Joshua A Boyce
Brigham And Women´s Hospital
Project start date: 2002-08-01
Project end date: 2012-12-31
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CystLT And P2Y Receptors And Lung Inflammation
Joshua A Boyce, Associate Professor Of Med Harvard Med S
Brigham And Women s Hospital Research Administration Boston, Ma 02115
Grant 5R01AI052353-05 from National Institute Of Allergy And Infectious Diseases IRG: LBPA
Abstract: Mast cells (MCs) are important in allergic diseases, tissue injury, and protection from infections. Indirect evidence suggests that the effector function of MCs is controlled by T cells, yet little is understood concerning this process in humans due to the limitations of human studies in vivo, and to technical difficulties in the isolation, purification, and maintenance in culture. Cysteinyl leukotrienes (cys-LTs), a class of peptide- conjugated lipids generated by MCs, signal through at least two known 7 transmembrane-spanning, G protein-coupled receptors (GPCR), termed the CysLT1 and CysLT2 receptors. Each is homologous to the purinergic (P2Y) receptors for nucleotides. Human MCs (hMCs) express the CysLT1 receptor, which unexpectedly mediates activation responses to both cys-LTs and to the extracellular nucleotide agonist uridine diphosphate (UDP). Furthermore, priming of hMCs with recombinant IL-4 preferentially enhances their sensitivity to both LTC4 and UDP without a concomitant change in CysLT1 receptor expression, suggesting that either a third cys-LT receptor (CysLT3) is induced by IL-4, or that the fundamental pharmacologic properties of CysLT1 are altered by IL-4 priming so as to selectively lower its threshold for LTC4 and UDP binding over LTD4. We cys-LTs and UDP induce the generation of multiple cytokines (EL-5, macrophage inflammatory protein 1beta, and tumor necrosis factor [TNF]- alpha) by IL-4-primed hMCs, and that blockade of endogenous cys- LT receptors or interference with endogenous cys-LT synthesis attenuate the generation of both IL-5 and TNF-alpha in response to cross-linking of the high-affinity Fc receptor for IgE (Fc epsilon RI). We propose the following hypotheses 1). Cys-LTs mediate both autocrine and paracrine functions of hMCs through at least two GPCRs that also bind uridine nucleotides, linking neurogenic signals and tissue injury to MCs and inflammation in asthma; 2) these same receptors permit MCs to initiate inflammatory responses to injury and infection in other tissues. Specific Aim 1 focuses on identifying the CysLT3 receptor and defining the mechanism(s) responsible for the observed IL-4- induced upregulation of hMC responses to UDP and LTC4. Specific Aim 2 uses site-directed mutagenesis to define the regions of the CysLT1 (and possible CysLT3) receptors involved in binding of LTD4, LTC4, and UDP, respectively. Specific Aim 3 will explore the functional role of the GPCRs for cys-LT and UDP in mediating MC-dependent inflammatory responses in vivo using targeted deletion of their respective genes.
Keywords: inflammation, leukocyte activation /transformation, leukotriene, mast cell, purinergic receptor, receptor expression, antibody receptor, asthma, autocrine, hormone receptor, macrophage inflammatory protein, paracrine, tumor necrosis factor alpha, uridine diphosphate, cord blood, human tissue, laboratory mouse, site directed mutagenesis, tissue /cell culture
Project start date: 2002-08-01
Project end date: 2007-07-14
5R01AI052353-05 (2006): $408079
5R01AI052353-04 (2005): $417900
5R01AI052353-03 (2004): $417900
5R01AI052353-02 (2003): $417900
CysLT And P2Y Receptors In Lung Inflammation
Joshua A Boyce, Associate Professor Of Med Harvard Med S
Brigham And Women´s Hospital
Grant 5R01AI052353-07 from National Institute Of Allergy And Infectious Diseases IRG: LCMI
Project start date: 2002-08-01
Project end date: 2012-12-31
Grants awarded to Joshua A Boyce
REGULATION OF PULMONARY PROSTAGLANDINS BY LEUKOTRIENE E4
Joshua A Boyce, Associate Professor
Brigham And Women´s Hospital, Research Administration, Boston, Ma 02115
Grant 1R01AI078908-01A1 from National Institute Of Allergy And Infectious Diseases
Abstract: This proposal seeks to determine the mechanisms and receptors by which leukotriene (LT)E4, the most stable ligand of the cysteinyl leukotrienes (cys-LTs), activates mast cells (MCs) and promotes pulmonary inflammation. An abundance of historical information supports the existence of a distinct receptor for LTE4 Our published studies demonstrate that LTE4 is the most potent cys-LT for both for human MC proliferation in vitro and potentiation of airway inflammation in vivo, and it activates human MCs by a mechanism independent of CysLT1R and CysLT2R. Our Preliminary Studies strongly indicate that the P2Y12 receptor, an adenosine diphosphate-responsive GPCR, is also the LTE4-reactive receptor (hereafter termed P2Y12/CysLT3R) mediating the effects of LTE4 on MCs in vitro and on lung pathology in vivo. The central hypothesis is that LTE4, the weakest agonist of the cys-LTs at their known receptors, is the most relevant cys-LT for promoting allergic mucosal inflammation. A corollary hypothesis is that the actions of LTE4 are mediated by a previously unrecognized cys-LT-reactive G protein-coupled receptor (GPCR), hereafter referred to as "P2Y12/CysLT3R". Aim 1 uses in vitro approaches to prove that P2Y12/CysLT3R is a true LTE4-reactive receptor. Aim 2 seeks to prove that P2Y12/CysLT3R accounts for the striking proinflammatory effects of LTE4 in the lung in vivo, and to identify the essential cellular targets of this effect. The findings are expected to have immediate implications for asthma pathophysiology and treatment
Keywords: 1-(4-chlorobenzoyl)-5-methoxy- 2-methyl-1-H-indole-3-acetic acid; 1-Phosphatidylinositol 3-Kinase; 11-Dehydroprostaglandin F2 alpha; 11-Dehydroprostaglandin F2alpha; 11-dehydro-TXB2; 11-dehydro-thromboxane B2; 11-dehydrothromboxane B2; 11-keto-thromboxane B2; 1H-Indole-3-acetic acid, 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-; 1H-Naphtho(2, 1-b)pyran-1-one, 5-(acetyloxy)-3-ethenyldodecahydro-6, 10, 10b-trihydroxy-3, 4a, 7, 7, 10a-pentamethyl-; 2-(Acetyloxy)benzoic Acid; 3`5`-cyclic ester of AMP; 3, 5 cyclic AMP synthetase; 5-Isothiocyanatofluorescein; 6, 8, 10, 14-Eicosatetraenoic acid, 5, 12-dihydroxy-, (S-(R*, S*-(E, Z, E, Z)))-; 7, 9, 11, 14-Eicosatetraenoic acid, 6-((2-amino-2-carboxyethyl)thio)-5-hydroxy-, (5S-(5R*, 6S*(S*), 7E, 9E, 11Z, 14Z))-; ADP Phosphohydrolase; ADP Receptors; ADPase; ANOVA; ATGN; ATP Diphosphohydrolase; ATP pyrophosphate-lyase (cyclizing); ATP-ADPase; ATP-Diphosphatase; ATP[{..}]1-phosphatidyl-1D-myo-inositol 3-phosphotransferase; ATP[{..