melanoma Cells.

Simple SummaryThis research investigates the disease relevance and therapeutic potential of cysteinyl leukotriene receptors in uveal melanoma (UM), a rare eye cancer that often spreads to the liver. Unfortunately, there are no therapies available to stop the spread of UM and patients are often faced with an extremely poor prognosis. We assess whether the cysteinyl leukotriene receptors (CysLT1 and CysLT2) are relevant to the progression of UM. Using UM patient samples, we identified that increased levels of CysLT1 in tumours is associated with reduced patient survival. Using UM cell lines and zebrafish models, we found that drugs targeting CysLT1, but not CysLT2, can alter hallmarks of cancer including cell growth, proliferation, and metabolism. This study is the first to examine the relationship of the CysLT receptors with clinical features of UM. Our data strengthen the importance of CysLT signalling in UM and suggest that antagonism of CysLT1 may be of therapeutic interest in the disease.Metastatic uveal melanoma (UM) is a rare, but often lethal, form of ocular cancer arising from melanocytes within the uveal tract. UM has a high propensity to spread hematogenously to the liver, with up to 50% of patients developing liver metastases. Unfortunately, once liver metastasis occurs, patient prognosis is extremely poor with as few as 8% of patients surviving beyond two years. There are no standard-of-care therapies available for the treatment of metastatic UM, hence it is a clinical area of urgent unmet need. Here, the clinical relevance and therapeutic potential of cysteinyl leukotriene receptors (CysLT1 and CysLT2) in UM was evaluated. High expression of CYSLTR1 or CYSLTR2 transcripts is significantly associated with poor disease-free survival and poor overall survival in UM patients. Digital pathology analysis identified that high expression of CysLT1 in primary UM is associated with reduced disease-specific survival (p = 0.012; HR 2.76; 95% CI 1.21–6.3) and overall survival (p = 0.011; HR 1.46; 95% CI 0.67–3.17). High CysLT1 expression shows a statistically significant (p = 0.041) correlation with ciliary body involvement, a poor prognostic indicator in UM. Small molecule drugs targeting CysLT1 were vastly superior at exerting anti-cancer phenotypes in UM cell lines and zebrafish xenografts than drugs targeting CysLT2. Quininib, a selective CysLT1 antagonist, significantly inhibits survival (p < 0.0001), long-term proliferation (p < 0.0001), and oxidative phosphorylation (p < 0.001), but not glycolysis, in primary and metastatic UM cell lines. Quininib exerts opposing effects on the secretion of inflammatory markers in primary versus metastatic UM cell lines. Quininib significantly downregulated IL-2 and IL-6 in Mel285 cells (p < 0.05) but significantly upregulated IL-10, IL-1β, IL-2 (p < 0.0001), IL-13, IL-8 (p < 0.001), IL-12p70 and IL-6 (p < 0.05) in OMM2.5 cells. Finally, quininib significantly inhibits tumour growth in orthotopic zebrafish xenograft models of UM. These preclinical data suggest that antagonism of CysLT1, but not CysLT2, may be of therapeutic interest in the treatment of UM.

Erratum to "Tetrandrine Ameliorates Airway Remodeling of Chronic Asthma by Interfering TGF-β1/Nrf-2/HO-1 Signaling Pathway-Mediated Oxidative Stress".

[This corrects the article DOI: 10.1155/2019/7930396.].

Tetrandrine Ameliorates Airway Remodeling of Chronic Asthma by Interfering TGF-β1/Nrf-2/HO-1 Signaling Pathway-Mediated Oxidative Stress.

