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BACKGROUND: Urticaria is characterized by inappropriate mast cell degranulation leading to the development of wheals and/or angioedema. Twin and family studies indicate that there is a substantial heritable component to urticaria risk. OBJECTIVE: Our aim was to identify genomic loci at which common genetic variation influences urticaria susceptibility. METHODS: Genome-wide association studies of urticaria (including all subtypes) from 3 European cohorts (UK Biobank, FinnGen, and the Trøndelag Health Study [HUNT]) were combined through statistical meta-analysis (14,306 urticaria cases and 650,664 controls). Cases were identified via electronic health care records from primary and/or secondary care. To identify putative causal variants and genes, statistical fine-mapping, colocalization, and interrogation of publicly available single-cell transcriptome sequencing resources were performed. RESULTS: Genome-wide significant associations (P < 5 × 10-8) were identified at 6 independent loci. These included 2 previously reported association signals at 1q44 and the human leucocyte antigen region on chromosome 6. Genes with expected or established roles in mast cell biology were associated with the 4 other genome-wide association signals (GCSAML, FCER1A, TPSAB1, and CBLB). Colocalization of association signals consistent with the presence of shared causal variants was observed between urticaria susceptibility and increased expression of GCSAML (posterior probability of colocalization [PPcoloc] = 0.89) and FCER1A (PPcoloc = 0.91) in skin. CONCLUSION: Common genetic variation influencing the risk of developing urticaria was identified at 6 genomic loci. The relationship between genes with roles in mast cell biology and several association signals implicates genetic variability of specific components of mast cell function in the development of urticaria.
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Angioedema , Urticaria , Humanos , Estudio de Asociación del Genoma Completo , Mastocitos , Urticaria/genética , Proteínas/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido SimpleRESUMEN
Skin immune homeostasis is a multi-faceted process where dermal dendritic cells (DDCs) are key in orchestrating responses to environmental stressors. We have previously identified CD141+CD14+ DDCs as a skin-resident immunoregulatory population that is vitamin-D3 (VitD3) inducible from monocyte-derived DCs (moDCs), termed CD141hi VitD3 moDCs. We demonstrate that CD141+ DDCs and CD141hi VitD3 moDCs share key immunological features including cell surface markers, reduced T cell stimulation, IL-10 production, and a common transcriptomic signature. Bioinformatic analysis identified the neuroactive ligand receptor pathway and the neuropeptide, urocortin 2 (UCN2), as a potential immunoregulatory candidate molecule. Incubation with VitD3 upregulated UCN2 in CD141+ DCs and UVB irradiation induced UCN2 in CD141+ DCs in healthy skin in vivo. Notably, CD141+ DDC generation of suppressive Tregs was dependent upon the UCN2 pathway as in vivo administration of UCN2 reversed skin inflammation in humanized mice. We propose the neuropeptide UCN2 as a novel skin DC-derived immunoregulatory mediator with a potential role in UVB and VitD3-dependent skin immune homeostasis.
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BACKGROUND: The emerging role of sphingosine-1-phosphate (S1P) in regulating smooth muscle functions has led to the exploration of the possibility that this sphingolipid could represent a potential therapeutic target in asthma and other lung diseases. Several studies in animal surrogates have suggested a role for S1P-mediated signaling in the regulation of airway smooth muscle (ASM) contraction, airway hyperresponsiveness, and airway remodeling, but evidence from human studies is lacking. OBJECTIVE: We sought to compare the responsiveness of the airways to S1P in healthy and asthmatic individuals in vivo, in isolated human airways ex vivo, and in murine airways dissected from healthy and house dust mite (HDM)-sensitized animals. METHODS: Airway responsiveness was measured by spirometry during inhalation challenges and by wire myography in airways isolated from human and mouse lungs. Thymidine incorporation and calcium mobilization assays were used to study human ASM cell responses. RESULTS: S1P did not induce contraction of airways isolated from healthy and HDM-exposed mice, nor in human airways. Similarly, there was no airway constriction observed in healthy and asthmatic subjects in response to increasing concentrations of inhaled S1P. However, a 30-minute exposure to S1P induced a significant concentration-dependent enhancement of airway reactivity to methacholine and to histamine in murine and human airways, respectively. HDM-sensitized mice demonstrated a significant increase in methacholine responsiveness, which was not further enhanced by S1P treatment. S1P also concentration-dependently enhanced proliferation of human ASM cells, an effect mediated through S1P receptor type 2, as shown by selective antagonism and S1P receptor type 2 small-interfering RNA knockdown. CONCLUSIONS: Our data suggest that S1P released locally into the airways may be involved in the regulation of ASM hyperresponsiveness and hyperplasia, defining a novel target for future therapies.
