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1.
Eur J Pharmacol ; 934: 175257, 2022 Nov 05.
Article in English | MEDLINE | ID: mdl-36116518

ABSTRACT

BACKGROUND: Cysteinyl-maresins, also known as maresin-conjugates in tissue regeneration (MCTRs), are recently discovered lipid mediators proposed to reduce airway inflammation. OBJECTIVE: To investigate the influence of MCTRs on IL-13-induced airway hyperresponsiveness in isolated human and mice airways. METHODS: Before responsiveness to contractile agonists were assessed in myographs, human small bronchi were cultured for 2 days and mouse tracheas were cultured for 1-4 days. During the culture procedure airways were exposed to interleukin (IL)-13 in the presence or absence of MCTRs. Signalling mechanisms were explored using pharmacologic agonists and antagonists, and genetically modified mice. RESULTS: IL-13 treatment increased contractions to histamine, carbachol and leukotriene D4 (LTD4) in human small bronchi, and to 5-hydroxytryptamine (5-HT) in mouse trachea. In both preparations, co-incubation of the explanted tissues with MCTR3 reduced the IL-13 induced enhancement of contractions. In mouse trachea, this inhibitory effect of MCTR3 was blocked by three different CysLT1 receptor antagonists (montelukast, zafirlukast and pobilukast) during IL-13 exposure. Likewise, MCTR3 failed to reduce the IL-13-induced 5-HT responsiveness in mice deficient of the CysLT1 receptor. However, co-incubation with the classical CysLT1 receptor agonist LTD4 did not alter the IL-13-induced 5-HT hyperreactivity. CONCLUSIONS: MCTR3, but not LTD4, decreased the IL-13-induced airway hyperresponsiveness by activation of the CysLT1 receptor. The distinct actions of the two lipid mediators on the CysLT1 receptor suggest an alternative signalling pathway appearing under inflammatory conditions, where this new action of MCTR3 implicates potential to inhibit airway hyperresponsiveness in asthma.


Subject(s)
Interleukin-13 , Leukotriene D4 , Humans , Mice , Animals , Leukotriene D4/pharmacology , Leukotriene D4/physiology , Interleukin-13/pharmacology , Serotonin , Carbachol/pharmacology , Histamine , Receptors, Leukotriene/metabolism , Leukotriene Antagonists
2.
J Cell Mol Med ; 18(9): 1785-96, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24909956

ABSTRACT

GPR17 is a G(i) -coupled dual receptor activated by uracil-nucleotides and cysteinyl-leukotrienes. These mediators are massively released into hypoxic tissues. In the normal heart, GPR17 expression has been reported. By contrast, its role in myocardial ischaemia has not yet been assessed. In the present report, the expression of GPR17 was investigated in mice before and at early stages after myocardial infarction by using immunofluorescence, flow cytometry and RT-PCR. Before induction of ischaemia, results indicated the presence of the receptor in a population of stromal cells expressing the stem-cell antigen-1 (Sca-1). At early stages after ligation of the coronary artery, the receptor was expressed in Sca-1(+) cells, and cells stained with Isolectin-B4 and anti-CD45 antibody. GPR17(+) cells also expressed mesenchymal marker CD44. GPR17 function was investigated in vitro in a Sca-1(+)/CD31(-) cell line derived from normal hearts. These experiments showed a migratory function of the receptor by treatment with UDP-glucose and leukotriene LTD4, two GPR17 pharmacological agonists. The GPR17 function was finally assessed in vivo by treating infarcted mice with Cangrelor, a pharmacological receptor antagonist, which, at least in part, inhibited early recruitment of GPR17(+) and CD45(+) cells. These findings suggest a regulation of heart-resident mesenchymal cells and blood-borne cellular species recruitment following myocardial infarction, orchestrated by GPR17.


Subject(s)
Mesenchymal Stem Cells/physiology , Myocardial Infarction/metabolism , Nerve Tissue Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Animals , Antigens, Ly/metabolism , Cell Movement , Hyaluronan Receptors , Leukocyte Common Antigens/metabolism , Leukotriene D4/pharmacology , Leukotriene D4/physiology , Membrane Proteins/metabolism , Mice, Inbred C57BL , Myocardial Infarction/pathology , Nerve Tissue Proteins/agonists , Purinergic P2Y Receptor Antagonists/pharmacology , Receptors, G-Protein-Coupled/agonists , Uridine Diphosphate Glucose/pharmacology , Uridine Diphosphate Glucose/physiology
3.
Blood ; 119(13): 3113-22, 2012 Mar 29.
Article in English | MEDLINE | ID: mdl-22323449

ABSTRACT

Generation of human monocyte-derived dendritic cells (DCs) for cancer vaccination involves ex vivo maturation in the presence of proinflammatory cytokines and prostaglandin E(2) (PGE(2)). Although the inclusion of PGE(2) during maturation is imperative for the induction of DC migration, PGE(2) has unfavorable effects on the immunostimulatory capacity of these cells. Like PGE(2), leukotrienes (LTs) are potent mediators of DC migration. We therefore sought to characterize the migratory and immunologic properties of DCs that matured in the presence of LTB(4), LTC(4), LTD(4), and PGE(2). Here, we demonstrate that DCs matured in the presence of LTC(4), but not LTB(4) or LTD(4), are superior to PGE(2)-matured DCs in stimulating CD4(+) T-cell responses and in inducing antigen-specific cytotoxic T lymphocytes (CTLs) in vitro without concomitant induction or recruitment of regulatory T cells (Tregs). LTC(4)-matured DCs migrate efficiently through layers of extracellular matrix and secrete higher levels of immunostimulatory IL-12p70 while producing reduced levels of immune-inhibitory IL-10, IL12p40, indoleamine-2,3-dioxidase, and TIMP-1 (tissue inhibitor of matrix metalloproteinases). Intracellular calcium mobilization and receptor antagonist studies reveal that, in contrast to LTD(4), LTC(4) did not signal through CysLTR(1) in DCs. Collectively, our data suggest that LTC(4) represents a promising candidate to replace PGE(2) in DC maturation protocols for cancer vaccination.