Background Imbalanced oxidative stress and antioxidant defense are involved in airway remodeling in asthma. It has been demonstrated that Tetrandrine has a potent role in antioxidant defense in rheumatoid arthritis and hypertension. However, the correlation between Tetrandrine and oxidative stress in asthma is utterly blurry. This study aimed to investigate the role of Tetrandrine on oxidative stress-mediated airway remolding. Materials and Methods Chronic asthma was established by ovalbumin (OVA) administration in male Wistar rats. Histopathology was determined by HE staining. Immunofluorescence was employed to detect the expression of α-SMA and Nrf-2. Level of oxidative stress and matrix metalloproteinases were examined by ELISA kits. Cell viability and cell cycle of primary airway smooth muscle cells (ASMCs) were evaluated by CCK8 and flow cytometry, respectively. Signal molecules were detected using western blot. Results Tetrandrine effectively impairs OVA-induced airway inflammatory and airway remodeling by inhibiting the expression of CysLT1 and CysLTR1. The increase of oxidative stress and subsequent enhancement of MMP9 and TGF-β1 expression were rescued by the administration of Tetrandrine in the rat model of asthma. In in vitro experiments, Tetrandrine markedly suppressed TGF-β1-evoked cell viability and cell cycle promotion of ASMCs in a dose-dependent manner. Furthermore, Tetrandrine promoted Nrf-2 nuclear transcription and activated its downstream HO-1 in vivo and in vitro. Conclusion Tetrandrine attenuates airway inflammatory and airway remodeling in rat model of asthma and TGF-β1-induced cell proliferation of ASMCs by regulating oxidative stress in primary ASMCs, suggesting that Tetrandrine possibly is an effective candidate therapy for asthma.

CYSLTR1 promotes adenoid hypertrophy by activating ERK1/2.

Cysteinyl leukotriene receptor 1 (CYSLTR1) serves a pivotal role in allergic reactions, which is one of the main causes of adenoid hypertrophy. The present study aimed to investigate the function of CYSLT1 within adenoid hypertrophy. A total of 40 patients with adenoid hypertrophy were recruited between January 2014 and January 2016 at the Children's Hospital of Hebei Province, China. The patients were divided into either the mild-moderate group or the severe group according to their disease severity. The expression of CYSLT1 in the adenoid tissue and whole blood of all patients and healthy controls was detected by reverse transcription-quantitative polymerase chain reaction. Associations between the expression level of CYSLT1 and the clinical characteristics of patients were analyzed. Primary human adenoid epithelial cells (HAECs) with CYSLT1 knockdown and overexpression were constructed. The levels of extracellular signal-regulated kinase (ERK)2 and phosphorylated-ERK1/2 in adenoid tissue and HAECs were detected by western blot analysis. The expression of CYSLT1 in adenoid tissue and whole blood of all patients with adenoid hypertrophy was significantly higher compared with the healthy controls (P<0.05). In addition, the expression level of CYSLT1 was significantly higher in the severe group compared with the mild-moderate group (P<0.05). The highest level of p-ERK1/2 in adenoid tissue was observed in the severe group, followed by the mild-moderate group and then the control group (P<0.05). CYSLT1 expression was positively associated with the severity of disease. CYSLT1 knockdown significantly decreased the level of p-ERK1/2 in HAECs (P<0.05), while CYSLT1 overexpression significantly increased the level of p-ERK1/2. It was concluded that CYSLT1 may contribute to the progression of adenoid hypertrophy by activating ERK1/2.

Genes involved in leukotriene synthesis pathway are dynamically regulated during lung development in Rhesus monkeys

BackgroundLeukotrienes play critical roles in many inflammatory lung diseases and several antagonists of their receptors have been used in the clinical settings. However, the physiological functions of leukotrienes in lung development are still unclear. MethodThe expression levels of 34 genes involved in leukotriene synthesis and function pathway in the lungs of Rhesus monkey during different developmental time points were determined on a MiSeq platform and analyzed by the reads per kilobase of transcript per million mapped reads (RPKM) method. ResultsThe results showed that the expression levels of PLA2G1B, PLA2G10, PLA2G2D, ALOX5, and ALOX5AP increased dramatically in the lung of Rhesus monkey, reflecting the changes in the pulmonary environment after delivery. Additionally, the different expression patterns between molecules related to LTB4 and LTC4 synthesis suggested distinct roles of LTB4 and LTC4 in lung development. Finally, the constant expression of CysLT1 during the development process provided new information to the pharmaceutical basis of the use of leukotriene receptor antagonists in the clinical setting. ConclusionThe expression levels of several key genes involved in leukotriene synthesis changed dramatically during lung development in Rhesus monkeys, suggesting the potential roles of leukotrienes in lung development in this animal model.

Enhanced cysteinyl-leukotriene type 1 receptor expression in T cells from house dust mite-allergic individuals following stimulation with Der p.