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Asma , Humanos , Ratones , Animales , Receptores de Esfingosina-1-Fosfato/metabolismo , Cloruro de Metacolina , Asma/metabolismo , Músculo Liso/metabolismo , Proliferación CelularRESUMEN
Airway inflammation and remodelling are important pathophysiologic features in asthma and other respiratory conditions. An intact epithelial cell layer is crucial to maintain lung homoeostasis, and this depends on intercellular adhesion, whilst damaged respiratory epithelium is the primary instigator of airway inflammation. The Coxsackievirus Adenovirus Receptor (CAR) is highly expressed in the epithelium where it modulates cell-cell adhesion stability and facilitates immune cell transepithelial migration. However, the contribution of CAR to lung inflammation remains unclear. Here we investigate the mechanistic contribution of CAR in mediating responses to the common aeroallergen, House Dust Mite (HDM). We demonstrate that administration of HDM in mice lacking CAR in the respiratory epithelium leads to loss of peri-bronchial inflammatory cell infiltration, fewer goblet-cells and decreased pro-inflammatory cytokine release. In vitro analysis in human lung epithelial cells confirms that loss of CAR leads to reduced HDM-dependent inflammatory cytokine release and neutrophil migration. Epithelial CAR depletion also promoted smooth muscle cell proliferation mediated by GSK3ß and TGF-ß, basal matrix production and airway hyperresponsiveness. Our data demonstrate that CAR coordinates lung inflammation through a dual function in leucocyte recruitment and tissue remodelling and may represent an important target for future therapeutic development in inflammatory lung diseases.
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Neumonía , Pyroglyphidae , Receptores Virales , Animales , Humanos , Ratones , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inflamación/metabolismo , Pulmón/metabolismo , Neumonía/metabolismo , Mucosa Respiratoria/metabolismo , Receptores Virales/metabolismoRESUMEN
Non-steroidal anti-inflammatory drugs (NSAIDs) and other eicosanoid pathway modifiers are among the most ubiquitously used medications in the general population. Their broad anti-inflammatory, antipyretic, and analgesic effects are applied against symptoms of respiratory infections, including SARS-CoV-2, as well as in other acute and chronic inflammatory diseases that often coexist with allergy and asthma. However, the current pandemic of COVID-19 also revealed the gaps in our understanding of their mechanism of action, selectivity, and interactions not only during viral infections and inflammation, but also in asthma exacerbations, uncontrolled allergic inflammation, and NSAIDs-exacerbated respiratory disease (NERD). In this context, the consensus report summarizes currently available knowledge, novel discoveries, and controversies regarding the use of NSAIDs in COVID-19, and the role of NSAIDs in asthma and viral asthma exacerbations. We also describe here novel mechanisms of action of leukotriene receptor antagonists (LTRAs), outline how to predict responses to LTRA therapy and discuss a potential role of LTRA therapy in COVID-19 treatment. Moreover, we discuss interactions of novel T2 biologicals and other eicosanoid pathway modifiers on the horizon, such as prostaglandin D2 antagonists and cannabinoids, with eicosanoid pathways, in context of viral infections and exacerbations of asthma and allergic diseases. Finally, we identify and summarize the major knowledge gaps and unmet needs in current eicosanoid research.
