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1.
Artigo em Inglês | MEDLINE | ID: mdl-31301373

RESUMO

BACKGROUND: 15-Lipoxygenase 1 (15LO1) is expressed in airway epithelial cells in patients with type 2-high asthma in association with eosinophilia. Chronic rhinosinusitis with nasal polyps (CRSwNP) is also associated with type 2 inflammation and eosinophilia. CCL26/eotaxin 3 has been reported to be regulated by 15LO1 in lower airway epithelial cells. However, its relation to 15LO1 in patients with CRSwNP or mechanisms for its activation are unclear. OBJECTIVE: We sought to evaluate 15LO1 and CCL26 expression in nasal epithelial cells (NECs) from patients with CRSwNP and healthy control subjects (HCs) and determine whether 15LO1 regulates CCL26 in NECs through extracellular signal-regulated kinase (ERK) activation. METHODS: 15LO1, CCL26, and phosphorylated ERK were evaluated in NECs from patients with CRSwNP and HCs. 15LO1/CCL26 and CCL26/cytokeratin 5 were colocalized by means of immunofluorescence. IL-13-stimulated NECs were cultured at an air-liquid interface with or without 15-lipoxygenase 1 gene (ALOX15) Dicer-substrate short interfering RNAs (DsiRNA) transfection, a specific 15LO1 enzymatic inhibitor, and 2 ERK inhibitors. Expression of 15LO1 and CCL26 mRNA and protein was analyzed by using quantitative RT-PCR, Western blotting, and ELISA. RESULTS: 15LO1 expression was increased in nasal polyp (NP) epithelial cells compared with middle turbinate epithelial cells from patients with CRSwNP and HCs. 15LO1 expression correlated with CCL26 expression and colocalized with CCL26 expression in basal cells of the middle turbinate and NPs from patients with CRSwNP. In primary NECs in vitro, IL-13 induced 15LO1 and CCL26 expression. 15LO1 knockdown and inhibition decreased IL-13-induced ERK phosphorylation and CCL26 expression. ERK inhibition (alone) similarly decreased IL-13-induced CCL26. Phosphorylated ERK expression was increased in NECs from CRSwNP subjects and positively correlated with both 15LO1 and CCL26 expression. CONCLUSIONS: 15LO1 expression is increased in NP epithelial cells and contributes to CCL26 expression through ERK activation. 15LO1 could be considered a novel therapeutic target for CRSwNP.

2.
Am J Respir Crit Care Med ; 200(7): 837-856, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31161938

RESUMO

Rationale: Gene expression of BAL cells, which samples the cellular milieu within the lower respiratory tract, has not been well studied in severe asthma.Objectives: To identify new biomolecular mechanisms underlying severe asthma by an unbiased, detailed interrogation of global gene expression.Methods: BAL cell expression was profiled in 154 asthma and control subjects. Of these participants, 100 had accompanying airway epithelial cell gene expression. BAL cell expression profiles were related to participant (age, sex, race, and medication) and sample traits (cell proportions), and then severity-related gene expression determined by correlating transcripts and coexpression networks to lung function, emergency department visits or hospitalizations in the last year, medication use, and quality-of-life scores.Measurements and Main Results: Age, sex, race, cell proportions, and medications strongly influenced BAL cell gene expression, but leading severity-related genes could be determined by carefully identifying and accounting for these influences. A BAL cell expression network enriched for cAMP signaling components most differentiated subjects with severe asthma from other subjects. Subsequently, an in vitro cellular model showed this phenomenon was likely caused by a robust upregulation in cAMP-related expression in nonsevere and ß-agonist-naive subjects given a ß-agonist before cell collection. Interestingly, ELISAs performed on BAL lysates showed protein levels may partly disagree with expression changes.Conclusions: Gene expression in BAL cells is influenced by factors seldomly considered. Notably, ß-agonist exposure likely had a strong and immediate impact on cellular gene expression, which may not translate to important disease mechanisms or necessarily match protein levels. Leading severity-related genes were discovered in an unbiased, system-wide analysis, revealing new targets that map to asthma susceptibility loci.