}]protein-tyrosine O-phosphotransferase; Accounting; Acetylsalicylic Acid; Acidophilic Leukocyte; Address; Adenosine 5`-(trihydrogen diphosphate); Adenosine 5`-Pyrophosphate; Adenosine Cyclic 3`, 5`-Monophosphate; Adenosine Cyclic Monophosphate; Adenosine Diphosphatase; Adenosine Diphosphate; Adenosine Pyrophosphate; Adenosine, cyclic 3`, 5`-(hydrogen phosphate); Adenyl Cyclase; Adenylate Cyclase; Adenylyl Cyclase; Adoptive Transfer; Aerosols; Age; Agonist; Alanine; Alanine, L-Isomer; Algorithms; Ali-esterase; Allergens; Allergic; Allergic Reaction; Allergy; Alum Adjuvant; Aminoacetic Acid; Amplifiers; Analysis of Variance; Animals; Antigenic Determinants; Antigens; Apyrase; Ascaridil; Aspergum; Aspiration, Respiratory; Aspirin; Assay; Asthma; Attenuated; Autocrine Communication; Autocrine Signaling; Autocrine Systems; Automobile Driving; B Cell Differentiation Factor I; B cell activating factor; B cell growth factor; B cell growth factor 2; B-Cell Differentiation Factor-1; B-Cell Growth Factor-1; B-Cell Growth Factor-I; B-Cell Growth Factor-II; B-Cell Proliferating Factor; B-Cell Stimulating Factor; B-Cell Stimulating Factor-1; B-Cell Stimulation Factor-1; B-Cell Stimulatory Factor-1; B-esterase; BAF; BALB/c; BCDF-1; BCGF; BCGF-1; BCGF-II; BCGF2; BCGF2 (B cell growth factor 2); BCSF 1; BSF-1; BSF1; BSF1 (B cell stimulating factor 1); Backcrossings; Base Pairing; Basophilic Granulocyte; Basophilic Histiocyte; Basophilic Leukocyte; Basophils; Basophils, Tissue; Behavior; Binding; Binding (Molecular Function); Binding Determinants; Binding Sites; Binetrakin; Bioassay; Biochemical; Biologic Assays; Biological Assay; Biotechnology; Bizzozero`s corpuscle/cell; Blinded; Blood Basophil; Blood Cells; Blood Coagulation Factor IV; Blood Eosinophil; Blood Neutrophil; Blood Platelets; Blood Polymorphonuclear Neutrophil; Blood Sample; Blood Segmented Neutrophil; Blood Serum; Blood specimen; Blood, Cord; Blotting, Western; Body Tissues; Bone Marrow; Bone Marrow Blood-Deriving Cell; Bone Marrow Blood-Forming Cell; Bone Marrow Cells; Bone structure of tibia; Borates; Boston; Bovine Serum Albumin; Bovine Species; Breathing; Bronchial Asthma; Buffers; C57BL/6 Mouse; CAP-hydrolyzing Enzyme; CCL1; CCL1 gene; CD117 Antigens; CHO Cells; CO2; COX; COX-1; COX-1 protein; COX-2; COX1; COX2; COX3; CRE Binding Protein; CREB; CREB Protein; CSIF; CSIF-10; CTLA-8; CTLA8; Ca++ element; Calcium; Capsaicin-Hydrolyzing Enzyme; Carbon Dioxide; Carbonic Anhydride; Carboxyesterase; Carboxylate Esterase; Carboxylester Lipase; Carboxylesterase B; Carboxylesterases; Carboxylic Ester Hydrolase; Carboxylic Ester Hydrolases; Carcinoma; Cardiac; Cattle; Cavia; Cell Aging; Cell Communication and Signaling; Cell Count; Cell Culture Techniques; Cell Growth in Number; Cell Line; Cell Lines, Strains; Cell Locomotion; Cell Migration; Cell Movement; Cell Multiplication; Cell Nucleus; Cell Number; Cell Proliferation; Cell Senescence; Cell Signaling; Cell division; CellLine; Cells; Cellular Aging; Cellular Infiltration; Cellular Migration; Cellular Proliferation; Characteristics; Chemicals; Chemosensitization; Chemosensitization/Potentiation; Chinese Hamster Ovary Cell; Chromatography, High Performance Liquid; Chromatography, High Pressure Liquid; Chromatography, High Speed Liquid; Chymase; City of Boston; Coagulation Factor IV; Coleonol; Combining Site; Complement; Complement Proteins; Complex; Computer Programs; Computer Simulation; Computer software; Computerized Models; Control Groups; Cromoglicic Acid; Cromoglycic Acid; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP Responsive Element Binding Protein; Cyclic AMP-Responsive DNA-Binding Protein; Cyclo-Oxygenase-1; Cyclooxygenase; Cyclooxygenase 3; Cys-LT; Cysteine; Cytokine Synthesis Inhibitory Factor; Cytokine formation-inhibiting factor (mouse clone F115 protein moiety reduced); Cytokines, Chemotactic; Cytology; Cytotoxic T-Lymphocyte-Associated Antigen 8; Cytotoxic T-Lymphocyte-Associated Serine Esterase 8; Data; Decaris; Deetjeen`s body; Defect; Dendritic Cells; Development; Dimerization; Dissociation; Disulfide Linkage; Dose; Douching, other than vaginal; Drivings, Automobile; Drug Administration, Topical; Drug Delivery; Drug Delivery Systems; Drug Targeting; Drug Targetings; Drug Therapy; Drugs; Dysfunction; EC 2.7.2-; EDF; EDTA; ELISA; EPH- and ELK-Related Tyrosine Kinase; EPH-and ELK-Related Kinase; EPHA8; Ecotrin; Edathamil; Edetic Acid; Effector Cell; Eicosanoids; Electronics; Empirin; Employee Strikes; Endothelial Cells; Enhancers; Entericin; Enzymatic Biochemistry; Enzyme Antagonist; Enzyme Inhibitor; Enzyme Inhibitor Agent; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Enzymes; Enzymology; Eo-CSF; Eosinophil Differentiation Factor; Eosinophilia; Eosinophilic Granulocyte; Eosinophilic Leukocyte; EphA8 Protein; Ephrin Type-A Receptor 8; Ephrin Type-A Receptor 8 Precursor; Epithelial Cells; Epithelial Neoplasms, Malignant; Epithelial Tumors, Malignant; Epithelium; Epitopes; Ergamisol; Ergamisole; Ester Hydrolase; Ethylenediaminetetraacetic Acid; Ethylenedinitrilotetraacetic Acid; Event; Exhibits; Extracellular Signal-Regulated Kinases; Extren; FITC; FRET; Factor IV; Fatty Acid Cyclooxygenase; Feedback; Femur; Fibroblasts; Figs; Figs - dietary; Fluorescein-5-isothiocyanate; Fluorescence; Fluorescence Resonance Energy Transfer; Flushing; Flushings; Forskolin; Friends; Functional disorder; G Protein-Complex Receptor; G alpha q Protein; G-Protein, Gq; G-Protein, Gq alpha Family; G-Protein-Coupled Receptors; GTP Binding Protein alpha Subunit, Gq; Galphaq Protein; Gamma Globulin, 7S; Gel; GeneHomolog; Generations; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic defect; Genotype; Glass; Glycine; Glycine, N-(S-(1-(4-carboxy-1-hydroxybutyl)-2, 4, 6, 9-pentadecatetraenyl)-L-cysteinyl)-, (R-(R*, S*-(E, E, Z, Z)))-; Glycine, N-(S-(1-(4-carboxy-1-hydroxybutyl)-2, 4, 6, 9-pentadecatetraenyl)-N-L-gamma-glutamyl-L-cysteinyl)-, (R-(R*, S*-(E, E, Z, Z)))-; Goblet Cells; Gq Protein; Guinea Pigs; H2O2; HEK3; HPLC; Half-Cystine; Hand; Hayem`s elementary corpuscle; Helminths; Hematopoietic; Heterophil Granulocyte; High Pressure Liquid Chromatography; Histologic; Histologically; Histology; Homo; Homolog; Homologous Chemotactic Cytokines; Homologous Gene; Homologue; Hour; Human; Human, General; Hydrogen Peroxide; Hydrogen Peroxide (H2O2); Hydroperoxide; Hypersensitivity; I-309; IFN; IL-10; IL-13; IL-17; IL-17A; IL-4; IL-5; IL10; IL10A; IL13; IL17; IL17 Protein; IL17A; IL4; IL4 Protein; IL5; INFLM; Ice; IgA enhancing factor; IgE; IgG; IgG1; Image; Immune Precipitation; Immune response; Immunoglobulin E; Immunoglobulin G; Immunol; Immunology; Immunology (Including BRMP); Immunology (NCI Program); Immunoprecipitation; In Situ; In Vitro; Inbred BALB C Mice; Incubated; Individual; Indocin; Indometacin; Indomethacin; Infection; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Bowel Disorder; Inflammatory Intestinal Disease; Inflammatory Intestinal Disorder; Inflammatory Response; Inhalation; Inhaling; Injection of therapeutic agent; Injections; Inspiration, Respiratory; Instruction; Intercrines; Interferons; Interleukin 10 Precursor; Interleukin 17 (Cytotoxic T-Lymphocyte-Associated Serine Esterase 8); Interleukin 17 Precursor; Interleukin 5 (Colony-Stimulating Factor, Eosinophil); Interleukin 5 Precursor; Interleukin-10; Interleukin-13; Interleukin-17; Interleukin-4; Interleukin-4 Precursor; Interleukin-5; Intermediary Metabolism; Intestinal; Intestinal Diseases, Parasitic; Intestinal Mucosa; Intestinal Neoplasia; Intestinal Neoplasms; Intestinal Tumor; Intestines; Intestines Neoplasms; Intracellular Communication and Signaling; Irrigation; Irrigation, other than vaginal; Isocarboxazid amidase; Isoenzymes; Isoforms; Isozymes; Journal Article; Journal Article (PT); Journal Article [Publication Type]; Ketrax; Knock-out; Knockout; L-Alanine; L-Cysteine; L-Lysine; L-Serine; LPA Receptors; LTB4; LTC4; LTD4; LTE4; Label; Lavage; Lecithinase C; Left lung; Lentiviral Vector; Lentivirus Vector; Leukotriene B-4; Leukotriene B4; Leukotriene C-4; Leukotriene C4; Leukotriene D-4; Leukotriene D4; Leukotriene E-4; Leukotriene E4; Leukotriene Receptor; Leukotrienes; Ligand Binding; Ligands; Light; Link; Lipid Rafts, Cell Membrane; Lipids; Liquid substance; Lung; Lymph node proper; Lymphocyte Stimulatory Factor 1; Lysine; Lysophosphatidic Acid Receptors; MAP Kinase Kinase 1; MAP Kinase Kinases; MAP Kinase Signaling System; MAP kinase; MAP2K1 Protein; MAPK; MAPK Kinase 1; MAPK Kinases; MAPKKs; MCGF-2; MEK-1 Protein Kinase; MEK1 Protein Kinase; METBL; MK-0571; MK-571; MKK-1 Protein Kinase; MKK1 Protein Kinase; MMCP-1; Mammals, Guinea Pigs; Mammals, Mice; Man (Taxonomy); Man, Modern; Manufacturer; Manufacturer