In order to determine the potential for allergen to modulate T cell expression of the CysLT1 receptor and responsiveness to leukotrienes, peripheral blood mononuclear cells from house dust mite-allergic or nonallergic individuals were incubated with D. pteronyssinus allergen (Der p). Baseline CysLT1 expression was similar in both groups of donors, but Der p significantly enhanced CysLT1 expression in CD4+ and CD8+ T cells of only allergic individuals and induced enhanced responsiveness of CD4+ T cells to LTD4 in terms of calcium mobilisation. This effect was prevented by the CysLT1 antagonist MK571. Der p also induced IL-4 and IL-10 production, and neutralizing antibody to IL-4 prevented both the enhanced CysLT1 expression and the enhanced responsiveness of T cells to LTD4 induced by Der p. In allergic individuals, Der p also induced T cell proliferation and a Th2-biased phenotype. Our data suggest that, in allergen-sensitized individuals, exposure to allergen can enhance T cell expression of CysLT1 receptors through a mechanism involving IL-4 production. This, in turn, would induce CD4+ T cell responsiveness to cysteinyl-leukotrienes and Th2 cell activation.

Cysteinyl Leukotriene Signaling Aggravates Myocardial Hypoxia in Experimental Atherosclerotic Heart Disease

BackgroundCysteinyl-leukotrienes (cys-LT) are powerful spasmogenic and immune modulating lipid mediators involved in inflammatory diseases, in particular asthma. Here, we investigated whether cys-LT signaling, in the context of atherosclerotic heart disease, compromises the myocardial microcirculation and its response to hypoxic stress. To this end, we examined Apoe−/− mice fed a hypercholesterolemic diet and analysed the expression of key enzymes of the cys-LT pathway and their receptors (CysLT1/CysLT2) in normal and hypoxic myocardium as well as the potential contribution of cys-LT signaling to the acute myocardial response to hypoxia.Methods and principal findingsMyocardial biopsies from Apoe−/− mice demonstrated signs of chronic inflammation with fibrosis, increased apoptosis and expression of IL-6, as compared to biopsies from C57BL/6J control mice. In addition, we found increased leukotriene C4 synthase (LTC4S) and CysLT1 expression in the myocardium of Apoe−/− mice. Acute bouts of hypoxia further induced LTC4S expression, increased LTC4S enzyme activity and CysLT1 expression, and were associated with increased extension of hypoxic areas within the myocardium. Inhibition of cys-LT signaling by treatment with montelukast, a selective CysLT1 receptor antagonist, during acute bouts of hypoxic stress reduced myocardial hypoxic areas in Apoe−/− mice to levels equal to those observed under normoxic conditions. In human heart biopsies from 14 patients with chronic coronary artery disease mRNA expression levels of LTC4S and CysLT1 were increased in chronic ischemic compared to non-ischemic myocardium, constituting a molecular basis for increased cys-LT signaling.ConclusionOur results suggest that CysLT1 antagonists may have protective effects on the hypoxic heart, and improve the oxygen supply to areas of myocardial ischemia, for instance during episodes of sleep apnea.

Reduced expression of the prostaglandin E2 receptor E-prostanoid 2 on bronchial mucosal leukocytes in patients with aspirin-sensitive asthma

Prostaglandin E(2) (PGE(2)) is thought to play a role in the pathogenesis of aspirin-sensitive asthma (ASA). We sought to extend our previous observations implicating impaired inflammatory cell responsiveness to PGE(2) as a pathogenetic mechanism in patients with aspirin-sensitive rhinosinusitis to the bronchial mucosa in patients with ASA. Immunohistochemistry was used to enumerate inflammatory cells and their expression of cysteinyl leukotriene receptors 1 and 2 (CysLT(1) and CysLT(2)) and the PGE(2) receptors E-prostanoid 1 to 4 (EP(1)-EP(4)) in bronchial biopsy specimens from patients with ASA, patients with aspirin-tolerant asthma, and control subjects (n= 15 in each group). Concentrations of PGE(2) in bronchoalveolar lavage fluid were measured by using ELISA. The effects of PGE(2) and EP receptor agonists on CD3/CD28-stimulated cytokine production by PBMCs were measured by using ELISA. Airways responsiveness to LTD(4)in vivo was measured in asthmatic patients by means of bronchial challenge. Compared with patients with aspirin-tolerant asthma, patients with ASA had increased bronchial mucosal neutrophil and eosinophil numbers but reduced percentages of T cells, macrophages, mast cells, and neutrophils expressing EP(2). Both groups showed increased bronchial sensitivity to inhaled LTD(4), but this did not correlate with mucosal expression of CysLT(1) or CysLT(2). Bronchoalveolar lavage fluid PGE(2) concentrations were comparable in all groups. In vitro PGE(2) inhibited cytokine production by PBMCs through EP(2) but not other PGE(2) receptors. Our data are consistent with the hypothesis that impaired inhibition of inflammatory leukocytes by PGE(2) acting through the EP(2) receptor has a role in the pathogenesis of ASA.