Subject(s)
Cell Movement/drug effects , Dendritic Cells/drug effects , Leukotriene C4/pharmacology , Lymphocyte Activation/drug effects , Antigens, Neoplasm/immunology , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cells, Cultured , Dendritic Cells/immunology , Dendritic Cells/metabolism , Dendritic Cells/physiology , Drug Evaluation, Preclinical , Humans , Leukotriene C4/metabolism , Leukotriene D4/metabolism , Leukotriene D4/physiology , Lymphocyte Activation/immunology , Monocytes/drug effects , Monocytes/immunology , Monocytes/metabolism , Monocytes/physiology , Receptors, Leukotriene/agonists , Receptors, Leukotriene/metabolism , Signal Transduction/drug effects , Signal Transduction/immunology , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunology , Up-Regulation/drug effects
4.
J Immunol ; 186(7): 4455-66, 2011 Apr 01.
Article in English | MEDLINE | ID: mdl-21357260

ABSTRACT

The conjunctiva is a mucous membrane that covers the sclera and lines the inside of the eyelids. Throughout the conjunctiva are goblet cells that secrete mucins to protect the eye. Chronic inflammatory diseases such as allergic conjunctivitis and early dry eye lead to increased goblet cell mucin secretion into tears and ocular surface disease. The purpose of this study was to determine the actions of the inflammatory mediators, the leukotrienes and the proresolution resolvins, on secretion from cultured rat and human conjunctival goblet cells. We found that both cysteinyl leukotriene (CysLT) receptors, CysLT(1) and CysLT(2,) were present in rat conjunctiva and in rat and human cultured conjunctival goblet cells. All leukotrienes LTB(4), LTC(4), LTD(4), and LTE(4), as well as PGD(2), stimulated goblet cell secretion in rat goblet cells. LTD(4) and LTE(4) increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), and LTD(4) activated ERK1/2. The CysLT(1) receptor antagonist MK571 significantly decreased LTD(4)-stimulated rat goblet cell secretion and the increase in [Ca(2+)](i). Resolvins D1 (RvD1) and E1 (RvE1) completely reduced LTD(4)-stimulated goblet cell secretion in cultured rat goblet cells. LTD(4)-induced secretion from human goblet cells was blocked by RvD1. RvD1 and RvE1 prevented LTD(4)- and LTE(4)-stimulated increases in [Ca(2+)](i), as well as LTD(4) activation of ERK1/2. We conclude that cysteinyl leukotrienes stimulate conjunctival goblet cell mucous secretion with LTD(4) using the CysLT(1) receptor. Stimulated secretion is terminated by preventing the increase in [Ca(2+)](i) and activation of ERK1/2 by RvD1 and RvE1.


Subject(s)
Conjunctiva/metabolism , Conjunctiva/pathology , Docosahexaenoic Acids/therapeutic use , Eicosapentaenoic Acid/analogs & derivatives , Goblet Cells/metabolism , Goblet Cells/pathology , Leukotriene D4/physiology , Leukotriene E4/physiology , Aged , Animals , Cells, Cultured , Docosahexaenoic Acids/biosynthesis , Docosahexaenoic Acids/physiology , Eicosapentaenoic Acid/biosynthesis , Eicosapentaenoic Acid/physiology , Eicosapentaenoic Acid/therapeutic use , Female , Humans , Inflammation Mediators/antagonists & inhibitors , Inflammation Mediators/physiology , Inflammation Mediators/therapeutic use , Leukotriene C4/antagonists & inhibitors , Leukotriene C4/physiology , Leukotriene D4/antagonists & inhibitors , Leukotriene E4/antagonists & inhibitors , Male , Middle Aged , Organ Culture Techniques , Rats , Rats, Sprague-Dawley , Receptors, Leukotriene/metabolism
5.
Pharmacology ; 85(5): 311-8, 2010.
Article in English | MEDLINE | ID: mdl-20453555

ABSTRACT

BACKGROUND: Histamine and cysteinyl leukotrienes are pivotal mast cell mediators which contribute considerably and likely complementary to the symptoms of allergic rhinitis. Currently, we sought to explore the direct actions of histamine and leukotriene D(4) (LTD(4)), a cysteinyl leukotriene, on porcine nasal arteries and veins. We also studied combined blocks of histamine and cysteinyl leukotrienes using loratadine and montelukast in an in vivo model of allergy-mediated nasal inflammation. METHODS: For the evaluation of the action of histamine and LTD(4) on arteries and veins, porcine nasal mucosa was isolated and cut into slices (100-300 microm thick). Real-time images of the nasal arteries and veins were recorded and vessel activities estimated by changes in cross-sectional area before and after the tested drugs. For the in vivo studies, the effect of loratadine and montelukast given alone and in combination was examined on upper airway inflammation in ovalbumin-sensitized and -challenged Brown Norway rats. RESULTS: Both histamine (0.001-10 micromol/l) and LTD(4) (0.001-10 micromol/l) produced a concentration-dependent increase in the lumen area of nasal mucosa arteries and veins. Histamine (0.01 micromol/l) alone produced a 24 and 12% increase in cross-sectional areas of arteries and veins, respectively. LTD(4) (0.001 micromol/l) alone increased artery and vein dilation by about 17 and 9%, respectively. Combination treatment with histamine (0.01 micromol/l) and LTD(4) (0.001 micromol/l) increased vessel dilation by 65% (arteries) and 26% (veins). In our in vivo Brown Norway rat studies, oral loratadine (0.01-10 mg/kg) and montelukast (0.01-10 mg/kg) significantly reduced antigen-induced total nasal inflammatory cell infiltration in a dose-dependent manner. The antiinflammatory dose-response curve of loratadine was shifted to the left when studied in combination with montelukast (0.01 mg/kg). Similarly, the dose-response characteristics of montelukast (0.01-10 mg/kg) was shifted in the presence of loratadine (0.01 mg/kg). CONCLUSION: Our studies support the position that histamine and cysteinyl leukotrienes may act collaboratively to elicit allergic nasal pathologies such as upper airway inflammation and nasal vessel dilation (which may translate into increased nasal mucosal engorgement). Furthermore, the current results are supportive of the hypothesis that combined treatment of allergic rhinitis with an H(1) receptor antagonist and a CysLT(1) receptor antagonist may have greater benefit than sole treatment with these agents alone.