Name; Marrow Basophil; Marrow Eosinophil; Marrow Mast Cell; Marrow Neutrophil; Marrow platelet; Mass Spectrum; Mass Spectrum Analysis; Mast Cell Growth Factor Receptor; Mast Cell Growth Factor-2; Mast-Cell Disease; Mathematical Model Simulation; Mathematical Models and Simulations; Measures; Measurin; Mediating; Mediator; Mediator of Activation; Mediator of activation protein; Medication; Medicine; Membrane; Membrane Microdomains; Messenger RNA; Metabolic Processes; Metabolism; Metaplasia; Metaplastic Change; Methods; Mice; Mice, Inbred BALB C; Mice, Mutant Strains; Microbiology, Virology, Parasitology; Microscopy; Mitogen-Activated Protein Kinase Kinase 1; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Modeling; Models, Computer; Molecular Interaction; Monitor; Motility; Motility, Cellular; Mouse, BALB C; Mucosa; Mucosal Inflammation; Mucosal Tissue; Mucositis; Mucous Membrane; Murine; Mus; Mutant Strains Mice; Mutate; Mutation; N, N`-1, 2-Ethanediylbis(N-(carboxymethyl)glycine); N-terminal; NH2-terminal; Names; Naproxen Esterase; Nebulizer; Neutrophilic Granulocyte; Neutrophilic Leukocyte; New England; Non-specific Carboxylesterase; Non-specific Esterase; Nonspecific Esterase; Nonvaginal irrigation; Nonvaginal lavage; Northeastern United States; Nuclear; Nucleotides; Nucleus; Orphan; Outcome; P500; PGD synthase; PGD synthetase; PGD2; PGD2 isomerase; PGD2 synthase; PGG/HS; PGH Synthase; PGH Synthase 1; PGHS-1; PGHS-2; PGR2 D-isomerase; PHS-2; PHS1; PI-3 Kinase; PI-3K; PI3-Kinase; PTGS1; PTGS2; PTGS2 gene; PTK; PVDF; Parasitic Intestinal Diseases; Parasitology; Parents; Pathologic; Pathologist; Pathology; Pathway interactions; Peripheral Blood Cell; Pharmaceutic Preparations; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Phase; Phenothiazin-5-ium, 3-amino-7-(dimethylamino)-2-methyl-, chloride; Phosphatidylinositol 3-Kinase; Phosphatidylinositol-3-OH Kinase; Phosphoinositide 3-Hydroxykinase; Phospholipase C; Phosphorylation; Photometry/Spectrum Analysis, Mass; Photoradiation; Physiologic; Physiologic pulse; Physiological; Physiology; Physiopathology; Platelets; Pneumonia; Pneumonitis; Polymorph; Polymorphonuclear Cell; Polymorphonuclear Leukocytes; Polymorphonuclear Neutrophils; Population; Potentiation; Preparation; Procaine Esterase; Procedures; Process; Production; Prosta-5, 13-dien-1-oic acid, 9, 15-dihydroxy-11-oxo-, (5Z, 9alpha, 13E, 15S)-; Prostaglandin D2; Prostaglandin G/H Synthase 1; Prostaglandin G/H Synthase and Cyclooxygenase; Prostaglandin H2 Synthase; Prostaglandin H2 Synthase 1; Prostaglandin-Endoperoxide Synthase 1; Prostaglandins; Prostanoids; Protein Dimerization; Protein Isoforms; Protein Phosphorylation; Protein Tyrosine Kinase; Protein Tyrosine Kinase EEK; Proteins; Proto-Oncogene Protein c-kit; Protocol; Protocols documentation; PtdINS3P; PtdIns 3-Kinase; PubMed; Publishing; Pulmonary Eosinophilia; Pulmonary Inflammation; Pulse; Puncture procedure; Punctures; RNA, Messenger; Radiolabeled; Reaction; Reactive Site; Reading; Receptor Protein; Receptors, Lysophosphatidic Acid; Recombinants; Recovery; Recovery of Function; Regulation; Relative; Relative (related person); Research Support; Resistance; Respiratory System, Lung; Reticuloendothelial System, Bone Marrow; Reticuloendothelial System, Lymph Node; Reticuloendothelial System, Platelets; Reticuloendothelial System, Spleen; Role; SCF Receptor; SCYA1; SIS cytokines; SISe; Saline; Saline Solution; Sampling; Science of Medicine; Senescence, Cellular; Senescence, Replicative; Serine; Serum; Signal Transduction; Signal Transduction Systems; Signaling; Simulation, Computer based; Slide; Software; Solaskil; Solid; Solutions; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Sphingolipid Microdomains; Sphingolipid-Cholesterol Rafts; Spleen; Staining method; Stainings; Stains; Stem Cell Factor Receptor; Sterility; Strepavidin; Streptavidin; Strikes; Strikes, Employee; Students; Sum; Surface; System; System, LOINC Axis 4; T cell replacing factor; T memory cell; T-Cell Growth Factor 2; T-Cell Replacing Factor; T-Cells; T-Lymphocyte; T. spiralis; TCA3; TM Domain; TP Receptors; Tail; Temperature; Testing; Therapeutic; Thick; Thickness; Thrombocytes; Thromboxa-5, 13-dien-1-oic acid, 9, 15-dihydroxy-11-oxo-, (5Z, 9alpha, 13E, 15S)-; Thromboxane Receptor; Thromboxanes; Thymus-Dependent Lymphocytes; Tibia; Time; Tissues; Tolonium chloride; Toluidine Blue; Toluidine Blue O; Topical application; Trachea; Trachea Proper; Tramisol; Trans-Activation (Genetics); Transactivation; Transcript; Transfection; Transformed Cell Line; Transmembrane Domain; Transmembrane Region; Trichina spiralis; Trichinella spiralis; Trichinelliasis; Trichinosis; Trimisol; Tumor of the Intestines; Tweens; Type I Phosphatidylinositol Kinase; Type III Phosphoinositide 3-Kinase; Tyrosine Kinase; Tyrosine-Protein Kinase Receptor EEK; Tyrosine-Specific Protein Kinase; Tyrosylprotein Kinase; Umbilical Cord Blood; Up-Regulation; Up-Regulation (Physiology); Upregulation; Variance Analyses; Veiled Cells; Veins; Viral; Virus; Viruses, General; Weight; Western Blotting; Western Blottings; Western Immunoblotting; Work; Worms, Parasitic; X Chromosome; adenosine 3`5` monophosphate; adenylcyclase; airway epithelium infalmmation; airway inflammation; alum; aluminum sulfate; anti-IgE; anti-IgEid; antigen challenge; autocrine; base; beta-trace protein; biological signal transduction; bovid; bovine; bowel; c kit; c-kit Protein; c-kit Receptor; cAMP; cAMP Response Element-Binding Protein; cAMP Responsive Element Binding Protein; calcium flux; calcium mobilization; carboxylesterase; cell age; cell engineering; cell motility; cell type; cellular engineering; cellular targeting; chemoattractant cytokine; chemokine; chloroacetate; chymase-1; chymotrypsin-like protease; clopidogrel; cohort; computational modeling; computational models; computational simulation; computer based models; computer program/software; computerized modeling; computerized simulation; congenic; cow; cromolyn; cultured cell line; cyclo-oxygenase I; cyclooxygenase 1; cysteinyl-leukotriene; cytokine; design; designing; driving; drug/agent; eosinocyte; eosinophil; epithelial carcinoma; esterase; ethene, 1, 1-difluoro-, homopolymer; experience; experiment; experimental research; experimental study; extracellular; fetal; fetal cord blood; fluid; fluorophore; functional recovery; gene product; genome mutation; hCOX-2; his-PG; host response; human subject; hydroxyaryl protein kinase; imaging; immunogen; immunoresponse; in silico; in vivo; indexing; inhibitor; inhibitor/antagonist; inspiration; intestine growth; irradiation; irrigation therapy; jejunum; journal article; kit Proto-Oncogene Protein; knock-down; lavage therapy; lipid raft; lipocalin-type PGD synthase; lipocalin-type PGDS; lipophosphodiesterase I; liquid; lymph gland; lymph nodes; mRNA; mast cell; mast cell hyperplasia; mast cell protease; mast cell protease 1; mast cell protease I; mast cell proteinase-1; mastocyte; mastocytosis; medical schools; member; membrane structure; memory T lymphocyte; methacholine; monomer; mouse mutant; neutrophil; p145(c-kit); p145c-kit; paraform; paraformaldehyde; parasitic gastrointestinal disorder; particle; passive sensitization; pathophysiology; pathway; phosphatidylcholine cholinephosphohydrolase; phosphatidylinositol 3-monophosphate; phosphatidylinositol 3-phosphate; polyhistidine; polyvinyldifluoride; polyvinylidene difluoride; polyvinylidene fluoride; prevent; preventing; progenitor; progesterone 11-hemisuccinate-(2-iodohistamine); prostaglandin D synthase; prostaglandin D2 synthase; prostaglandin D2-isomerase; prostaglandin H synthase-1; prostaglandin H2 D-isomerase; prostaglandin R2 D-isomerase; prostaglandin endoperoxide D-isomerase; protein blotting; protein expression; pulmonary; purinoceptor P2Y1; radiolabel; radiotracer; receptor; receptor, P2Y1; reconstitute; reconstitution; release of sequestered calcium ion into cytoplasm; research study; resistant; response; senescence; sex; shRNA; short hairpin RNA; skeletal muscle protease; small hairpin RNA; social role; sterile; thrombocyte/platelet; thymus derived lymphocyte; tibia; time interval; topical administration; topical drug application; topically applied; tyrosyl protein kinase; vector; virtual simulation; windpipe