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Asma , Tratamiento Farmacológico de COVID-19 , Hipersensibilidad , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , Asma/tratamiento farmacológico , Consenso , Eicosanoides/metabolismo , Humanos , Hipersensibilidad/tratamiento farmacológico , Inflamación/tratamiento farmacológico , SARS-CoV-2RESUMEN
Intracellular calcium mobilization can be measured using several methods varying in indicator dyes and devices used. In this chapter, we describe the fluorescence-based method (FLIPR Calcium 4 Assay) developed by Molecular Devices for a FlexStation and routinely used in our laboratory for detecting intracellular calcium changes. The assay is designed to study calcium mobilization induced by majority of GPCRs and calcium channels and allows for simultaneous concentration-dependent analysis of several receptor agonists and antagonists, useful in receptor characterization and drug discovery projects.
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Bioensayo/métodos , Canales de Calcio/metabolismo , Calcio/metabolismo , Descubrimiento de Drogas/métodos , Fluorometría/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Receptores Acoplados a Proteínas G/metabolismo , Células Cultivadas , Humanos , Transducción de SeñalRESUMEN
Eicosanoids are biologically active lipid mediators, comprising prostaglandins, leukotrienes, thromboxanes, and lipoxins, involved in several pathophysiological processes relevant to asthma, allergies, and allied diseases. Prostaglandins and leukotrienes are the most studied eicosanoids and established inducers of airway pathophysiology including bronchoconstriction and airway inflammation. Drugs inhibiting the synthesis of lipid mediators or their effects, such as leukotriene synthesis inhibitors, leukotriene receptors antagonists, and more recently prostaglandin D2 receptor antagonists, have been shown to modulate features of asthma and allergic diseases. This review, produced by an European Academy of Allergy and Clinical Immunology (EAACI) task force, highlights our current understanding of eicosanoid biology and its role in mediating human pathology, with a focus on new findings relevant for clinical practice, development of novel therapeutics, and future research opportunities.
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Asma , Hipersensibilidad , Asma/etiología , Consenso , Eicosanoides , Humanos , LeucotrienosRESUMEN
Decades of costly failures in translating drug candidates from preclinical disease models to human therapeutic use warrant reconsideration of the priority placed on animal models in biomedical research. Following an international workshop attended by experts from academia, government institutions, research funding bodies, and the corporate and non-governmental organisation (NGO) sectors, in this consensus report, we analyse, as case studies, five disease areas with major unmet needs for new treatments. In view of the scientifically driven transition towards a human pathways-based paradigm in toxicology, a similar paradigm shift appears to be justified in biomedical research. There is a pressing need for an approach that strategically implements advanced, human biology-based models and tools to understand disease pathways at multiple biological scales. We present recommendations to help achieve this.
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Investigación Biomédica , Descubrimiento de Drogas , Enfermedad de Alzheimer , Animales , Asma , Trastorno del Espectro Autista , Enfermedades Autoinmunes , Consenso , Fibrosis Quística , Humanos , Hepatopatías , Modelos AnimalesRESUMEN
Leukotriene E4 (LTE4) the most stable of the cysteinyl leukotrienes (cysLTs) binds poorly to classical type 1 (CysLT1) and 2 (CysLT2) receptors although it induces potent responses in human airways in vivo, such as bronchoconstriction, airway hyperresponsiveness and inflammatory cell influx suggesting the presence of a novel receptor that preferentially responds to LTE4. To identify such a receptor two human mast cell lines, LAD2 and LUVA, were selected that differentially responded to LTE4 when analysed by intracellular signalling and gene expression. Comparative transcriptome analysis and recombinant gene overexpression experiments revealed CysLT1 as a receptor responsible for potent LTE4-induced response in LAD2 but not in LUVA cells, an observation confirmed further by gene knockdown and selective inhibitors. Lentiviral overexpression of CysLT1 in LUVA cells augmented intracellular calcium signalling induced by LTE4 but did not restore full agonist responses at the gene expression level. Our data support a model where both an increased expression of Gαq-coupled CysLT1, and sustained intracellular calcium mobilisation and extracellular signal-regulated kinase (Erk) activation, are required for LTE4-mediated regulation of gene expression in human cells. Our study shows for the first time that CysLT1 expression is critically important for responsiveness to LTE4 within a human cell system.