Subject(s)
Cysteine/physiology , Histamine/physiology , Leukotrienes/physiology , Nasal Mucosa/blood supply , Nasal Mucosa/drug effects , Rhinitis/drug therapy , Acetates/pharmacology , Acetates/therapeutic use , Animals , Cyclopropanes , Cysteine/antagonists & inhibitors , Dose-Response Relationship, Drug , Drug Synergism , Drug Therapy, Combination , Female , Histamine H1 Antagonists, Non-Sedating/pharmacology , Histamine H1 Antagonists, Non-Sedating/therapeutic use , Hypersensitivity/drug therapy , In Vitro Techniques , Leukotriene Antagonists/pharmacology , Leukotriene Antagonists/therapeutic use , Leukotriene D4/antagonists & inhibitors , Leukotriene D4/physiology , Loratadine/pharmacology , Loratadine/therapeutic use , Male , Nasal Mucosa/pathology , Neutrophil Infiltration/drug effects , Quinolines/pharmacology , Quinolines/therapeutic use , Rats , Rats, Inbred BN , Rhinitis/immunology , Sulfides , Sus scrofa
6.
J Immunol ; 182(11): 6789-98, 2009 Jun 01.
Article in English | MEDLINE | ID: mdl-19454674

ABSTRACT

Cytokines and chemokines control hematopoietic stem and progenitor cell (HPC) proliferation and trafficking. However, the role of nonpeptide mediators in the bone marrow microenvironment has remained elusive. Particularly CysLT(1), a G protein-coupled receptor recognizing inflammatory mediators of the cysteinyl leukotriene family, is highly expressed in HPCs. We therefore analyzed the effects of its ligands on human CD34(+) HPCs. The most potent CysLT(1) ligand, LTD(4), rapidly and significantly up-regulated alpha(4)beta(1) and alpha(5)beta(1) integrin-dependent adhesion of both primitive and committed HPC. LTD(4)-triggered adhesion was inhibited by specific CysLT(1) antagonists. The effects of other CysLT(1) ligands were weak (LTC(4)) or absent (LTE(4)). In serum-free liquid cultures supplemented with various hematopoietic cytokines including IL-3, only LTD(4) significantly augmented the expansion of HPCs in a dose-dependent manner comparable to that of peptide growth factors. LTC(4) and LTE(4) were less effective. In CD34(+) cell lines and primary HPCs, LTD(4) induced phosphorylation of p44/42 ERK/MAPK and focal adhesion kinase-related tyrosine kinase Pyk2, which is linked to integrin activation. Bone marrow stromal cells produced biologically significant amounts of cysteinyl leukotrienes only when hematopoietic cells were absent, suggesting a regulatory feedback mechanism in the hematopoietic microenvironment. In contrast to antagonists of the homing-related G protein-coupled receptor CXCR4, administration of a CysLT(1) antagonist failed to induce human CD34(+) HPC mobilization in vivo. Our results suggest that cysteinyl leukotriene may contribute to HPC retention and proliferation only when cysteinyl leukotriene levels are increased either systemically during inflammation or locally during marrow aplasia.


Subject(s)
Cell Adhesion , Cell Proliferation , Hematopoietic Stem Cells/cytology , Integrin alpha4beta1/metabolism , Integrin alpha5beta1/metabolism , Leukotriene D4/physiology , Receptors, Leukotriene/agonists , Antigens, CD34 , Bone Marrow Cells , Cell Communication , Cells, Cultured , Humans , Phosphorylation , Stromal Cells , Up-Regulation/physiology
7.
J Immunol ; 182(6): 3819-26, 2009 Mar 15.
Article in English | MEDLINE | ID: mdl-19265161

ABSTRACT

Lipoxins (LX) are a class of eicosanoid that possesses a wide spectrum of antiinflammatory and proresolution bioactions. Here we have investigated the impact of the endogenously produced eicosanoid LXA(4) on endothelial cell inflammatory, proliferative, and antigenic responses. Using HUVECs we demonstrate that LXA(4) inhibits vascular endothelial growth factor (VEGF)-stimulated inflammatory responses including IL-6, TNF-alpha, IFN-gamma and IL-8 secretion, as well as endothelial ICAM-1 expression. Interestingly, LXA(4) up-regulated IL-10 production from HUVECs. Consistent with these antiinflammatory and proresolution responses to LXA(4), we demonstrate that LXA(4) inhibited leukotriene D(4) and VEGF-stimulated proliferation and angiogenesis as determined by tube formation of HUVECs. We have explored the underlying molecular mechanisms and demonstrate that LXA(4) pretreatment is associated with the decrease of VEGF-stimulated VEGF receptor 2 (KDR/FLK-1) phosphorylation and downstream signaling events including activation of phospholipase C-gamma, ERK1/2, and Akt.