Relevance: Mast cells are crucial to allergic reactions and important in asthma. Cysteinyl leukotrienes are important chemical mediators of asthma and allergy. This Project will define the ways that cysteinyl leukotrienes, especially leukotriene E4, work to promote mast cell development and inflammation in the lung. The project will also reveal whether a particular molecule called P2Y12 might be a useful new drug target for asthma treatment
Project start date: 2009-07-17
Project end date: 2011-06-30
Budget start date: 17-JUL-2009
Budget end date: 30-JUN-2010
PFA/PA: PA-07-070
1R01AI078908-01A1 (2009): $396681
Chemical Mediators Of Acute Pulmonary Disorders
Joshua A Boyce, Associate Professor Of Med Harvard Med S
Brigham And Women s Hospital Research Administration Boston, Ma 02115
Grant 5P01HL036110-22 from National Heart, Lung, And Blood Institute IRG: HLBP
Abstract: This is a continuing, collaborative effort to understand the regulation of mast cell (MC)-associated effector functions and the relevance of these processes to asthma. MCs are unique among hematopoietic effector cells for their constitutive residence in the lung and other vascularized tissues, and their prominent contribution to both afferent and efferent phases of innate and adaptive immune responses. The central hypothesis of this Program Project is that MCs, by virtue of their constitutive and inducible effector pathways, form a functional bridge between innate and acquired immune responses and the clinical expression of asthma. The availability of purified recombinant MC tryptases and the development of mouse strains lacking the enzymes necessary to synthesize serglyin proteoglycans permits study of the role of proteoglycans in regulating the diverse functions of tryptases in vitro and in vivo. A novel in vitro approach will be used to define the mechanism by which GP49B1, a counterregulatory receptor bearing immunotyrosine-based inhibitory motifs (ITIMs), regulates MC activation through mechanistically diverse receptors to limit MC-dependent pathology in both allergic and innate immune esponses. The genes on mouse chromosomes 2 and 6 that interact to confer intrinsic MC-dependent AHR in A/J mice will be identified, and the MC-dependency of this phenotype will be confirmed with adoptive transfer of MCs into A/J mice rendered MC-deficient due to a mutation in the c-kit tyrosine kinase. Mouse strains lacking hematopoietic PGD2 synthase (PGDS) and LTC4synthase (LTC4S), respectively, as well as knockout strains lacking each receptor for PGD2 and LTC4, will be used to define the complementary and counterregulatory functions of these eicosanoids in vitro and in vivo. The role of the bronchoprotective eicosanoid, PGE2, and inducible PGE2 synthases in aspirin intolerant asthma (AIA) will be studied using a novel in vitro approach to the development of human MCs (hMCs) from well-characterized donors with AIA. Abnormalities in relevant synthases and PGE receptors will prompt resequencing for discovery of polymorphic variants. The receptors and mechanisms responsible for the inhibitory effects of PGE2 on hMC activation in vitro will be defined. These studies collectively provide information critical to understanding the biochemical and genetic regulation of key MC-associated effector functions, and defining their role in the control of intrinsic AHR, inflammation, and tissue repair subsequent to both allergic and innate immune responses that likely contribute to the pathophysiology of asthma.
Keywords: acute disease /disorder, cytokine, mast cell, respiratory hypersensitivity, clinical research
Project start date: 1997-09-01
Project end date: 2010-05-31
5P01HL036110-22 (2007): $2316444
5P01HL036110-21 (2006): $2320759
2P01HL036110-20A1 (2005): $2237772
Phenotypic Regulation Of Human Mast Cells
Joshua A Boyce, Associate Professor Of Med Harvard Med S
Brigham And Women s Hospital Research Administration Boston, Ma 02115
Grant 2P01AI031599-090012 from National Institute Of Allergy And Infectious Diseases IRG: ZAI1
Abstract: Mast cells initiate inflammatory reactions in asthma and allergic diseases. Circulating human progenitor mast cells (hPrMC) migrate to tissues where they mature into mature mast cells (hMC). The effector capabilities of hMC show inter-tissue heterogeneity, reflecting the microenvironmental regulation. Cord blood mononuclear cells cultured in the triad of recombinant human stem cell factor (rhSCF), rhIL-6, and rhIL-10 (SCR/IL-6/IL- 10) develop into hPrMC characterized cytofluorographically by monophasic expression of c-kit and an IL-3 receptor (IL-3R); greater than 70 percent of the hPrMC are immunoreactive for chymase and carboxypeptidase A (CPA) expression. SCF-driven proliferation of hPrMC is augmented by rhIL-6 or rhIL-3, but no rhIL-2, rhG-CSF or rhM-CSF, supporting their commitment to the mast cell lineage. rhIL-3 strikingly and reversibly attenuates the rhSCF-dependent steady-state expression of tryptase mRNA and protein by hPrMC independent of proliferation. An additional attenuating effect of rhGM-CSF on rhSCF-dependent tryptase expression indicates a functional GM-CSFR on hPrMC, while rhIL-9 counteracts the suppressive effects of rhIL-3. The IL-3R is lost with progressive maturation of hPrMC to hMC, which are characterized by uniform, strong metachromasia with toluidine blue dye, and strong immunoreactivity for tryptase and chymase. hMC heterogeneity likely reflects the interactions between cytokine receptors with their respective ligands at critical phases of hPrMC development. Specific Aim 1 focuses on the surface expression of receptors for T cell-derived cytokines on hPrMC and hMC, including the alpha subunits of IL-3R, IL-4R, IL- 5R, IL-6R, IL-9R, IL-13R, and GM-CSFR; the betac of IL-3R, GM- CSFR and IL-5R; the common gamma subunit of the IL-4R and IL-9R; and c-kit at 3 and 9 wk of development (reflecting homogeneous hPrMC and hMC, respectively) and at a transitional point of 6 wk. The dose-dependent effects of the corresponding cytokines will be examined on cell viability, proliferation, ultrastructural phenotype, and FcepsilonRI-mediated exocytosis. Specific Aim 2 focuses on the regulation of protease expression in hPrMC and hMC by rhSCF, which induces tryptase, and explores the counteractive effects of rhIL-3 and rhGM-CSF at transcriptional and post- transcriptional levels, with additional studies of rhIL-9 and other T cell-derived growth factors previously implicated in the regulation of mast cell proteases. Specific Aim 3 focuses on the roles of these growth factors in determining the arachidonic acid metabolism phenotype of hPrMC and hMC for their generation of leukotriene (LT)C4 and prostaglandin (PG)D2 in response to IgE- dependent stimulation. Cytokine-induced alterations in LTC4 or PGD2 generation will be further studied in terms of changes in the level(s) of the functional and immunodetectable proteins and corresponding mRNA transcripts of the 5-lipoxygenase (LO)/LTC4 synthase and the PGH synthase/PGD2 synthase pathways, respectively, of hPrMC and hMC.