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Regulación de la Expresión Génica , Leucotrieno E4/metabolismo , Receptores de Leucotrienos/agonistas , Receptores de Leucotrienos/metabolismo , Calcio/metabolismo , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Leucotrieno E4/farmacología , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Leucotrienos/genética , Transducción de Señal/efectos de los fármacos , TranscriptomaRESUMEN
Intracellular calcium mobilization can be measured using several methods varying in indicator dyes and devices used. In this chapter, we describe the fluorescence-based method (FLIPR Calcium 4 Assay) developed by Molecular Devices for a FlexStation and routinely used in our laboratory for detecting intracellular calcium changes. The assay is designed to study calcium mobilization induced by majority of GPCRs and calcium channels and allows for simultaneous concentration-dependent analysis of several receptor agonists and antagonists, useful in receptor characterization and drug discovery projects.
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Calcio/metabolismo , Ensayos Analíticos de Alto Rendimiento , Miocitos del Músculo Liso/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Bioensayo , Bradiquinina/farmacología , Calcimicina/farmacología , Ionóforos de Calcio/farmacología , Señalización del Calcio , Descubrimiento de Drogas , Colorantes Fluorescentes , Histamina/farmacología , Humanos , Cinética , Lisofosfolípidos/farmacología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Cultivo Primario de Células , Juego de Reactivos para Diagnóstico , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Sistema Respiratorio/citología , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacologíaAsunto(s)
Bronquios/efectos de los fármacos , Calcitriol/farmacología , Interleucinas/genética , ARN Mensajero/genética , Receptores de Superficie Celular/genética , Bronquios/citología , Bronquios/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Eosinófilos/citología , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Regulación de la Expresión Génica , Humanos , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/antagonistas & inhibidores , Interleucinas/inmunología , Mastocitos/citología , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Cultivo Primario de Células , ARN Mensajero/inmunología , Receptores de Superficie Celular/agonistas , Receptores de Superficie Celular/inmunología , Transducción de Señal , SolubilidadRESUMEN
BACKGROUND: Common human diseases are caused by the complex interplay of genetic susceptibility as well as environmental factors. Due to the environment's influence on the epigenome, and therefore genome function, as well as conversely the genome's facilitative effect on the epigenome, analysis of this level of regulation may increase our knowledge of disease pathogenesis. METHODS: In order to identify human-specific epigenetic influences, we have performed a novel genome-wide DNA methylation analysis comparing human, chimpanzee and rhesus macaque. RESULTS: We have identified that the immunological Leukotriene B4 receptor (LTB4R, BLT1 receptor) is the most epigenetically divergent human gene in peripheral blood in comparison with other primates. This difference is due to the co-ordinated active state of human-specific hypomethylation in the promoter and human-specific increased gene body methylation. This gene is significant in innate immunity and the LTB4/LTB4R pathway is involved in the pathogenesis of the spectrum of human inflammatory diseases. This finding was confirmed by additional neutrophil-only DNA methylome and lymphoblastoid H3K4me3 chromatin comparative data. Additionally we show through functional analysis that this receptor has increased expression and a higher response to the LTB4 ligand in human versus rhesus macaque peripheral blood mononuclear cells. Genome-wide we also find human species-specific differentially methylated regions (human s-DMRs) are more prevalent in CpG island shores than within the islands themselves, and within the latter are associated with the CTCF motif. CONCLUSIONS: This result further emphasises the exclusive nature of the human immunological system, its divergent adaptation even from very closely related primates, and the power of comparative epigenomics to identify and understand human uniqueness.