Enhanced CysLT1 Expression In T Cells From House Dust Mite-Allergic Individuals Following Stimulation With Der P1

Enhanced CysLT1 Expression In T Cells From House Dust Mite-Allergic Individuals Following Stimulation With Der P1

Concentration-Dependent Noncysteinyl Leukotriene Type 1 Receptor-Mediated Inhibitory Activity of Leukotriene Receptor Antagonists

The use of cysteinyl leukotriene receptor antagonists (LTRAs) for asthma therapy has been associated with a significant degree of interpatient variability in response to treatment. Some of that variability may be attributable to noncysteinyl leukotriene type 1 receptor (CysLT(1))-mediated inhibitory mechanisms that have been demonstrated for this group of drugs. We used a model of CysLT(1) signaling in human monocytes to characterize CysLT(1)-dependent and -independent anti-inflammatory activity of two chemically different, clinically relevant LTRAs (montelukast and zafirlukast). Using receptor-desensitization experiments in monocytes and CysLT(1)-transfected HEK293 cells and IL-10- and CysLT(1) small interfering RNA-induced downregulation of CysLT(1) expression, we showed that reported CysLT(1) agonists leukotriene D(4) and UDP signal through calcium mobilization, acting on separate receptors, and that both pathways were inhibited by montelukast and zafirlukast. However, 3-log greater concentrations of LTRAs were required for the inhibition of UDP-induced signaling. In monocytes, UDP, but not leukotriene D(4), induced IL-8 production that was significantly inhibited by both drugs at micromolar concentrations. At low micromolar concentrations, both LTRAs also inhibited calcium ionophore-induced leukotriene (leukotriene B(4) and leukotriene C(4)) production, indicating 5-lipoxygenase inhibitory activities. We report herein that montelukast and zafirlukast, acting in a concentration-dependent manner, can inhibit non-CysLT(1)-mediated proinflammatory reactions, suggesting activities potentially relevant for interpatient variability in response to treatment. Higher doses of currently known LTRAs or new compounds derived from this class of drugs may represent a new strategy for finding more efficient therapy for bronchial asthma.

CysLT1 expression and function are downregulated during dendritic cell maturation with zymosan: Role of IL-10 and prostaglandins

CysLT1 expression and function are downregulated during dendritic cell maturation with zymosan: Role of IL-10 and prostaglandins

Expression of cysLT1 and cysLT2 Receptor in Chronic Hyperplastic Eosinophilic Sinusitis

Elevated production of cysteinyl leukotrienes (cysLTs) from sinus tissues and abundant sinus eosinophils are characteristic features of chronic hyperplastic eosinophilic sinusitis (CHS). CysLTs exert their action through G-protein-coupled receptors named cysLTs receptor type I (cysLT1R) and type II (cysLT2R). These expressions of cysLT receptors in the sinus mucosa have yet to be clarified and the relationship between eosinophilia and the expression of these receptors remains obscure. We compared the expressions of cysLT1R and cysLT2R in the sinus mucosa in patients with CHS, non-eosinophilic chronic sinusitis (NECS), and control sinus tissues; and analyzed the correlation between the expression of CysLTRs and the presence of sinus eosinophils by immunohistochemistry and real-time PCR. A significantly higher percentage of eosinophils expressing cysLT2R protein was observed in patients with CHS compared with NECS and controls. In addition, cysLT2R mRNA expression in CHS was significantly higher than in NECS and controls. Furthermore, a positive correlation was observed between cysLT2R mRNA expression and the number of infiltrated eosinophils. In contrast, the cysLT1R mRNA expression did not differ significantly among these groups. The effect of cysLTs on sinus eosinophils may be mediated through the cysLT2R in patients with CHS. These results may suggest the therapeutic benefit of cysLT2R antagonists in CHS.