Keywords: cytokine receptor, hypersensitivity, inflammation, mast cell, receptor expression, colony stimulating factor, eicosanoid metabolism, endopeptidase, fatty acid synthase, immunoglobulin E, interleukin 13, interleukin 3, interleukin 4, interleukin 5, interleukin 6, interleukin 9, leukotriene, lipoxygenase, prostaglandin, prostaglandin endoperoxide synthase, clinical research, human subject
Project start date: 1991-09-01
Project end date: 2003-08-31
ROLE OF PGE2 IN HUMAN MAST CELL BIOLOGY
Joshua A Boyce, Associate Professor
Brigham And Women´s Hospital, Research Administration, Boston, Ma 02115
Abstract: Mast cells (MCs) initiate allergic responses and are involved in innate protection from infections. MC activation through the high-affinity Fc receptor for IgE (FcsRI) induces de novo synthesis of two major eicosanoids cysteinyl leukotrienes (cysLTs), formed by the 5-lipoxygenase/leukotriene C4 synthase {5-LO/LTC4S) pathway, and prostaglandin (PG) D2, a product of the PGH synthase (PGHS)/PGD synthase pathway sequence. Both cysLTs and PGD2 act through specific receptor systems to mediate MC-dependent bronchconstriction, leukocyte recruitment, and airway hyperresponsiveness in vivo. Another eicosanoid, PGE2l is markedly bronchoprotective in both allergic and aspirin-intolerant asthma (AIA). Preliminary data now reveal that cord blood-derived human MCs (hMCs) respond to stimulation with staphylococcal peptidoglyan (PGN), a ligand for toll-like receptor (TLR) 2, and to poly IC, a ligand for TLR3, with delayed, sustained secretion of PGE2. PGN induces expression of mRNAfor both PGHS-2 and microsomal PGE2 synthase-1 (M-PGES-1), along with the corresponding proteins. Notably, exogenous PGE2 markedly inhibits cysLT and PGD2 generation by hMCs, and substantially inhibits the production of tumor necrosis factor (TNF-a) and IL-5 in response to either FcsRI crosslinkage or stimulation with PGN. We hypothesize that 1. Innate and adaptive immune responses elicit contrasting profiles of eicosanoid generation from MCs, with PGHS-2 and M-PGES-1 being inducible in each; 2. PGE& through more than one EP receptor, limits consequences of MC activation in an autocrine orparacrine manner; and 3. AIA involves dysregulation of inducible PGE2 synthase function. We therefore propose the following Specific Aims 1) to define the terminal synthases responsible for the sustained phase of PGE2 synthesis in hMCs activated through different transmembrane stimuli, 2) to define the receptors and biochemical mechanisms responsible for PGE2-mediated inhibition of hMC activation, and 3) to determine whether defects in the inducible PGE2 synthesis system underlie AIA
Keywords: (5Z, 8Z, 11Z, 14Z)-Icosa-5, 8, 11, 14-tetraenoate, hydrogen-donor[{..}]oxygen oxidoreductase; 1-Phosphatidylinositol 3-Kinase; 11-Dehydroprostaglandin F2 alpha; 11-Dehydroprostaglandin F2alpha; 2-(Acetyloxy)benzoic Acid; 3`5`-cyclic ester of AMP; 5` Untranslated Regions; 5`-Inosinic acid, homopolymer, complex with 5`-cytidylic acid homopolymer (1[{..}]1); 5`UTR; 5-Lipoxygenase; ATF; ATP[{..}]1-phosphatidyl-1D-myo-inositol 3-phosphotransferase; Abbreviations; Acetylsalicylic Acid; Acute; Adenosine; Adenosine Cyclic 3`, 5`-Monophosphate; Adenosine Cyclic Monophosphate; Adenosine Cyclic Monophosphate-Dependent Protein Kinases; Adenosine, cyclic 3`, 5`-(hydrogen phosphate); Affinity; Airway Hyper-responsiveness; Allergens; Allergic; Antibodies; Arachidonate 5-Lipoxygenase; Arachidonate[{..}]oxygen 5-oxidoreductase; Arachidonic Acid 5-Lipoxygenase; Arachidonic Acid Cyclooxygenase; Aspergum; Aspirin; Asthma; Autocrine Systems; B Cell Differentiation Factor I; B cell activating factor; B cell growth factor; B cell growth factor 2; B-Cell Differentiation Factor-1; B-Cell Growth Factor-1; B-Cell Growth Factor-I; B-Cell Growth Factor-II; B-Cell Proliferating Factor; B-Cell Stimulating Factor; B-Cell Stimulating Factor-1; B-Cell Stimulation Factor-1; B-Cell Stimulatory Factor-1; BAF; BCDF-1; BCGF; BCGF-1; BCGF-II; BCGF2; BCGF2 (B cell growth factor 2); BCSF 1; BSF-1; BSF1; BSF1 (B cell stimulating factor 1); Basophilic Histiocyte; Basophils, Tissue; Binetrakin; Biochemical; Blood leukocyte; Blood, Cord; Body Tissues; Bone Marrow; Bronchial Asthma; Bronchioalveolar Lavage; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; CD 23 Antigens; CD23 Antigens; COX; COX-2; COX-2 protein; COX2; COX2 enzyme; CRE; CRE Binding Protein; CREB; CREB Protein; CREB1; CREB1 gene; Cachectin; Cachectin-Tumor Necrosis Factor; Carboxypeptidase A; Cell Communication and Signaling; Cell Signaling; Cellular biology; Chemicals; Chromatography; Chromatography / Separation Science; Chymase; Ciclosporin; CsA; Cyclic AMP; Cyclic AMP Response Element; Cyclic AMP Response Element-Binding Protein; Cyclic AMP Responsive Element Binding Protein; Cyclic AMP-Dependent Protein Kinases; Cyclic AMP-Responsive DNA-Binding Protein; Cyclo-Oxygenase; Cyclo-Oxygenase-2; Cyclooxygenase; Cyclosporin A; Cyclosporine; Cyclosporine A; Cys-LT; CysLT(2) receptor; CysLT2 receptor; DIF; Data; Defect; Dinoprostone; Disease; Disorder; Douching, other than vaginal; Dysfunction; EC 2.7; EC 2.7.2-; EC 3.1.1.4; EDF; ERK MAP Kinases; Ecotrin; Eicosanoids; Elements; Empirin; Entericin; Enzymes; Eo-CSF; Eosinophil Differentiation Factor; Extracellular Signal Regulated Kinases; Extracellular Signal-Regulated Kinase Gene; Extracellular Signal-Regulated Kinases; Extracellular Signal-Regulated MAP Kinases; Extren; FEV1; FEV1%VC; Fatty Acid Cyclo-Oxygenase; Fc Receptor; FcR; Forced Expiratory Volume 1 Test; Forced Expiratory Volume in 1 Second; Functional disorder; G Protein-Complex Receptor; G-Protein-Coupled Receptors; GFP; Generalized Growth; Generations; Glycine, N-(S-(1-(4-carboxy-1-hydroxybutyl)-2, 4, 6, 9-pentadecatetraenyl)-N-L-gamma-glutamyl-L-cysteinyl)-, (R-(R*, S*-(E, E, Z, Z)))-; Green Fluorescent Proteins; Growth; Hematopoietic; Histamine-Sensitizing Factor; Human; Human, General; Hydroperoxide Cyclase; IAP Pertussis Toxin; IL-4; IL-5; IL4; IL4 Protein; IL5; IgA enhancing factor; IgE; IgE Receptors; Immune Globulins; Immune response; Immunity, Innate; Immunity, Native; Immunity, Natural; Immunity, Non-Specific; Immunoglobulin E; Immunoglobulin E Receptor; Immunoglobulin Enhancer-Binding Protein; Immunoglobulins; Immunoglobulins / Antibodies; In Vitro; Infection; Inflammatory; Interleukin 5 (Colony-Stimulating Factor, Eosinophil); Interleukin 5 Precursor; Interleukin-4; Interleukin-4 Precursor; Interleukin-5; Interleukins; Intracellular Communication and Signaling; Irrigation; Irrigation, other than vaginal; Islet-Activating Protein; Kinases; LPS; LTA4 Synthase; LTC4; LTC4 synthase; Lavage; Lavage, Bronchopulmonary; Lecithinase A2; Leukocytes; Leukotriene A Synthase; Leukotriene A4 Synthase; Leukotriene A4 Synthetase; Leukotriene C-4; Leukotriene C4; Leukotrienes; Ligands; Lipopolysaccharides; Liquid substance; Lung Lavage; Lung diseases; Lymphocyte Stimulatory Factor 1; Lymphocytosis-Promoting Factor; MAP Kinase Gene; MAP kinase; MAP-ERK Kinase; MAPK; MAPK ERK Kinases; MCGF-2; MEKs; MGF Stem Cell Factor; MMCP-1; MTGN; Mammals, Mice; Man (Taxonomy); Man, Modern; Marrow Mast Cell; Marrow leukocyte; Mast Cell Growth Factor; Mast Cell Growth Factor-2; Measurin; Mediating; Mediator; Mediator of Activation; Mediator of activation protein; Mice; Mitogen-Activated Protein Kinase Gene; Mitogen-Activated Protein Kinases; Mitogens; Murein; Murine; Mus; NF-AT proteins; NF-kB; NF-kappa B; NF-kappaB; NFAT proteins; NFATC proteins; NFKB; Natural Immunity; Nonvaginal irrigation; Nonvaginal lavage; Nuclear; Nuclear Factor kappa B; Nuclear Transcription Factor NF-kB; PFT/FEV1; PGD receptor; PGD synthase; PGD synthetase; PGD2; PGD2 isomerase; PGD2 receptor; PGD2 synthase; PGDP receptor; PGE Receptors; PGE2; PGE2 Receptors; PGE2 alpha; PGE2alpha; PGG/HS; PGH Synthase; PGH Synthase 2; PGH2 Synthetase; PGHS-2; PGHS2; PGR2 D-isomerase; PHS II; PHS-2; PI-3 Kinase; PI-3K; PI3-Kinase; PKA; PLA2; PTGS2; PTGS2 gene; Parents; Pathogenesis; Pathway interactions; Patients; Peptidoglycan; Pertussigen; Pertussis Toxin; Phase; Phosphatidylinositol 3-Kinase; Phosphatidylinositol-3-OH Kinase; Phosphoinositide 3-Hydroxykinase; Phospholipase A2; Phosphotransferases; Physiologic; Physiological; Physiopathology; Play; Poly I-C; Poly(I).poly(C); Poly(rI).Poly(rC); Polyinosinic acid[{..