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Leukotriene E4 (LTE4), the most stable of the cysteinyl leukotrienes (cysLTs), binds poorly to classical type 1 and 2 cysLT receptors although in asthmatic individuals it may potently induce bronchial constriction, airway hyperresponsiveness and inflammatory cell influx to the lung. A recent study has suggested that the purinergic receptor P2Y12 is required for LTE4 mediated pulmonary inflammation in a mouse model of asthma and signals in response to cysLTs. The aim of the study was to characterise the responsiveness of human P2Y12 to cysteinyl leukotrienes. Models of human CysLT1, CysLT2 and P2Y12 overexpressed in HEK293, CHO cells and human platelets were used and responsiveness to different agonists was measured using intracellular calcium, cAMP and ß-arrestin recruitment assays. CysLTs induced concentration dependent calcium mobilisation in cells overexpressing CysLT1 and CysLT2 but failed to induce any calcium response in cells expressing P2Y12 or P2Y12+ Gα16. In contrast, selective P2Y12 agonists ADP and 2-MeS-ADP induced specific calcium flux in cells expressing P2Y12+ Gα16. Similarly, specific response to 2-MeS-ADP, but not to cysLTs was also observed in cells expressing P2Y12 when intracellular cAMP and ß-arrestin signalling were analysed. Platelets were used as a model of human primary cells expressing P2Y12 to analyse potential signalling and cell activation through P2Y12 receptor or receptor heterodimers but no specific LTE4 responses were observed. These results show that LTE4 as well as other cysLTs do not activate intracellular signalling acting through P2Y12 and suggest that another LTE4 specific receptor has yet to be identified.
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Cisteína/metabolismo , Leucotrieno E4/metabolismo , Leucotrienos/metabolismo , Receptores de Leucotrienos/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Animales , Arrestinas/metabolismo , Plaquetas/metabolismo , Células CHO , Calcio/metabolismo , Cricetulus , AMP Cíclico/metabolismo , Cisteína/agonistas , Células HEK293 , Humanos , Inflamación , Leucotrienos/agonistas , Ratones , Activación Plaquetaria , Agonistas del Receptor Purinérgico P2Y/farmacología , Transducción de Señal , Tionucleótidos/farmacología , beta-ArrestinasRESUMEN
BACKGROUND: Allergic asthma is characterized by reversible airway obstruction and bronchial hyperresponsiveness associated with T(H)2 cell-mediated inflammation. Cysteinyl leukotrienes (CysLTs) are potent lipid mediators involved in bronchoconstriction, mucus secretion, and cell trafficking in asthmatic patients. Recent data have implicated CysLTs in the establishment and amplification of T(H)2 responses in murine models, although the precise mechanisms are unresolved. OBJECTIVES: Preliminary microarray studies suggested that human T(H)2 cells might selectively express cysteinyl leukotriene receptor 1 (CYSLTR1) mRNA. We sought to establish whether human T(H)2 cells are indeed a CysLT target cell type. METHODS: We examined the expression of CYSLTR1 using real-time PCR in human T(H)1 and T(H)2 cells. We functionally assessed cysteinyl leukotriene receptor 1 protein (CysLT(1)) expression using calcium flux, cyclic AMP, and chemotaxis assays. RESULTS: We show that human T(H)2 cells selectively express CYSLTR1 mRNA at high levels compared with T(H)1 cells after in vitro differentiation from naive precursors. Human T(H)2 cells are selectively responsive to CysLTs in a calcium flux assay when compared with T(H)1 cells with a rank order of potency similar to that described for CysLT(1) (leukotriene [LT] D(4) > LTC(4) > LTE(4)). We also show that LTD(4)-induced signaling in T(H)2 cells is mediated through CysLT(1) coupled to G(α)q and G(α)i proteins, and both pathways can be completely inhibited by selective CysLT(1) antagonists. LTD(4) is also found to possess potent chemotactic activity for T(H)2 cells at low nanomolar concentrations. CONCLUSIONS: These findings suggest a novel mechanism of action for CysLTs in the pathogenesis of asthma and provide a potential explanation for the anti-inflammatory effects of CysLT(1) antagonists.