Subject(s)
Angiogenesis Inhibitors/physiology , Endothelium, Vascular/pathology , Endothelium, Vascular/physiopathology , Inflammation Mediators/physiology , Lipoxins/physiology , Cell Proliferation , Cells, Cultured , Coculture Techniques , Cytokines/biosynthesis , Cytokines/physiology , Endothelium, Vascular/immunology , Humans , Inflammation Mediators/antagonists & inhibitors , Inflammation Mediators/metabolism , Leukotriene D4/physiology , Neovascularization, Pathologic/immunology , Phosphorylation/immunology , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/metabolism
8.
Mol Immunol ; 46(6): 1204-11, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19223078

ABSTRACT

In alveolar macrophages, leukotriene (LT) B(4) and cysteinyl LTs (LTC(4), LTD(4) and LTE(4)) both enhance Fc gamma receptor (Fc gammaR)-mediated phagocytosis. In the present study we investigated the role of specific PKC isoforms (PKC-alpha and -delta), the MAP kinases p38 and ERK 1/2, and PI3K in mediating the potentiation of Fc gammaR-mediated phagocytosis induced by addition of leukotrienes to the AMs. It was found that exogenously added LTB(4) and LTD(4) both enhanced PKC-delta and -alpha phosphorylation during Fc gammaR engagement. Studies with isoform-selective inhibitors indicated that exogenous LTB(4) effects were dependent on both PKC-alpha and -delta, while LTD(4) effects were exclusively due to PKC-delta activation. Although both exogenous LTB(4) and LTD(4) enhanced p38 and ERK 1/2 activation, LTB(4) required only ERK 1/2, while LTD(4) required only p38 activation. Activation by both LTs was dependent on PI3K activation. Effects of endogenous LTs on kinase activation were also investigated using selective LT receptor antagonists. Endogenous LTB(4) contributed to Fc gammaR-mediated activation of PKC-alpha, ERK 1/2 and PI3K, while endogenous cysLTs contributes to activation of PKC-delta, p38 and PI3K. Taken together, our data show that the capacities of LTB(4) and LTD(4) to enhance Fc gammaR-mediated phagocytosis reflect their differential activation of specific kinase programs.


Subject(s)
Leukotriene B4/physiology , Leukotriene D4/physiology , Macrophages, Alveolar/immunology , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase C/metabolism , Receptors, IgG/physiology , Animals , Cells, Cultured , Female , Leukotriene B4/pharmacology , Leukotriene D4/pharmacology , Macrophages, Alveolar/drug effects , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Phagocytosis , Phosphoinositide-3 Kinase Inhibitors , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Protein Kinase C/antagonists & inhibitors , Rats , Rats, Wistar , Receptors, IgG/immunology
9.
J Immunol ; 182(1): 530-7, 2009 Jan 01.
Article in English | MEDLINE | ID: mdl-19109185

ABSTRACT

Eicosanoid lipid mediators, including prostaglandin E(2) (PGE(2)) and leukotrienes (LTs) B(4) and D(4), are produced in abundance in the infected lung. We have previously demonstrated that individually, PGE(2) suppresses while both classes of LTs augment alveolar macrophage (AM) innate immune functions. In this study, we sought to more appropriately model the milieu at a site of infection by studying the in vitro effects of these lipid mediators on Fc gammaR-mediated phagocytosis when they are present in combination. Consistent with their individual actions, both LTB(4) and LTD(4) opposed the suppressive effect of PGE(2) on phagocytosis, but only LTB(4) did so by mitigating the stimulatory effect of PGE(2) on intracellular cAMP production. Unexpectedly, we observed that IgG-opsonized targets themselves elicited a dose-dependent reduction in intracellular cAMP in AMs, but this was not observed in peritoneal macrophages or elicited peritoneal neutrophils; this effect in AMs was completely abolished by treatment with the LT synthesis inhibitor AA861, the BLT receptor 1 antagonist CP 105,696, and the G alpha i inhibitor pertussis toxin. Of two downstream cAMP effectors, protein kinase A and exchange protein activated by cAMP, the ability of PGE(2) to activate the latter but not the former was abrogated by both LTs B(4) and D(4). Taken together, our results indicate that both classes of LTs oppose the immune suppressive actions of PGE(2), with the stimulatory actions of LTB(4) reflecting combinatorial modulation of intracellular cAMP and those of LTD(4) being cAMP independent.


Subject(s)
Cell Communication/immunology , Cyclic AMP/metabolism , Dinoprostone/physiology , Leukotriene B4/physiology , Macrophages, Alveolar/immunology , Macrophages, Alveolar/metabolism , Phagocytosis/immunology , Animals , Cells, Cultured , Combinatorial Chemistry Techniques , Cyclic AMP/physiology , Dinoprostone/metabolism , Drug Synergism , Female , Leukotriene B4/metabolism , Leukotriene D4/physiology , Rats , Rats, Wistar , Receptors, IgG/antagonists & inhibitors , Receptors, IgG/physiology , Signal Transduction/immunology , Up-Regulation/immunology
10.
Am J Respir Cell Mol Biol ; 39(2): 227-34, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18323532