}]polycytidylic acid; Polyinosinic-Polycytidylic Acid; Polymorphism, Single Base; Polyribose Inosin-Cytidil; Production; Programs (PT); Programs [Publication Type]; Prosta-5, 13-dien-1-oic acid, 11, 15-dihydroxy-9-oxo-, (5Z, 11alpha, 13E, 15S)-; Prosta-5, 13-dien-1-oic acid, 9, 15-dihydroxy-11-oxo-, (5Z, 9alpha, 13E, 15S)-; Prostaglandin Cyclo-Oxygenase; Prostaglandin Cyclooxygenase; Prostaglandin D2; Prostaglandin E Receptor; Prostaglandin E2; Prostaglandin E2 alpha; Prostaglandin E2alpha; Prostaglandin Endoperoxide Synthetase; Prostaglandin G-H Synthase; Prostaglandin G/H Synthase 2; Prostaglandin G/H Synthase and Cyclooxygenase; Prostaglandin H Synthase; Prostaglandin H2 Synthase 2; Prostaglandin H2 Synthetase; Prostaglandin Synthase; Prostaglandin Synthetase; Prostaglandin-Endoperoxide Synthase; Prostaglandin-Endoperoxide Synthase 2; Prostaglandins; Prostaglandins H; Prostanoids; Protein Kinase A; Proteins; PtdIns 3-Kinase; Pulmonary Diseases; Pulmonary Disorder; Pulmonary Function Test/Forced Expiratory Volume 1; RNA, Small Interfering; Reaction; Receptor Protein; Receptors, IgE; Recombinants; Respiratory Disease; Respiratory Disorder; Respiratory System Disease; Respiratory System Disorder; Reticuloendothelial System, Bone Marrow; Reticuloendothelial System, Leukocytes; Reverse Transcription; Role; SNP; SNPs; Sandimmun; SangCya; Signal Transduction; Signal Transduction Systems; Signaling; Single Nucleotide Polymorphism; Small Interfering RNA; Source; Steel Factor; Stem Cell Factor; Stimulus; System; System, LOINC Axis 4; T cell replacing factor; T-Cell Growth Factor 2; T-Cell Replacing Factor; TIL4; TLR protein; TLR2; TLR2 receptor; TLR3; TLR3 gene; TNF; TNF (unspecified); TNF A; TNF Receptor Ligands; TNF gene; TNF-alpha; TNFSF2; Threonine/Tyrosine Protein Kinase; Thromboxane A2 Receptor; Tissue Growth; Tissues; Toll-Like Receptor 2; Toll-like receptors; Toll/Interleukin 1 Receptor-Like 4; Toll/Interleukin 1 Receptor-Like Protein 4; Transcription Factor NF-kB; Transphosphorylases; Tumor Necrosis Factor; Tumor Necrosis Factor Family Protein; Tumor Necrosis Factor Gene; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Type I Phosphatidylinositol Kinase; Type III Phosphoinositide 3-Kinase; UDP; UTRs; Umbilical Cord Blood; Untranslated Regions; Urd; Uridine; Uridine 5`-(trihydrogen diphosphate); Uridine Diphosphate; Uridine Pyrophosphate; Variant; Variation; White Blood Cells; White Cell; activating transcription factor; adenosine 3`5` monophosphate; airway hyper-reactivity; airway hyperreactivity; airway hyperresponsiveness; antibody receptor; autocrine; beta-trace protein; biological signal transduction; bronchopulmonary lavage therapy; c-kit Ligand; cAMP; cAMP Response Element; cAMP Response Element-Binding Protein; cAMP Responsive Element Binding Protein; cAMP-Dependent Protein Kinases; cell biology; cell type; chymase-1; chymotrypsin-like protease; cyclo-oxygenase II; cyclooxygenase 2; cysteinyl leukotriene receptor 2; cysteinyl-leukotriene; cytokine; disease/disorder; epsilon Fc Receptors; extracellular; extracellular signal related kinase; fetal cord blood; fluid; gene product; hCOX-2; host response; immunoresponse; in vivo; irrigation therapy; kappa B Enhancer Binding Protein; kit Ligand; lavage therapy; lecithinase A; leukotriene A4-glutathione S-leukotrienyltransferase; leukotriene C4 synthetase; leukotriene-C4 synthase; lipocalin-type PGD synthase; lipocalin-type PGDS; liquid; lung disorder; mRNA Leader Sequences; mast cell; mast cell protease; mast cell protease 1; mast cell protease I; mast cell proteinase-1; mastocyte; modulator protein; neoral; nuclear factor kappa beta; nuclear factor of activated T-cells, cytoplasmic; nuclear factors of activated T-cells; ontogeny; pathophysiology; pathway; phosphatase inhibitor 2; phosphatidase; phosphatidolipase; phosphatidylcholine 2 acylhydrolase; phosphoprotein phosphatase inhibitor-2; poly IC; polyinosate.polycytidylate; progenitor; programs; prostaglandin D receptor; prostaglandin D synthase; prostaglandin D2 receptor; prostaglandin D2 synthase; prostaglandin D2-isomerase; prostaglandin DP receptor; prostaglandin H synthase-2; prostaglandin H2 D-isomerase; prostaglandin R2 D-isomerase; prostaglandin endoperoxide D-isomerase; protein phosphatase inhibitor-2; receptor; response; sandimmune; siRNA; skeletal muscle protease; social role; transcription factor NF-AT; tumor necrosis factor (unspecified); unspecified interleukin; white blood cell; white blood corpuscle
Budget start date: 1-JUN-2009
Budget end date: 31-MAY-2010
5P01HL036110-24_0017 (2009): $516823
Sponsored Links Excellgen http://Excellgen.com
CystLT And P2Y Receptors And Lung Inflammation
Joshua A Boyce, Associate Professor Of Med Harvard Med S
Brigham And Women s Hospital Research Administration Boston, Ma 02115
Grant 1R01AI052353-01 from National Institute Of Allergy And Infectious Diseases IRG: LBPA
Abstract: Mast cells (MCs) are important in allergic diseases, tissue injury, and protection from infections. Indirect evidence suggests that the effector function of MCs is controlled by T cells, yet little is understood concerning this process in humans due to the limitations of human studies in vivo, and to technical difficulties in the isolation, purification, and maintenance in culture. Cysteinyl leukotrienes (cys-LTs), a class of peptide- conjugated lipids generated by MCs, signal through at least two known 7 transmembrane-spanning, G protein-coupled receptors (GPCR), termed the CysLT1 and CysLT2 receptors. Each is homologous to the purinergic (P2Y) receptors for nucleotides. Human MCs (hMCs) express the CysLT1 receptor, which unexpectedly mediates activation responses to both cys-LTs and to the extracellular nucleotide agonist uridine diphosphate (UDP). Furthermore, priming of hMCs with recombinant IL-4 preferentially enhances their sensitivity to both LTC4 and UDP without a concomitant change in CysLT1 receptor expression, suggesting that either a third cys-LT receptor (CysLT3) is induced by IL-4, or that the fundamental pharmacologic properties of CysLT1 are altered by IL-4 priming so as to selectively lower its threshold for LTC4 and UDP binding over LTD4. We cys-LTs and UDP induce the generation of multiple cytokines (EL-5, macrophage inflammatory protein 1beta, and tumor necrosis factor [TNF]- alpha) by IL-4-primed hMCs, and that blockade of endogenous cys- LT receptors or interference with endogenous cys-LT synthesis attenuate the generation of both IL-5 and TNF-alpha in response to cross-linking of the high-affinity Fc receptor for IgE (Fc epsilon RI). We propose the following hypotheses 1). Cys-LTs mediate both autocrine and paracrine functions of hMCs through at least two GPCRs that also bind uridine nucleotides, linking neurogenic signals and tissue injury to MCs and inflammation in asthma; 2) these same receptors permit MCs to initiate inflammatory responses to injury and infection in other tissues. Specific Aim 1 focuses on identifying the CysLT3 receptor and defining the mechanism(s) responsible for the observed IL-4- induced upregulation of hMC responses to UDP and LTC4. Specific Aim 2 uses site-directed mutagenesis to define the regions of the CysLT1 (and possible CysLT3) receptors involved in binding of LTD4, LTC4, and UDP, respectively. Specific Aim 3 will explore the functional role of the GPCRs for cys-LT and UDP in mediating MC-dependent inflammatory responses in vivo using targeted deletion of their respective genes.