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Cisteína/farmacología , Factores Inmunológicos/farmacología , Leucotrienos/farmacología , Receptores de Leucotrienos/genética , Células Th2/inmunología , Señalización del Calcio/inmunología , Quimiotaxis/efectos de los fármacos , Quimiotaxis/inmunología , Cisteína/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Factores Inmunológicos/metabolismo , Leucotrieno D4/farmacología , Leucotrienos/metabolismo , Unión Proteica/efectos de los fármacos , Receptores de Leucotrienos/metabolismo , Células Th2/efectos de los fármacos , Células Th2/metabolismoRESUMEN
The use of cysteinyl leukotriene receptor antagonists (LTRAs) for asthma therapy has been associated with a significant degree of interpatient variability in response to treatment. Some of that variability may be attributable to noncysteinyl leukotriene type 1 receptor (CysLT(1))-mediated inhibitory mechanisms that have been demonstrated for this group of drugs. We used a model of CysLT(1) signaling in human monocytes to characterize CysLT(1)-dependent and -independent anti-inflammatory activity of two chemically different, clinically relevant LTRAs (montelukast and zafirlukast). Using receptor-desensitization experiments in monocytes and CysLT(1)-transfected HEK293 cells and IL-10- and CysLT(1) small interfering RNA-induced downregulation of CysLT(1) expression, we showed that reported CysLT(1) agonists leukotriene D(4) and UDP signal through calcium mobilization, acting on separate receptors, and that both pathways were inhibited by montelukast and zafirlukast. However, 3-log greater concentrations of LTRAs were required for the inhibition of UDP-induced signaling. In monocytes, UDP, but not leukotriene D(4), induced IL-8 production that was significantly inhibited by both drugs at micromolar concentrations. At low micromolar concentrations, both LTRAs also inhibited calcium ionophore-induced leukotriene (leukotriene B(4) and leukotriene C(4)) production, indicating 5-lipoxygenase inhibitory activities. We report herein that montelukast and zafirlukast, acting in a concentration-dependent manner, can inhibit non-CysLT(1)-mediated proinflammatory reactions, suggesting activities potentially relevant for interpatient variability in response to treatment. Higher doses of currently known LTRAs or new compounds derived from this class of drugs may represent a new strategy for finding more efficient therapy for bronchial asthma.
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Acetatos/farmacología , Inhibición de Migración Celular/inmunología , Quimiotaxis de Leucocito/inmunología , Antagonistas de Leucotrieno/farmacología , Quinolinas/farmacología , Receptores de Leucotrienos/fisiología , Compuestos de Tosilo/farmacología , Calcio/fisiología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/inmunología , Línea Celular , Inhibición de Migración Celular/efectos de los fármacos , Células Cultivadas , Quimiotaxis de Leucocito/efectos de los fármacos , Ciclopropanos , Humanos , Indoles , Mediadores de Inflamación/farmacología , Mediadores de Inflamación/fisiología , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo , Modelos Inmunológicos , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Fenilcarbamatos , Sulfuros , Sulfonamidas , Uridina Difosfato/fisiologíaRESUMEN
Leukotriene (LT) E(4) mediates many of the principal features of bronchial asthma, such as bronchial constriction, hyperresponsiveness, eosinophilia, and increased vascular permeability. Furthermore, it is the most stable of the cysteinyl leukotrienes (CysLTs) and can be active at the site of release for a prolonged time after its synthesis. There might be several reasons why LTE(4) has been forgotten. LTE(4) demonstrated low affinity for CysLT(1) and CysLT(2) receptors in equilibrium competition assays. It was less potent than other CysLTs in functional assays, such as calcium flux, in cells transfected with CysLT(1) and CysLT(2). The introduction of CysLT(1) antagonists into clinical practice diverted interest into CysLT(1)-related mechanisms, which were mediated mainly by LTD(4). However, experiments with animal models and human studies have revealed that LTE(4) has unique characteristics that cannot be explained by the current knowledge of CysLT(1) and CysLT(2). These activities include its potency relative to other CysLTs to increase airway responsiveness to histamine, to enhance eosinophilic recruitment, and to increase vascular permeability. Asthmatic airways also demonstrate marked in vivo relative hyperresponsiveness to LTE(4), especially in patients with aspirin-sensitive respiratory disease. This has stimulated a search for additional LT receptors that would respond preferentially to LTE(4) stimulation.