3.
JCI Insight ; 4(5)2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30730306

RESUMO

Although type-2-induced (T2-induced) epithelial dysfunction is likely to profoundly alter epithelial differentiation and repair in asthma, the mechanisms for these effects are poorly understood. A role for specific mucins, heavily N-glycosylated epithelial glycoproteins, in orchestrating epithelial cell fate in response to T2 stimuli has not previously been investigated. Levels of a sialylated MUC4ß isoform were found to be increased in airway specimens from asthmatic patients in association with T2 inflammation. We hypothesized that IL-13 would increase sialylation of MUC4ß, thereby altering its function and that the ß-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) would regulate the sialylation. Using human biologic specimens and cultured primary human airway epithelial cells (HAECs),we demonstrated that IL-13 increases ST6GAL1-mediated sialylation of MUC4ß and that both were increased in asthma, particularly in sputum supernatant and/or fresh isolated HAECs with elevated T2 biomarkers. ST6GAL1-induced sialylation of MUC4ß altered its lectin binding and secretion. Both ST6GAL1 and MUC4ß inhibited epithelial cell proliferation while promoting goblet cell differentiation. These in vivo and in vitro data provide strong evidence for a critical role for ST6GAL1-induced sialylation of MUC4ß in epithelial dysfunction associated with T2-high asthma, thereby identifying specific sialylation pathways as potential targets in asthma.

4.
J Allergy Clin Immunol ; 143(6): 2075-2085.e10, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30639343

RESUMO

BACKGROUND: Genetic and genomic data increasingly point to the airway epithelium as critical to asthma pathogenesis. Epithelial growth factor (EGF) family members play a fundamental role in epithelial differentiation, proliferation, and repair. Although expression of erythroblastosis oncogene B2 (ErbB2) mRNA, an EGF family receptor, was reported to be lower in asthmatic patients, little is understood about its functional role. OBJECTIVE: We sought to determine whether decreased ErbB2 activation in freshly isolated human airway epithelial cells (HAECs) from asthmatic patients associated with impaired wound closure in vitro. METHODS: An in vitro scratch-wound model of air-liquid interface cultured and freshly isolated HAECs were compared between HAECs from healthy control subjects (HCs) and asthmatic patients in relation to ErbB2. RESULTS: Freshly brushed HAECs from asthmatic patients had impaired ErbB2 activation compared with those from HCs. In an in vitro scratch-wound model, HAECs from asthmatic patients showed delayed wound closure compared with HAECs from HCs. Cell proliferation, as assessed based on [3H] thymidine incorporation after wounding, and expression or activation of ErbB2 and cyclin D1 at the leading edge of the wound were lower in HAECs from asthmatic patients and HCs. A selective ErbB2 tyrosine kinase inhibitor, mubritinib, impaired wound closure and decreased cyclin D1 expression in healthy HAECs, with less effect on cells from asthmatic patients, supporting diminished activity in asthmatic patients. CONCLUSION: These results implicate a primary defect in the ErbB2 pathway as constraining epithelial repair processes in asthmatic patients. Restoration of homeostatic ErbB2 function should be considered a novel asthma therapeutic target.

5.
Am J Respir Cell Mol Biol ; 53(6): 844-52, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25945802

RESUMO

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, exists in several isoforms, which differentially impacts neuronal and immune cell survival and differentiation. The role of BDNF and its isoforms in asthma remains unclear. The objectives of this study were to compare the BDNF protein isoforms and specific splice variant expression in sputum and bronchoscopic samples from healthy control subjects and participants with asthma, and to relate these changes to findings in IL-13-stimulated human airway epithelial cells. Sputum and bronchoscopic samples from healthy control subjects and participants with asthma were evaluated for BDNF protein (ELISA and Western blot) and BDNF mRNA (gel and quantitative real-time PCR) in relation to asthma severity and type 2 inflammatory processes. BDNF mRNA was measured in cultured primary human airway epithelial cells after IL-13 stimulation. Total BDNF protein differed among the groups, and its mature isoform was significantly higher in sputum from subjects with severe asthma compared with healthy control subjects (overall P = 0.008, P = 0.027, respectively). Total BDNF was higher in those with elevated fractional exhaled nitric oxide and sputum eosinophilia. In vitro, IL-13 increased BDNF exon VIb splice variant and the ratio to BDNF common exon IX mRNA (P < 0.001, P = 0.003, respectively). Epithelial brushing exon VIb mRNA and total BDNF protein differed among the groups and were higher in subjects with severe asthma than in healthy control subjects (overall P = 0.01, P = 0.02, respectively). The mature BDNF isoform and the exon VIb splice variant are increased in human asthmatic airways. The in vitro increase in response to IL-13 suggests that type 2 cytokines regulate BDNF levels and activity in asthma.