Poster Round 5.1: Inflammation and Host Response

Background Helicobacter pylori-induced corpus-predominant gastritis appears to carry the highest risk for developing gastric cancer. The alteration of arachidonic acid metabolism, especially via the cyclooxygenase-2 (COX-2) pathway, was shown to play an important role in gastric carcinogenesis. Whether the 5-lipoxygenase (5-LOX) pathway is also involved in gastric carcinogenesis is not known. Aim To evaluate the effects of H. pylori eradication in subjects with corpus-predominant gastritis with respect to histology and the expression of 5-lipoxygenase and leukotriene receptors. Methods Patients with H. pylori-induced corpus-predominant gastritis received triple therapy for H. pylori eradication. Five years later, esophagogastroduodenoscopy with biopsies was repeated. The expression of 5-LOX, leukotriene B4 receptor (BLT)1, and cysteinyl-leukotriene receptor (CysLT)1 was analyzed by immunohistochemistry. COX-2 expression was assessed as internal control. Results Twelve patients were found to have a persistent H. pylori infection 5 years after the eradication therapy and were matched for age and sex to those with successful eradication in a 1 : 1 ratio. None of the study participants developed intraepithelial neoplasia or gastric cancer. Gastritis improved only in patients with successful eradication therapy. The expression of CysLT1 was decreased, and that of BLT1 was increased in patients with successful eradication (p < .05), but not in those with persistent H. pylori infection. The expression of 5-LOX was unchanged in both patients with successful eradication and those with eradication failure. COX-2 was significantly down-regulated only in subjects with successful H. pylori eradication (p < .05). Conclusions H. pylori eradication led to a resolution of corpus-predominant gastritis, and restored the expression of the enzymes/receptors involved in the COX/5-LOX pathway. In this way, the risk for developing gastric cancer may be reduced.

G-CSF Priming before HA Chemotherapy Increased Apoptosis and Chemotaxis in Myelodysplastic Syndromes Cell Line MUTZ-1 by Activating the MEK/ERK Dependent Signal Pathway.

Myelodysplastic syndrome (MDS) represents a heterogenous group of clonal stem cell disorders with qualitative and quantitative abnormalities of blood cells and a high probability of evolving to acute leukemia. Intensive induction chemotherapy in order to reduce the malignant clone and reconstruct normal hematopoiesis is a classic therapy for MDS, especially high risk MDS. Homoharringtonine (HHT), one of the alkaloids from a Chinese natural plant, Cephalotaxus, incombination with cytarabine (A-rac) (HA chemotherapy) has been used successfully in the treatment of MDS. It has been recently reported that the effect of HA chemotherapy could be enhanced by granulocyte colony stimulating factor (G-CSF) (HAG chemotherapy). But the mechanism underlying the enhanced effectiveness in HAG remains unknown. We investigated the effect and mechanism of HAG chemotherapy on the apoptosis and chemotaxis in MDS cell line MUTZ-1. Cells were divided into HA group (HHT + Ara-C) and HAG group (treated with G-CSF before HA). Growth inhibition was measured by MTT method and 50% inhibiting concentration (IC50) was calculated. Cell cycle and apoptosis were measured by flow cytometry. Mitochondria membrane electric potential was determined by confocal microscopy. The mRNA expression of the chemokine receptor CXCR4 and the cysteinyl leukotriene receptor Cys-LT1 were detected by RT-PCR. The protein expression of Cys-LT1 and signaling molecular in MUTZ-1 cells was detected by western blot. The chemotaxsis of MUTZ-1 cells was detected by transwell migration assay. We found that the percentage of apoptotic MUTZ-1 cells increased and the mitochondria membrane electric potential in the cells decreased significantly more in HAG group than in HA group. The percentage in S phase of the cell cycle increased after exposure to G-CSF for 4 hours, and at its peak after 6 hours. Cys-LT1 mRNA and protein expression and chemotactic migration increased more in HAG group than in HA group, while no significant change was observed in CXCR4 expression. G-CSF activated both mitogen activated protein kinase kinase (MEK1/2) and extracellular signal-regulated kinase (ERK1/2). Chemotactic migration was partly blocked by the MEK1/2 inhibitor U0126. These results indicated that G-CSF priming before HA chemotherapy increased apoptosis and chemotaxis in MDS cell line MUTZ-1, and MEK/ERK signaling pathway is involved in G-CSF-induced chemotaxis.