Keywords: inflammation, leukocyte activation /transformation, leukotriene, mast cell, purinergic receptor, receptor expression, antibody receptor, asthma, autocrine, hormone receptor, macrophage inflammatory protein, paracrine, tumor necrosis factor alpha, uridine diphosphate, cord blood, human tissue, laboratory mouse, site directed mutagenesis, tissue /cell culture
Project start date: 2002-08-01
Project end date: 2007-05-31
1R01AI052353-01 (2002): $375696
Lysophosphatidic Acid Receptors In Mast Cell Biology
Joshua A Boyce, Associate Professor Of Med Harvard Med S
Brigham And Women s Hospital Research Administration Boston, Ma 02115
Grant 2P01AI031599-130002 from National Institute Of Allergy And Infectious Diseases IRG: ZAI1
Abstract: Mast cells (MCs) are important effectors of allergic diseases, tissue injury, and protection from infections. Their development and activation are critical events in allergic inflammation and resultant diseases. Comparatively little is understood about the mechanisms that control MC development and their non-lgE-mediated activation in humans. We recently demonstrated that cysteinyl leukotrienes (cys-LTs) initiate cytokine generation by cord blood-derived human MCs (hMCs) through an interleukin (IL)-4-inducible receptor that is shared with the nucleotide uridine diphosphate. Lysophosphatidic acid (LPA), a lipid that is constitutively present in serum and tissue fluids, is unique among lipids for its behavior as a growth factor for several stromal and vascular cells, and is produced in increased quantities in malignancy, tissue injury, and inflammation. Preliminary studies indicate that hMCs express mRNA encoding all three known LPA receptors (LPA1R, LPA2R, and LPA3R, formerly designated Edg2, Edg4, and Edg7 receptors, respectively). Moreover, LPA supplementation exerts a striking, dose-dependent increase in the numbers of hMCs arising in serum-free cultures of cord blood mononuclear cells developing in the presence of stem cell factor (SCF), IL-6, and IL-10. LPA also increases the expression of c-kit, tryptase, and chymase by hMCs and enhances the development of metachromatic secretory granules. Furthermore, LPA behaves as an agonist for the exocytosis of histamine at higher doses, contrasting sharply with the activation response to cys-LTs. We hypothesize that 1) LPA facilitates basal SCF-dependent MC development and enhances it in inflammation; 2) LPARs in hMCs signal through pathways that differ from those initiated by receptors for cys-LTs, accounting for their distinct respective profiles as secretagogues; and 3) LPA is a natural ligand for MC expansion and activation and can be responsible for MC-dependent effector responses in vivo. Specific Aim 1 explores the mechanistic basis for the observed effects of LPA on MC growth and development in vitro. Specific Aim 2 contrasts the post-receptor-signaling pathways that account for differential activation responses of hMCs stimulated with LPA vs. those induced by cys-LTs. Specific Aim 3 explores the functional role of the G protein coupled receptors for LPARs in mediating MC development and MC-dependent inflammatory responses in vivo using targeted deletion of their respective genes.
Keywords: cell surface receptor, inflammation, lysophospholipid, mast cell, receptor expression, G protein, biological signal transduction, leukotriene, phosphatidylinositol 3 kinase, protooncogene, receptor coupling, stem cell factor, SDS polyacrylamide gel electrophoresis, flow cytometry, human tissue, laboratory mouse, tissue /cell culture, transgenic animal, western blotting
Project start date: 2003-09-01
Project end date: 2008-08-31
The Role Of Mast Cells In Lung Inflammation
Joshua A Boyce, Associate Professor Of Med Harvard Med S
Brigham And Women s Hospital Research Administration Boston, Ma 02115
Grant 5R01AI048802-05 from National Institute Of Allergy And Infectious Diseases IRG: LBPA
Abstract: Mast cells (MC) are critical effector cells in asthma and allergy. They develop in situ from bone marrow-derived committed progenitors (PrMC), which traffic from the circulation to various tissues. Mature MC can rapidly redistribute to mucosal surfaces and to secondary lymphoid tissues in response to inflammatory stimuli. Neither the homing of PrMC nor the redistribution of mature MC are well-understood, but both processes are pertinent to a variety of immune responses and diseases. An in vitro culture system permits the first derivation of virtually pure human (h)PrMC from cord blood mononuclear cells, using the triad of recombinant human stem cell factor (SCF), IL-6, and IL-10. These hPrMC express 4 functional cell surface chemokine receptors; CXCR2, CXCR4, CCR3, and CCR5. With continued culture, the hPrMC develop into mature hMC that are functionally and immunochemically similar to their in vivo counterparts. These hMC express CCR3, but lack CXCR2, CXCR4, or CCR5 on their surface. Preliminary study now reveals that both mature cultured hMC and nasal polyp hMC posses intracellular stores of both CXCR4 and CCR3. CCR3 redistributes to the hMC plasma membrane within 1 h of IgE-dependent activation, while strong de novo steady-state expression of mRNA encoding CCR7 is induced by 6 h. Thus hMC sequentially alter both their expression and distribution of chemokine receptors in response to activation signals. Based on a yeast 2-hybrid assay to detect interacting proteins, both CCR3 and CXCR4 associate with WAIT (WD-repeat protein associated with integrin tails)-1, a protein that also appears to localize to the granules of hMC under basal conditions. Finally, co-stimulation of hMC with both IgE receptor cross-linkage and the recombinant CCR3 ligand eotaxin-1 selectively amplifies the generation of IL-13 and induces IL-4 production de novo. Specific Aim 1 focuses on defining the storage site(s) of pre-formed CCR3 and CXCR4 in hPrMC and hMC derived in vitro, modulation of the distribution and function of these receptors in response to IgE-dependent and non-IgE- dependent activation signals, and the functional relevance of the inducible CCR7, which may facilitate the movement of hMC to regional lymph nodes. Specific Aim 2 focuses on the potential functions of WAIT-1 in controlling the localization and function(s) of CCR3 and CXCR4. Specific Aim 3 explores the mechanisms by which CCR3 controls cytokine generation by hMC. These studies, carried out in non-transformed hPrMC and hMC, have potential implications for the control of hMC distribution and effector function in asthma.
Keywords: chemokine, chemotaxis, cytokine receptor, inflammation, mast cell, protein biosynthesis, receptor expression, biological signal transduction, cell migration, cellular pathology, interleukin 10, interleukin 13, interleukin 4, interleukin 6, stem cell factor, clinical research, cord blood, human tissue, tissue /cell culture
Project start date: 2002-05-01
Project end date: 2008-04-30
5R01AI048802-05 (2006): $367500
5R01AI048802-04 (2005): $376719
5R01AI048802-03 (2004): $377084
5R01AI048802-02 (2003): $377421
1R01AI048802-01A2 (2002): $376860
LIPIDOMIC AND TRANSCRIPTOME SIGNATURES IN ASPIRIN-EXACERBATED RESPIRATORY DISEASE
Joshua A Boyce
Brigham And Women´s Hospital, Research Administration, Boston, Ma 02115
Grant 1R21AI082369-01A1 from National Institute Of Allergy And Infectious Diseases
Abstract: Aspirin-exacerbated respiratory disease (AERD) is characterized by persistent chronic inflammation of the upper and lower airways leading to chronic rhinosinusitis, nasal polyposis, and asthma. When individuals with AERD ingest aspirin or other inhibitors of cyclooxygenase 1 they develop acute worsening of their rhinosinusitis accompanied by acute bronchoconstriction that may be life-threatening. AERD affects ~10% of adults with asthma, yet the fundamental underlying mechanisms remain unknown. Biochemically, AERD is characterized by abnormalities in eicosanoid generation and function. There is increased generation of cysteinyl leukotrienes at baseline and further release in response to inhibition of cyclooxygenase 1. The bronchoconstrictor response to leukotriene E4 is increased. Respiratory tissues of individuals with AERD show increased expression of the type 1 cysteinyl leukotriene receptor and diminished expression of cyclooxygenase 2 and the type 2 receptor for prostaglandin E2. Our preliminary data indicate that respiratory tissue from individuals with AERD avidly metabolize arachidonic acid to at least three major metabolites, one of which we identified at 15-HETE; the other 2 metabolites have not been identified. This strikingly abnormal eicosanoid profile indicates the involvement of previously unrecognized arachidonate metabolites in the pathobiology of AERD. The abnormalities in eicosanoid generation and responsiveness in AERD lack a coherent explanation, and state-of-the-art technology has not been applied to their investigation. We propose