Assuntos
Asma/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Adulto , Asma/patologia , Fator Neurotrófico Derivado do Encéfalo/genética , Brônquios/metabolismo , Células Cultivadas , Éxons , Feminino , Expressão Gênica , Humanos , Interleucina-13/metabolismo , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Índice de Gravidade de Doença , Escarro/metabolismo , Adulto Jovem
6.
J Allergy Clin Immunol ; 135(5): 1144-53.e1-9, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25819984

RESUMO

BACKGROUND: ß2-Adrenergic receptor (ß2AR) agonists are critical treatments for asthma. However, receptor desensitization can lead to loss of therapeutic effects. Although desensitization to repeated use of ß2-agonists is well studied, type 2 inflammation could also affect ß2AR function. OBJECTIVE: We sought to evaluate the effect of the type 2 cytokine IL-13 on ß2AR desensitization in human airway epithelial cells (HAECs) and determine whether 15-lipoxygenase-1 (15LO1) binding with phosphatidylethanolamine-binding protein 1 (PEBP1) contributes to desensitization through release of G protein receptor kinase 2 (GRK2). METHODS: HAECs in air-liquid interface culture with or without IL-13 (48 hours) or isoproterenol hydrochloride (ISO; 30 minutes) pretreatment were stimulated with ISO (10 minutes). Cyclic adenosine 3, 5-monophosphate (cAMP) levels were measured using ELISA, and ß2AR and GRK2 phosphorylation was measured using Western blotting. Short interfering RNA was used for 15LO1 knockdown. Interactions of GRK2, PEBP1, and 15LO1 were detected by means of immunoprecipitation/Western blotting and immunofluorescence. HAECs and airway tissue from control subjects and asthmatic patients were evaluated for I5LO1, PEBP1, and GRK2. RESULTS: Pretreatment with ISO or IL-13 decreased ISO-induced cAMP generation compared with ISO for 10 minutes alone paralleled by increases in ß2AR and GRK2 phosphorylation. GRK2 associated with PEBP1 after 10 minutes of ISO in association with low phosphorylated GRK2 (pGRK2) levels. In contrast, in the presence of IL-13 plus ISO (10 minutes), binding of GRK2 to PEBP1 decreased, whereas 15LO1 binding and pGRK2 levels increased. 15LO1 knockdown restored ISO-induced cAMP generation. These findings were recapitulated in freshly brushed HAECs from cells and tissue of asthmatic patients. CONCLUSION: IL-13 treatment of HAECs leads to ß2AR desensitization, which involves 15LO1/PEBP1 interactions to free GRK2, and allows it to phosphorylate (and desensitize) ß2ARs, suggesting that the beneficial effects of ß2-agonists could be blunted in patients with type 2 associated asthma.


Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Interleucina-13/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Mucosa Respiratória/metabolismo , Adulto , Araquidonato 15-Lipoxigenase/genética , Asma/diagnóstico , Asma/genética , Asma/imunologia , Asma/metabolismo , Estudos de Casos e Controles , AMP Cíclico/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Interleucina-13/farmacologia , Isoproterenol/farmacologia , Masculino , Pessoa de Meia-Idade , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Fosforilação , Ligação Proteica , Mucosa Respiratória/efeitos dos fármacos
7.
J Allergy Clin Immunol ; 135(2): 386-94, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25312760

RESUMO

BACKGROUND: Severe asthma (SA) can involve both innate and type 2 cytokine-associated adaptive immunity. Although IL-27 has been reported to potentiate TH1 responses (including the chemokine CXCL9) and suppress TH2 responses, its function in asthmatic patients is unknown. OBJECTIVE: We sought to evaluate IL-27 expression in human asthma alone and in combination with type 2 immunity to determine the relationship to disease severity and CXCL9 expression. We also sought to model these interactions in vitro in human bronchial epithelial cells. METHODS: Bronchoalveolar lavage cells from 87 participants were evaluated for IL-27 mRNA and protein alone and in association with epithelial CCL26 (a marker of type 2 activation) in relation to asthma severity and CXCL9 mRNA. Human bronchial epithelial cells cultured at the air-liquid interface and stimulated with IL-27 (1-100 ng/mL) with or without IL-13 (1 ng/mL) were evaluated for CXCL9 expression by using quantitative real-time PCR and ELISA. Phosphorylated and total signal transducer and activator of transcription (STAT) 1/3 were detected by means of Western blotting. Small interfering RNA knockdown of STAT1 or STAT3 was performed. RESULTS: Bronchoalveolar lavage cell IL-27 mRNA and protein levels were increased in asthmatic patients. Patients with evidence for type 2 pathway activation had higher IL-27 expression (P = .02). Combined IL-27 and CCL26 expression associated with more SA and higher CXCL9 expression (P = .004 and P = .007 respectively), whereas IL-27 alone was associated with milder disease. In vitro IL-13 augmented IL-27-induced CXCL9 expression, which appeared to be due to augmented STAT1 activation and reduced STAT3 activation. CONCLUSIONS: IL-27, in combination with a type 2/CCL26 signature, identifies a more SA phenotype, perhaps through combined effects of IL-27 and IL-13 on STAT signaling. Understanding these interactions could lead to new targets for asthma therapy.