ABSTRACT

Leukotriene (LT)D(4) is suggested to play a role in airway remodeling, which is characterized by fibrogenesis and airway smooth muscle cell hyperplasia. In this study, we investigated the effects of LTD(4) on the expression of furin, a proprotein convertase involved in the maturation/activation of several substrates implicated in the remodeling processes. HEK293 cells stably transfected with the CysLT1 receptor were used to study the transcriptional regulation of furin by LTD(4). Stimulation of the cells with LTD(4) resulted in a time- and concentration-dependent induction of furin mRNA and protein expression. The study of furin gene (fur) promoters P1, P1A, and P1B revealed a selective transactivation of the P1 promoter by LTD(4). Mutations in the activator protein (AP)-1-binding element of the P1 promoter resulted in the partial loss of transactivation by LTD(4). Binding of AP-1 transcription factor to fur P1 promoter after stimulation with LTD(4) was demonstrated by electrophoretic mobility shift assay, and supershift assays indicated the formation of c-Jun/c-Fos complexes. LTD(4) induced the maturation of the furin substrates membrane-type 1 matrix metalloproteinase and transforming growth factor-beta1, which was inhibited by the furin inhibitor alpha1-PDX. Finally, LTD(4) induced furin gene expression in monocytic THP-1 cells, which was abrogated using a selective CysLT1 receptor antagonist and inhibitors of the mitogen-activated protein kinases MEK-1, p38, and JunK. Our data show for the first time that LTD(4), via the CysLT1 receptor, can transcriptionally activate furin production with consequent maturation of furin substrates relevant to airway remodeling. These findings suggest that CysLT1 is involved in remodeling processes through modulation of furin transcription.


Subject(s)
Furin/biosynthesis , Leukotriene D4/pharmacology , Receptors, Leukotriene/physiology , Cell Line , Humans , Leukotriene D4/physiology , Matrix Metalloproteinase 14/metabolism , Mitogen-Activated Protein Kinases/metabolism , Promoter Regions, Genetic , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Signal Transduction , Transcriptional Activation , Transforming Growth Factor beta1/metabolism , Up-Regulation
11.
Pulm Pharmacol Ther ; 21(2): 276-84, 2008.
Article in English | MEDLINE | ID: mdl-17611132

ABSTRACT

The bronchoconstriction caused by inhaled neurokinin A (NKA) in patients with asthma is indirect. The mediators involved in NKA-induced bronchoconstriction are unknown. Studies with various H1 receptor antagonists were negative, making an important contribution of histamine unlikely. To study the role of cysteinyl leukotrienes in neurokinin A-induced bronchoconstriction, we performed a randomised, double-blind, cross-over, placebo controlled trial in 12 patients with mild to moderate asthma. Zafirlukast and matching placebo were given orally, 40 mg the evening before and 40 mg the morning of assessment. In one period NKA was administered, in the other period leukotriene D4 (LTD4). Increasing concentrations of NKA and LTD4 were inhaled from a 30 L bag, after nebulization via a Mallinckrodt nebuliser. The difference between log10PC20LTD4 after treatment with placebo or zafirlukast was highly significant (p<0.0001). A trend was observed towards a difference between log10PC20 neurokinin A after treatment with placebo or zafirlukast (p=0.0741). The dose ratio for the neurokinin A provocation was 4.4 and for the LTD4 provocation 67.7. In conclusion, zafirlukast had a large inhibitory effect on LTD4-induced bronchoconstriction, but offered only limited protective effect against neurokinin A-induced bronchoconstriction. We suggest that leukotrienes play a limited role in the bronchoconstrictor effect of neurokinin A in patients with asthma.


Subject(s)
Asthma/physiopathology , Bronchoconstriction/drug effects , Leukotriene Antagonists/pharmacology , Leukotriene D4/pharmacology , Neurokinin A/pharmacology , Tosyl Compounds/pharmacology , Adult , Arachidonate 5-Lipoxygenase/genetics , Bronchial Provocation Tests , Cross-Over Studies , Dose-Response Relationship, Drug , Double-Blind Method , Glutathione Transferase/genetics , Humans , Indoles , Leukotriene D4/antagonists & inhibitors , Leukotriene D4/physiology , Neurokinin A/antagonists & inhibitors , Neurokinin A/physiology , Phenylcarbamates , Polymorphism, Genetic , Sulfonamides
12.
J Immunol ; 179(8): 5454-61, 2007 Oct 15.
Article in English | MEDLINE | ID: mdl-17911632

ABSTRACT

Leukotrienes (LTs) are lipid mediators implicated in asthma and other inflammatory diseases. LTB(4) and LTD(4) also participate in antimicrobial defense by stimulating phagocyte functions via ligation of B leukotriene type 1 (BLT1) receptor and cysteinyl LT type 1 (cysLT1) receptor, respectively. Although both Galpha(i) and Galpha(q) proteins have been shown to be coupled to both BLT1 and cysLT1 receptors in transfected cell systems, there is little known about specific G protein subunit coupling to LT receptors, or to other G protein-coupled receptors, in primary cells. In this study we sought to define the role of specific G proteins in pulmonary alveolar macrophage (AM) innate immune responses to LTB(4) and LTD(4). LTB(4) but not LTD(4) reduced cAMP levels in rat AM by a pertussis toxin (PTX)-sensitive mechanism. Enhancement of FcgammaR-mediated phagocytosis and bacterial killing by LTB(4) was also PTX-sensitive, whereas that induced by LTD(4) was not. LTD(4) and LTB(4) induced Ca(2+) and intracellular inositol monophosphate accumulation, respectively, highlighting the role of Galpha(q) protein in mediating PTX-insensitive LTD(4) enhancement of phagocytosis and microbicidal activity. Studies with liposome-delivered G protein blocking Abs indicated a dependency on specific Galpha(q/11) and Galpha(i3) subunits, but not Galpha(i2) or G(beta)gamma, in LTB(4)-enhanced phagocytosis. The selective importance of Galpha(q/11) protein was also demonstrated in LTD(4)-enhanced phagocytosis. The present investigation identifies differences in specific G protein subunit coupling to LT receptors in antimicrobial responses and highlights the importance of defining the specific G proteins coupled to heptahelical receptors in primary cells, rather than simply using heterologous expression systems.