to use two state-of-the-art techniques to address the fundamental mechanisms of AERD. We will use sequential liquid chromatography/tandem mass spectrometry to provide a comprehensive analysis of the lipid mediators generated by nasal polyp tissue from individuals with AERD compared to aspirin-tolerant asthmatics with chronic rhinosinusitis. We will combine this with genome-wide analysis of the transcriptome of nasal polyps from the same individuals. This combined approach will allow us to evaluate the gene expression signature in AERD for enrichment of defined functional gene networks that underlie the inflammatory process and the abnormalities in lipid mediator generation. Success will lead to a significant break-through in our understanding of AERD, will spur the development of new treatments for AERD, and will allow the development of novel diagnostic tools that do rely on potentially dangerous in vivo provocation tests with aspirin. Asthma is a common disease that affects about 15% of the population of the United States, and about one in every ten adults with asthma develops life-threatening attacks of asthma when they ingest aspirin or similar analgesics. These individuals suffer from debilitating sinus disease and severe asthma. The aim of this proposal is to use state-of-the art analyses of the lipid mediators that are generated and the genes that are expressed in the airways of individuals with aspirin-exacerbated respiratory disease in order to provide key new insights into the mechanisms of this disease, its diagnosis, and its treatment
Keywords: 2-(Acetyloxy)benzoic Acid; 21+ years old; 5, 6, 15-tri-HETE; 5, 6, 15-triHETE; 5, 6, 15-trihydroxy-7, 9, 11, 13-eicosatetraenoic acid; 7, 9, 11, 14-Eicosatetraenoic acid, 6-((2-amino-2-carboxyethyl)thio)-5-hydroxy-, (5S-(5R*, 6S*(S*), 7E, 9E, 11Z, 14Z))-; Accessory Sinuses; Acetylsalicylic Acid; Acute; Address; Adult; Affect; Agonist; Analgesic Agents; Analgesic Drugs; Analgesic Preparation; Analgesics; Angioedema; Angioneurotic Edema; Anodynes; Anti-Inflammatories; Anti-Inflammatory Agents; Anti-inflammatory; Antiinflammatories; Antiinflammatory Agents; Antinociceptive Agents; Antinociceptive Drugs; Arachidonic Acids; Arts; Aspergum; Aspiration, Respiratory; Aspirin; Asthma; Blood leukocyte; Body Tissues; Breathing; Bronchial Asthma; Bronchial Constriction; Bronchial-Constricting Agents; Bronchoconstriction; Bronchoconstrictor Agents; Bronchoconstrictors; COX; COX-1; COX-1 protein; COX-2 protein; COX1; COX2; COX2 enzyme; COX3; Cardiovascular; Cardiovascular Body System; Cardiovascular system; Cardiovascular system (all sites); Cessation of life; Chromatography, High Performance Liquid; Chromatography, High Pressure Liquid; Chromatography, High Speed Liquid; Chronic; Clinical; Cyclo-Oxygenase-1; Cyclo-Oxygenase-2; Cyclooxygenase; Cyclooxygenase 3; Cys-LT; Data; Death; Development; Diagnosis; Diagnostic; Dinoprostone; Disease; Disorder; Dose; EP2 receptor; Ecotrin; Eicosanoid Receptor; Eicosanoids; Empirin; Employee Strikes; Entericin; Extren; Fatty Acid Cyclooxygenase; Flushing; Flushings; Frequencies (time pattern); Frequency; Future; GWAS; Gene Expression; Gene Expression Profile; Generations; Genes; Glucocorticoids; Goals; HETE; HPLC; High Pressure Liquid Chromatography; Human, Adult; Hydroxyeicosatetraenoic Acids; INFLM; Individual; Inflammation; Inflammation Mediators; Inflammatory; Ingestion; Inhalation; Inhaling; Injection of therapeutic agent; Injections; Inspiration, Respiratory; Intermediary Metabolism; Investigation; LTC4 synthase; LTE4; LXA4; Lead; Leukocytes; Leukotriene E-4; Leukotriene E4; Leukotrienes; Life; Lipids; Liquid Chromatography; Lung diseases; METBL; Marrow leukocyte; Measurin; Metabolic; Metabolic Processes; Metabolism; Methods and Techniques; Methods, Other; Morbidity; Morbidity - disease rate; Multiple Polyps; Nasal; Nasal Cavity Polyp; Nasal Polyps; Nasal Sinuses; Nasal cavity/Paranasal; Nasal cavity/Paranasal sinuses; Nose; Nose, Nasal Passages; Olfaction; Olfactions; Oral; Organ System, Cardiovascular; PGE2; PGE2 alpha; PGE2alpha; PGH Synthase; PGH Synthase 1; PGH Synthase 2; PGHS-1; PGHS-2; PGHS2; PHS II; PHS1; PTGS1; PTGS2; Pain; Painful; Paranasal Sinuses; Pathway interactions; Pb element; Phase; Polyps; Population; Pressure; Pressure- physical agent; Process; Production; Prosta-5, 13-dien-1-oic acid, 11, 15-dihydroxy-9-oxo-, (5Z, 11alpha, 13E, 15S)-; Prostaglandin E2; Prostaglandin E2 alpha; Prostaglandin E2alpha; Prostaglandin G/H Synthase 1; Prostaglandin G/H Synthase 2; Prostaglandin G/H Synthase and Cyclooxygenase; Prostaglandin H2 Synthase; Prostaglandin H2 Synthase 1; Prostaglandin H2 Synthase 2; Prostaglandin-Endoperoxide Synthase 1; Prostaglandin-Endoperoxide Synthase 2; Prostaglandins; Prostanoids; Pulmonary Diseases; Pulmonary Disorder; QOL; Quality of life; Quincke`s Edema; Receptor Protein; Relative; Relative (related person); Respiratory Disease; Respiratory Disorder; Respiratory Mucosa; Respiratory System Disease; Respiratory System Disorder; Respiratory System, Nose, Nasal Passages; Reticuloendothelial System, Leukocytes; Risk; Sampling; Signal Pathway; Sinus; Smell; Smell Perception; Stream; Strikes; Strikes, Employee; Symptoms; Syndrome; Techniques; Technology; Testing; Therapeutic Glucocorticoid; Tissues; Transcript; United States; Urticaria, Giant; Vascular, Heart; White Blood Cells; White Cell; adult human (21+); airway epithelium infalmmation; airway inflammation; analgesia; arachidonate; arachidonic metabolism; chronic rhinosinusitis; circulatory system; cyclo-oxygenase I; cyclo-oxygenase II; cyclooxygenase 1; cyclooxygenase 2; cysteinyl leukotriene receptor; cysteinyl-leukotriene; disease/disorder; eicosanoid metabolism; gastrointestinal; gene expression signature; genome wide association scan; genome wide association studies; genome wide association study; genome-wide; genome-wide analysis; genome-wide scan; genomewide association scan; genomewide association studies; genomewide association study; genomewide scan; heavy metal Pb; heavy metal lead; in vivo; inhibitor; inhibitor/antagonist; insight; inspiration; leukotriene A4-glutathione S-leukotrienyltransferase; leukotriene C4 synthetase; leukotriene-C4 synthase; lipid mediator; lipoxin A4; lung disorder; new diagnostics; next generation diagnostics; novel; novel diagnostics; pathway; peripheral blood; polyposis; pressure; prevent; preventing; prostaglandin EP2 receptor; prostaglandin H synthase-1; prostaglandin H synthase-2; public health relevance; receptor; receptor expression; respiratory; response; rhinosinusitis; success; tandem mass spectrometry; tool; transcriptome; urinary; white blood cell; white blood corpuscle; whole genome association studies; whole genome association study
Relevance: Asthma is a common disease that affects about 15% of the population of the United States, and about one in every ten adults with asthma develops life-threatening attacks of asthma when they ingest aspirin or similar analgesics. These individuals suffer from debilitating sinus disease and severe asthma. The aim of this proposal is to use state-of-the art analyses of the lipid mediators that are generated and the genes that are expressed in the airways of individuals with aspirin-exacerbated respiratory disease in order to provide key new insights into the mechanisms of this disease, its diagnosis, and its treatment
Project start date: 2009-08-01
Project end date: 2011-07-31
Budget start date: 1-AUG-2009
Budget end date: 31-JUL-2010
PFA/PA: PA-06-181
1R21AI082369-01A1 (2009): $266500
Joshua A Boyce
Brigham And Women´s Hospital
Project start date: 2008-04-01
Project end date: 2016-03-31
CysLt And P2Y Receptors And Lung Inflammation
Joshua A Boyce, Associate Professor Of Med Harvard Med S
Brigham And Women s Hospital Research Administration Boston, Ma 02115
Grant 2R56AI052353-06A1 from National Institute Of Allergy And Infectious Diseases IRG: LCMI
Keywords: inflammation, lung, receptor, clinical research
Project start date: 2002-08-01
Project end date: 2008-01-14
2R56AI052353-06A1 (2007): $418609
Sponsored Links Excellgen http://Excellgen.com
FUNCTION OF IMMATURE HYPODENSE HUMAN EOSINOPHILS
Joshua A Boyce, Associate Professor Of Med Harvard Med S
Brigham And Women´s Hospital
research Administration
boston, Ma 02115
Grant 5K11AI001304-05 from National Institute Of Allergy And Infectious Diseases IRG: AITC
Keywords: asthma, cellular pathology, eczema, eosinophil, granulocyte, hypersensitivity, rhinitis cellular immunity, corticosteroid, fatty acid biosynthesis, fatty acid synthase, leukopoiesis, leukotriene, lipoxygenase, programmed cell death, superoxide human tissue, immunocytochemistry, tissue /cell culture
Project start date: 1996-04-01
Project end date: 2001-03-31
5K11AI001304-05 (2000): $128520
5K11AI001304-04 (1999): $91800
5K11AI001304-03 (1998): $91800
5K11AI001304-02 (1997): $81000
1K11AI001304-01A1 (1996): $81000