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Asma/inmunología , Hiperreactividad Bronquial/inmunología , Hipersensibilidad a las Drogas/inmunología , Leucotrieno E4/inmunología , Animales , Aspirina/efectos adversos , Asma/tratamiento farmacológico , Hiperreactividad Bronquial/tratamiento farmacológico , Hipersensibilidad a las Drogas/tratamiento farmacológico , Histamina , Humanos , Leucotrieno C4/inmunología , Leucotrieno D4/inmunología , Cloruro de Metacolina , Receptores de Leucotrienos/inmunología , Piel/efectos de los fármacos , Piel/inmunología , Piel/patologíaRESUMEN
Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) activation is a regulatory step in the control of arachidonic acid (AA) liberation for eicosanoid formation. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator involved in the regulation of many important proinflammatory processes and has been found in the airways of asthmatic subjects. We investigated the mechanism of S1P-induced AA release and determined the involvement of cPLA(2)alpha in these events in A549 human lung epithelial cells. S1P induced AA release rapidly within 5 min in a dose- and time-dependent manner. S1P-induced AA release was inhibited by the cPLA(2)alpha inhibitors methyl arachidonyl fluorophosphonate (MAFP) and pyrrolidine derivative, by small interfering RNA-mediated downregulation of cPLA(2)alpha, and by inhibition of S1P-induced calcium flux, suggesting a significant role of cPLA(2)alpha in S1P-mediated AA release. Knockdown of the S1P3 receptor, the major S1P receptor expressed on A549 cells, inhibited S1P-induced calcium flux and AA release. The S1P-induced calcium flux and AA release was associated with sphingosine kinase 1 (Sphk1) expression and activity. Furthermore, Rho-associated kinase, downstream of S1P3, was crucial for S1P-induced cPLA(2)alpha activation. Our data suggest that S1P acting through S1P3, calcium flux, and Rho kinase activates cPLA(2)alpha and releases AA in lung epithelial cells. An understanding of S1P-induced cPLA(2)alpha activation mechanisms in epithelial cells may provide potential targets to control inflammatory processes in the lung.