Subject(s)
GTP-Binding Proteins/metabolism , Macrophage Activation/immunology , Macrophages, Alveolar/immunology , Receptors, Leukotriene/metabolism , Animals , Cells, Cultured , Cyclic AMP/antagonists & inhibitors , Cyclic AMP/metabolism , Down-Regulation/immunology , Female , GTP-Binding Protein alpha Subunit, Gi2/biosynthesis , GTP-Binding Protein alpha Subunit, Gi2/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/biosynthesis , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , GTP-Binding Proteins/biosynthesis , GTP-Binding Proteins/physiology , Intracellular Fluid/metabolism , Leukotriene B4/antagonists & inhibitors , Leukotriene B4/physiology , Leukotriene D4/antagonists & inhibitors , Leukotriene D4/physiology , Macrophages, Alveolar/metabolism , Rats , Rats, Wistar , Receptors, Leukotriene/physiology , Toxoids/pharmacology
13.
Med Res Rev ; 27(4): 469-527, 2007 Jul.
Article in English | MEDLINE | ID: mdl-16894531

ABSTRACT

Cysteinyl-leukotrienes (cysteinyl-LTs), that is, LTC4, LTD4, and LTE4, trigger contractile and inflammatory responses through the specific interaction with G protein-coupled receptors (GPCRs) belonging to the purine receptor cluster of the rhodopsin family, and identified as CysLT receptors (CysLTRs). Cysteinyl-LTs have a clear role in pathophysiological conditions such as asthma and allergic rhinitis (AR), and have been implicated in other inflammatory conditions including cardiovascular diseases, cancer, atopic dermatitis, and urticaria. Molecular cloning of human CysLT1R and CysLT2R subtypes has confirmed most of the previous pharmacological characterization and identified distinct expression patterns only partially overlapping. Interestingly, recent data provide evidence for the immunomodulation of CysLTR expression, the existence of additional receptor subtypes, and of an intracellular pool of CysLTRs that may have roles different from those of plasma membrane receptors. Furthermore, genetic variants have been identified for the CysLTRs that may interact to confer risk for atopy. Finally, a crosstalk between the cysteinyl-LT and the purine systems is being delineated. This review will summarize and attempt to integrate recent data derived from studies on the molecular pharmacology and pharmacogenetics of CysLTRs, and will consider the therapeutic opportunities arising from the new roles suggested for cysteinyl-LTs and their receptors.


Subject(s)
Asthma/physiopathology , Leukotriene C4/physiology , Leukotriene D4/physiology , Leukotriene E4/physiology , Membrane Proteins/physiology , Receptors, Leukotriene/physiology , Adult , Animals , Asthma/drug therapy , Cardiovascular Diseases/physiopathology , Child , Child, Preschool , Dermatitis, Atopic/drug therapy , Dermatitis, Atopic/etiology , Female , Humans , Hydroxyurea/adverse effects , Hydroxyurea/analogs & derivatives , Leukotriene Antagonists/adverse effects , Leukotriene Antagonists/therapeutic use , Membrane Proteins/drug effects , Membrane Proteins/genetics , Pharmacogenetics , Receptors, Leukotriene/drug effects , Receptors, Leukotriene/genetics , Receptors, Purinergic/physiology , Recombinant Proteins/pharmacology , Rhinitis, Allergic, Seasonal/drug therapy , Rhinitis, Allergic, Seasonal/physiopathology , SRS-A/biosynthesis , Tissue Distribution
14.
J Allergy Clin Immunol ; 117(5): 1155-62, 2006 May.
Article in English | MEDLINE | ID: mdl-16675346

ABSTRACT

BACKGROUND: Dendritic cells (DCs) acquire, during their maturation, the expression of the chemokine receptor CCR7 and the ability to migrate to lymph nodes in response to CC chemokine ligand 19 (CCL19). This migration is impaired in mice lacking the leukotriene (LT) C4 transporter and restored by addition of exogenous LTC4. OBJECTIVE: To define the role of LT in human DC function, we studied the expression and function of the cysteinyl-leukotriene (CysLT) receptors during DC differentiation from monocytes and subsequent maturation. METHODS: Receptor expression was measured by flow cytometry and real-time PCR. Responsiveness to LTD4 stimulation was assessed by calcium flux and chemotaxis. RESULTS: Maturation of DC with LPS, a classic Toll-like receptor 4 agonist, reduced CysLT receptor 1 (CysLT1) expression by 50%, whereas CysLT receptor 2 expression was increased. In contrast, the Toll-like receptor 3 agonist poly inosinic and cytidylic acid (polyI:C) had no effect on receptor expression. Downregulation of CysLT1 expression by LPS could not be mimicked by TNF-alpha alone or in combination with IL-1beta or IL-6. It was, however, prevented by inhibitors of COX and could be reproduced by a combination of TNF-alpha and prostaglandin E2. Immature DCs and DCs matured with polyI:C, but not with LPS, responded to LTD4 with a robust cytosolic calcium flux, which was prevented by the CysLT1 antagonist montelukast. LTD4 induced DC chemotaxis and enhanced DC migration in response to CCL19 in DCs matured with polyI:C, but only weakly in DCs matured with LPS. CONCLUSION: Our data suggest that human DCs may differentially respond to leukotriene, depending on their maturational stimuli. CLINICAL IMPLICATIONS: Our study demonstrates that some microbial agents can reduce the migration of dendritic cells in response to leukotrienes, with potential for differential involvement of these cells in allergic inflammation.