The G Protein-Coupled Receptor cysLT1 Is Expressed in Acute Myeloid Leukemia Mediating Cell Migration and Survival.

Previous studies have shown that the G protein-coupled receptor (GPCR) cysLT1, which recognizes inflammatory lipid mediators (cysteinyl-leukotrienes), is expressed in immature hematopoietic and tumor (eg. colon cancer) cells. In the present study, we analyzed leukemic blasts from 19 patients with newly diagnosed acute myeloid leukemia for analysis of expression and function of cysLT1. By RT-PCR, cysLT1 mRNA was found in all primary AML cells. Quantitative TaqMan PCR demonstrated high levels particularly in acute myeloblastic leukemia without maturation (FAB M1). There was also a positive correlation between the cysLT1 mRNA level and the expression of CD34 and CD-117 (c-kit), indicating that high cysLT1 expression corresponds to an AML phenotype resembling normal hematopoietic progenitor cells. CysLT1 was functionally active in AML blasts, as demonstrated by intracellular calcium fluxes and actin polymerization induced by the ligand LTD4. Similar to mRNA expression, strongest responses were seen in AML FAB M1. As LTD4 can be produced in the bone marrow by stromal cells and may contribute to bone marrow infiltration of AML, chemotaxis was analyzed. Surprisingly, already low concentrations (10nM) induced significant chemotaxis of AML blasts, while higher concentrations (up to 1 uM) were less effective in a dose-dependent manner. Incubation of myeloid leukemic cell lines (eg. KG1a) and primary AML blasts with the specific cysLT1 antagonist MK571 resulted in significantly reduced viability after 48 h in a dose-dependent manner (10nM-10uM), suggesting also an autocrine function of cysLT1 ligands. To explore signal transduction pathways involved in leukemic cell proliferation and chemotaxis, we found that in AML cell lines, LTD4 induced phosphorylation of Erk/MAP kinase, wich is related to proliferation, and Pyk2, which represents a focal adhesion kinase-like signaling molecule that links GPCRs with cell migration, while the Akt pathway was not involved. We conclude that cysLT1 is consistently expressed in acute myeloid leukemia, and mediates both chemotactic and proliferative responses. Therefore, cysLT1 antagonists, which are already used in the treatment of allergy, may improve the effectiveness of antileukemic therapy.

Expression of eicosanoid receptors subtypes and eosinophilic inflammation: implication on chronic rhinosinusitis

BackgroundEicosanoid receptors are G-protein-coupled receptors playing an important immunomodulatory role in airway diseases. However, there is little information on the expression of these receptors and their link with eosinophilic inflammation in paranasal sinus diseases. We aimed with this study to investigate the tissue expression of leukotrienes and prostaglandin E2 receptors in chronic rhinosinusitis patients and the link of this regulation with eosinophilic inflammation.MethodsSamples were prepared from nasal tissue of patients with chronic rhinosinusitis without nasal polyps (CRS, n = 11), with nasal polyps (CRS-NP, n = 13) and healthy subjects (Controls, n = 6). mRNA expression of CysLT1, CysLT2, BLT1, BLT2, E-prostanoid receptors (EP1, EP2, EP3, EP4) and sol-IL-5Rα was determined by real-time PCR. Concentrations of PGE2, LTC4/D4/E4, LTB4 and sol-IL-5Rα were determined by ELISA and of ECP by ImmunoCap. Protein expression and tissue localization of eicosanoid receptors and activated eosinophils were evaluated by immunohistochemistry.ResultsCysLT1 mRNA expression was significantly increased in CRS-NP compared to CRS and controls, and CRS compared to controls, whereas CysLT2 mRNA was enhanced in both CRS groups without differences between them. Levels of both receptors correlated to the number of activated eosinophils, sol-IL-5Rα, ECP and LTC4/D4/E4 concentrations in the disease groups. PGE2 protein concentrations and prostanoid receptors EP1 and EP3 were down-regulated in the CRS-NP tissue vs. CRS and controls, whereas EP2 and EP4 expression was enhanced in CRS and CRS-NP patients vs. controls. No differences in BLT receptors were observed between patients and controls.ConclusionCyLTs receptors are up-regulated in nasal polyp tissue and their expression correlate with eosinophilic inflammation supporting previous results. Eicosanoid receptors mRNA pattern observed suggests that down-regulation of EP1 and EP3 in CRS-NP and up-regulation EP2 and EP4 in CRS and CRS-NP groups may have some role in the development of the diseases and their regulation may not be directly linked to eosinophil activation but involve post-transcriptional events mainly related to other inflammatory cell sources.