Asunto(s)
Ácido Araquidónico/metabolismo , Asma/enzimología , Fosfolipasas A2 Grupo IV/metabolismo , Pulmón/enzimología , Lisofosfolípidos/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Mucosa Respiratoria/enzimología , Esfingosina/análogos & derivados , Animales , Ácidos Araquidónicos/farmacología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Eicosanoides/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Organofosfonatos/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol) , ARN Interferente Pequeño/farmacología , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Esfingosina/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
The immunoregulatory cytokine IL-10 plays an essential role in down-modulating adaptive and innate immune responses leading to chronic inflammatory diseases. In contrast, cysteinyl leukotrienes (cysLTs), important proinflammatory mediators of cell trafficking and innate immune responses, are thought to enhance immune reactions in the pathogenesis of diseases, such as bronchial asthma, atherosclerosis, and pulmonary fibrosis. The aim of this study was to determine the IL-10 regulatory role in cysLT-induced activation of human monocytes and monocyte-derived dendritic cells. Herein we show that cysLT-induced activation and chemotaxis of human monocytes and monocyte-derived immature dendritic cells (iDC) are inhibited by IL-10 pretreatment. IL-10 down-regulated cysLT type 1 and 2 receptors' mRNA in a time- and concentration-dependent fashion. cysLT-induced activation of monocytes and iDCs measured by intracellular calcium flux and immediate-early gene expression (FBJ murine osteosarcoma viral oncogen homolog B and early growth response-2) was potently decreased by IL-10 and by the cysLT antagonist MK571. Chemotaxis of monocytes and iDCs to increasing concentrations of leukotriene D(4) (LTD(4)) was also inhibited by IL-10. LTD(4) enhanced iDC migration in response to CCL5. IL-10 selectively inhibited LTD(4)-induced chemotaxis without affecting migration to CCL5. These data indicate that cysLT-induced activation of human monocytes and dendritic cells may be specifically inhibited by IL-10, suggesting a direct link between the 5-lipoxygenase proinflammatory pathway and IL-10 regulatory mechanisms. Antileukotriene therapies may reproduce some regulatory mechanisms played by IL-10 in inflammatory processes.
Asunto(s)
Cisteína/metabolismo , Células Dendríticas/inmunología , Interleucina-10/metabolismo , Leucotrienos/metabolismo , Monocitos/inmunología , Receptores de Leucotrienos/metabolismo , Inhibición de Migración Celular , Quimiocina CCL5/inmunología , Quimiocina CCL5/metabolismo , Quimiotaxis , Quimiotaxis de Leucocito , Cisteína/inmunología , Células Dendríticas/metabolismo , Humanos , Interleucina-10/inmunología , Leucotrieno D4/inmunología , Leucotrieno D4/metabolismo , Leucotrienos/inmunología , Monocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Receptores de Leucotrienos/deficiencia , Receptores de Leucotrienos/genéticaRESUMEN
BACKGROUND: Cysteinyl leukotrienes (CysLTs) are important mediators of innate immune responsiveness and chronic inflammatory diseases. CysLTs acting through CysLT receptors can influence the migration and activity of cells, such as eosinophils, monocytes, and dendritic cells. OBJECTIVE: We sought to determine the gene expression signature of human monocytes in response to CysLTs and to elucidate the signaling pathways involved in monocyte activation. METHODS: Gene expression was analyzed by using oligonucleotide microarrays. Responsiveness to CysLTs was assessed by using real-time PCR, calcium flux, kinase activation, and chemotaxis assays. RESULTS: CysLT type 1 receptor (CysLTR(1)) transcript 1 is predominantly expressed in human monocytes, and CysLTs signal through CysLTR(1) in these cells. Several immediate-early genes, including early growth response 2 and 3, FBJ murine osteosarcoma viral oncogene homolog B, activating transcription factor 3, and nuclear receptor subfamily 4 were significantly induced by leukotriene (LT) D(4). This effect was mediated by CysLTR(1) coupled to the G protein alpha inhibitory subunit, activation of phospholipase C, and inositol-1,4,5-triphosphate and store-operated calcium channels. LTD(4) induced p38 mitogen-activated protein kinase phosphorylation, a pathway also involved in the regulation of immediate-early gene expression in monocytes. LTD(4) stimulated monocyte chemotactic activity that was fully blocked by a selective CysLTR(1) inhibitor, MK571, and pertussis toxin, suggesting that CysLTR(1) coupled to the G protein alpha inhibitory subunit is a dominant functional pathway in human monocytes. CONCLUSION: Our data show that CysLTs acting through CysLTR(1) can significantly influence the activation and migration of human monocytes and that these effects can be fully inhibited by CysLTR(1) antagonists.