Subject(s)
Dendritic Cells/metabolism , Leukotriene C4/physiology , Leukotriene D4/physiology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Receptors, Leukotriene/genetics , Receptors, Leukotriene/metabolism , Toll-Like Receptors/agonists , Calcium/metabolism , Cells, Cultured , Chemotaxis, Leukocyte/immunology , Cysteine/physiology , Dendritic Cells/immunology , Humans , Leukotriene C4/biosynthesis , Leukotriene D4/biosynthesis , Leukotrienes/physiology
15.
Oncogene ; 25(50): 6660-5, 2006 Oct 26.
Article in English | MEDLINE | ID: mdl-16715140

ABSTRACT

The cysteinyl leukotriene1 (CysLT1) receptor (CysLT1R) enhances survival and proliferation of intestinal cells via distinct pathways. Here, we have demonstrated that there is significant endogenous production of CysLTs from both non-tumour- and tumour-derived intestinal epithelial cells. Treatment of two non-tumour cell lines, Int 407 and IEC-6, with CysLT1R antagonists led to shrinkage and detachment of cells, confirmed as apoptotic cell death, and a dose-dependent reduction in proliferation. However, in the tumour intestinal cell lines Caco-2, SW480, HCT-116 and HT-29, treatment with CysLT1R antagonists significantly reduced proliferation, but had no effect on apoptosis. A unique characteristic of intestinal cancer cells is the presence of nuclear CysLT1Rs, which are inaccessible to receptor antagonists. In these cells, inhibition of the endogenous production of CysLTs indirectly, by 5-lipoxygenase inhibition, impaired CysLT1R signalling throughout the cell, and resulted in apoptosis of the tumour cells. These data reveal the existence of constitutive CysLT1R signalling that mediates both survival and proliferation in intestinal cells. Importantly, we propose that tumour-derived intestinal cells are resistant to CysLT1R antagonist-induced apoptosis, a phenomena that could be explained by nuclear CysLT1R signalling.


Subject(s)
Intestinal Mucosa/metabolism , Leukotriene D4/physiology , Membrane Proteins/metabolism , Receptors, Leukotriene/metabolism , Apoptosis/drug effects , Autocrine Communication/physiology , Caco-2 Cells , Cell Line , Cell Proliferation/drug effects , Cell Survival/physiology , HCT116 Cells , HT29 Cells , Humans , Leukotriene D4/biosynthesis , Lipoxygenase Inhibitors , Membrane Proteins/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction
16.
J Neurosci ; 26(15): 4004-14, 2006 Apr 12.
Article in English | MEDLINE | ID: mdl-16611817

ABSTRACT

Drosophila larval neuromuscular junction (NMJ) is a well established preparation enabling quantitative analyses of synaptic physiology at identifiable synapses. Here, we report the first characterization of synaptic long-term depression (LTD) at the Drosophila NMJ. LTD can be reliably induced by specific patterns of tetanic stimulation, and the level of LTD depends on both stimulus frequency and Ca2+ concentration. We provide evidence that LTD is likely a result of presynaptic changes. Through screening of targeted mutants with defects in memory or signal transduction pathways, we found that LTD is strongly reduced in the akt mutants. This defect can be rescued by acutely induced expression of the normal akt transgene, suggesting that altered LTD is not attributable to developmental abnormalities and that Akt is critical for the induction of LTD. Our study also indicates that the molecular mechanisms of LTD are distinct from that of short-term synaptic plasticity, because akt mutants showed normal short-term facilitation and posttetanic potentiation, whereas LTD was unaffected in mutants that exhibit defective short-term synaptic plasticity, such as dunce and rutabaga. The characterization of LTD allows genetic analysis of the molecular mechanisms of long-term synaptic plasticity in Drosophila and provides an additional assay for studying functions of genes pertaining to synaptic and behavioral plasticity.


Subject(s)
Leukotriene D4/physiology , Neuromuscular Junction/physiology , Proto-Oncogene Proteins c-akt/physiology , Animals , Calcium/physiology , Calcium Chloride/pharmacology , Drosophila/growth & development , Larva/physiology , Neuromuscular Junction/drug effects , Proto-Oncogene Proteins c-akt/drug effects
17.
J Biol Chem ; 281(10): 6776-84, 2006 Mar 10.
Article in English | MEDLINE | ID: mdl-16407243

ABSTRACT

Increased levels of the inflammatory mediator leukotriene D4 (LTD4) are present at sites of inflammatory bowel disease, and such areas also exhibit an increased risk for subsequent cancer development. It is known that LTD4 affects the expression of many proteins that influence survival and proliferation of intestinal epithelial cells. We demonstrate here that after LTD4 exposure, beta-catenin translocates to the nucleus where it signals activation of the TCF/LEF family of transcription factors. These events are mediated via a phosphatidylinositol 3-kinase-dependent phosphorylation of the inhibitory Ser-9 residue of glycogen synthase kinase 3beta. We also show that in the presence of LTD4, free beta-catenin translocates to the mitochondria where it associates with the cell survival protein Bcl-2. We hypothesize that LTD4 may enhance cell survival via activation of beta-catenin signaling, in particular, by promoting the association of beta-catenin with Bcl-2 in the mitochondria. Similar to Wnt-1 signaling, LTD4 signals an increased level of free beta-catenin and elevated TCF/LEF promotor activity. This work in intestinal epithelial cells further lends credence to the idea that inflammatory signaling pathways are intrinsically linked with potential oncogenic signals involved in cell survival and apoptosis.