Leukotriene D4 enhances immunoglobulin production in CD40-activated human B lymphocytes

Cysteinyl-leukotrienes (cysLTs) are lipid mediators recognized for their role in asthma and allergic inflammation. CysLT1, one of the receptors for cysLTs, is expressed, among others, in bronchial smooth muscle cells, phagocytes, and B lymphocytes. The potential role of CysLT1 in B lymphocytes remains unexplored. We examined the modulation of expression and function of CysLT1 in human B lymphocytes. Human B lymphocytes were purified from peripheral blood and analyzed by means of RT-PCR and flow cytometry, after culture with IL-4 and anti-CD40 antibody or CD154-transfected fibroblasts. Functional responses to leukotriene (LT) D4 in terms of cytosolic calcium flux, proliferation, and immunoglobulin production were also examined. B lymphocytes expressed CysLT1 at both the mRNA and protein levels. Two-fold to 3-fold enhancement of CysLT1 expression was observed after B-cell exposure to a combination of activating anti-CD40 antibody and IL-4. The expression of CysLT1 was also enhanced when B lymphocytes were cocultured with CD154-transfected fibroblasts in the presence of IL-4. Moreover, IL-4 and CD40-activated B lymphocytes showed an increased responsiveness to LTD4 in terms of cytosolic calcium flux, which was totally prevented by the selective CysLT1 antagonist montelukast. Stimulation of IL-4 and CD40-activated B lymphocytes with picomolar concentrations of LTD4 induced mature epsilon transcripts and upregulated IgE and IgG production 2-fold to 3-fold. We have demonstrated that expression of CysLT1 can be upregulated in B lymphocytes after stimulation with CD154, with consequent increased responsiveness of the cells to LTD4 in terms of immunoglobulin production. This study provides evidence that cysLTs generated during allergic reactions can affect B-cell function and enhance IgE and IgG production, which may further contribute to the allergic process.

Lipoxin, leukotriene, and PDGF receptors cross-talk to regulate mesangial cell proliferation.

The lipoxygenase-derived leukotrienes (LTs) are important proinflammatory lipid mediators. Lipoxins (LXs), more recently described lipoxygenase products, modulate many proinflammatory actions of LTs and have impressive proresolution properties. Mesangial cell (MC) proliferation is a central event in the pathogenesis of glomerulonephritis. LTD4-induced proliferation of mesangial cells is modulated by LXA4. Here, we demonstrate that LXA4 inhibits PDGF- and LTD4-stimulated proliferation through modulation of platelet-derived growth factor receptor beta (PDGFRbeta) activation. Specifically, we demonstrate that LTD4 transactivates the PDGFRbeta, a process associated with c-src recruitment and ras activation. We demonstrate expression of cysLT1 and cysLT2 receptors in MCs. LTD4-induced c-src activation was insensitive to pertussis toxin and the cysLT1 receptor antagonist Zafirlukast but was blocked by the nonselective antagonist Pobilukast. We show that LXA4 inhibits LTD4-stimulated activation of the PDGFRbeta and that LXA4 modulates PDGF-BB-stimulated tyrosine phosphorylation of the PDGFRb and subsequent mitogenic events. Furthermore, expression of recombinant LXA4 receptor (ALXR) in CHOK1 cells was associated with an attenuation of serum-stimulated proliferation. These data demonstrate that LXA4 receptor (ALXR) activation is accompanied by antimitogenic effects coupled with inactivation of growth factor receptors, highlighting the complex cross-talk between G protein-coupled receptors and receptor tyrosine kinases in an inflammatory milieu. These data elaborate on the profile of cell signaling events that underpin the anti-inflammatory and proresolution bioactions of LX.