Subject(s)
Apoptosis/physiology , Leukotriene D4/physiology , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction/physiology , beta Catenin/metabolism , Cell Line , Cell Survival , Cell Transformation, Neoplastic/metabolism , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Intestinal Mucosa/enzymology , Intestinal Mucosa/metabolism , Microscopy, Fluorescence , Mitochondria/metabolism , Protein Transport , Proto-Oncogene Proteins c-bcl-2/genetics , TCF Transcription Factors/biosynthesis , TCF Transcription Factors/genetics , beta Catenin/genetics
18.
Bull Acad Natl Med ; 190(7): 1511-8; discussion 1518-21, 2006 Oct.
Article in French | MEDLINE | ID: mdl-17450683

ABSTRACT

Leukotrienes are potent inflammatory mediators synthesized locally within the cardiovascular system through the 5-lipoxygenase pathway of arachidonic acid metabolism. The leukotrienes, consisting of dihydroxy leukotriene LTB4 and the cysteinyl leukotrienes LTC4, LTD4 and LTE4, act by targeting cell surface receptors expressed on inflammatory cells and on structural cells of vessel walls. LTB, induces leukocyte activation and chemotaxis via high- and low-affinity receptor subtypes (BLT1 and BLT2), respectively. Recently, BLT, receptors were found on human vascular smooth muscle cells, inducing their migration and proliferation. Cysteinyl leukotrienes are vasoconstrictors and induce endothelium-dependent vascular responses through the CysLT, and CysLT2 receptor subtypes. There is also pharmacological evidence for the existence of further CysLT receptor subtypes. Taken together, experimental and genetic studies suggest a major role of leukotrienes in atherosclerosis and in its ischemic complications such as acute coronary syndromes and stroke. Furthermore, the effects on vascular smooth muscle cells suggest a role in the vascular remodeling observed after coronary angioplasty, as well as in aortic aneurysm. Further experimental and clinical studies are needed to determine the potential of therapeutic strategies targeting the leukotriene pathway in cardiovascular disease.


Subject(s)
Cardiovascular Diseases/drug therapy , Cardiovascular Diseases/physiopathology , Leukotriene Antagonists/therapeutic use , Leukotrienes/physiology , Receptors, Leukotriene/physiology , Angioplasty, Balloon, Coronary , Animals , Aortic Aneurysm/etiology , Aortic Aneurysm/physiopathology , Arachidonic Acid/metabolism , Atherosclerosis/physiopathology , Cardiovascular Diseases/etiology , Cardiovascular Diseases/metabolism , Cell Movement , Coronary Restenosis/physiopathology , Disease Models, Animal , Guinea Pigs , Humans , Hypertension/physiopathology , Leukotriene B4/metabolism , Leukotriene B4/physiology , Leukotriene C4/metabolism , Leukotriene C4/physiology , Leukotriene D4/metabolism , Leukotriene D4/physiology , Leukotriene E4/metabolism , Leukotriene E4/physiology , Leukotrienes/blood , Leukotrienes/metabolism , Leukotrienes/urine , Mice , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/physiology , Rats , Stroke/physiopathology
19.
Clin Immunol ; 117(2): 177-81, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16109507

ABSTRACT

Fibroblasts play an important role in the repair and remodeling processes following injury. Leukotriene D4 (LTD4) is a potent mediator in inflammatory processes, but the direct effect of cysteinyl leukotrienes on fibroblast migration remains unelucidated. In this study, the effect of the LTD4 on normal human lung fibroblasts (NHLF) chemotaxis induced by human plasma fibronectin (HFn) was investigated using the modified Boyden's chamber technique. LTD4 potentiated NHLF chemotaxis to HFn in concentration-dependent manner. A specific cysteinyl leukotriene receptor type 1 antagonist, pranlukast inhibited this effect, indicating that LTD4 affected cell migration via its specific receptor. The potentiating effect of LTD4 on fibroblast chemotaxis was completely abolished by pertussis toxin (PTX), suggesting that LTD4-induced effect was dependent on PTX-sensitive Gi/o signaling. These findings suggest that LTD4 has a potential to augment fibroblast chemotaxis, and to contribute to regulation of the wound healing and following remodeling in fibrotic processes of the lung.


Subject(s)
Adjuvants, Immunologic/pharmacology , Chemotaxis/immunology , Fibroblasts/physiology , Fibronectins/physiology , Leukotriene D4/physiology , Cells, Cultured , Dose-Response Relationship, Drug , Flow Cytometry , Humans , Lung/cytology , Lung/physiology , Pertussis Toxin/pharmacology , U937 Cells
20.
Biochem Soc Trans ; 33(Pt 4): 698-700, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16042577

ABSTRACT

The inflammatory mediator LTD4 (leukotriene D4) is present at high levels in many inflammatory conditions, and areas of chronic inflammation have an increased risk for subsequent cancer development. We demonstrate here that following LTD4 stimulation, beta-catenin is translocated to the nucleus, triggering the transcriptional activity of the TCF (T-cell factor)/LEF (lymphoid enhancer factor) family of transcription factors. These events are dependent on phosphoinositide-3 kinase activation and glycogen synthase kinase inhibition. Our data suggest that, similar to Wnt signalling, LTD4 increases free beta-catenin and targets it to the nucleus.


Subject(s)
Leukotriene D4/physiology , Oncogenes , Transcription, Genetic , Gene Expression Regulation , Humans , Inflammation/physiopathology , Inflammatory Bowel Diseases/genetics , Protein Transport
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