Assuntos
Asma/imunologia , Asma/metabolismo , Imunidade , Interleucina-27/metabolismo , Adolescente , Adulto , Idoso , Asma/diagnóstico , Asma/genética , Biomarcadores/metabolismo , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Quimiocina CCL26 , Quimiocina CXCL9/metabolismo , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Citocinas/genética , Citocinas/metabolismo , Feminino , Volume Expiratório Forçado , Expressão Gênica , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Interleucina-27/genética , Masculino , Pessoa de Meia-Idade , Óxido Nítrico , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Fatores de Transcrição STAT/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Adulto Jovem
8.
J Allergy Clin Immunol ; 131(6): 1504-12, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23506843

RESUMO

BACKGROUND: Bronchoalveolar lavage (BAL) fluid prostaglandin D2(PGD2) levels are increased in patients with severe, poorly controlled asthma in association with epithelial mast cells (MCs). PGD2, which is generated by hematopoietic prostaglandin D synthase (HPGDS), acts on 3 G protein-coupled receptors, including chemoattractant receptor-homologous molecule expressed on TH2 lymphocytes (CRTH2) and PGD2 receptor 1 (DP1). However, much remains to be understood regarding the presence and activation of these pathway elements in asthmatic patients. OBJECTIVE: We sought to compare the expression and activation of PGD2 pathway elements in bronchoscopically obtained samples from healthy control subjects and asthmatic patients across a range of disease severity and control, as well as in relation to TH2 pathway elements. METHODS: Epithelial cells and BAL fluid were evaluated for HPGDS (quantitative real-time PCR/immunohistochemistry [IHC]) and PGD2 (ELISA/liquid chromatography mass spectrometry) in relation to levels of MC proteases. Expression of the 2 inflammatory cell receptors DP1 and CRTH2 was evaluated on luminal cells. These PGD2 pathway markers were then compared with asthma severity, level of control, and markers of TH2 inflammation (blood eosinophils and fraction of exhaled nitric oxide). RESULTS: Confirming previous results, BAL fluid PGD2 levels were highest in patients with severe asthma (overall P = .0001). Epithelial cell compartment HPGDS mRNA and IHC values differed among groups (P = .008 and P < .0001, respectively) and correlated with MC protease mRNA. CRTH2 mRNA and IHC values were highest in patients with severe asthma (P = .001 and P = .0001, respectively). Asthma exacerbations, poor asthma control, and TH2 inflammatory markers were associated with higher PGD2, HPGDS, and CRTH2 levels. CONCLUSION: The current study identifies coordinated upregulation of the PGD2 pathway in patients with severe, poorly controlled, TH2-high asthma despite corticosteroid use.


Assuntos
Asma/imunologia , Asma/metabolismo , Prostaglandina D2/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Transdução de Sinais , Adulto , Asma/prevenção & controle , Líquido da Lavagem Broncoalveolar/imunologia , Estudos de Casos e Controles , Feminino , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/genética , Lipocalinas/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores Imunológicos/genética , Receptores de Prostaglandina/genética , Mucosa Respiratória/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Regulação para Cima , Adulto Jovem
9.
Thorax ; 67(12): 1061-6, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23015684

RESUMO

BACKGROUND: Eosinophilic inflammation is implicated in asthma. Eotaxin 1-3 regulate eosinophil trafficking into the airways along with other chemotactic factors. However, the epithelial and bronchoalveolar lavage (BAL) cell expression of these chemokines in relation to asthma severity and eosinophilic phenotypes has not been addressed. OBJECTIVE: To measure the expression of the three eotaxin isoforms in bronchoscopically obtained samples and compare them with clinically relevant parameters between normal subjects and patients with asthma. METHODS: Normal subjects and patients with asthma of varying severity recruited through the Severe Asthma Research Program underwent clinical assessment and bronchoscopy with airway brushing and BAL. Eotaxin 1-3 mRNA/protein were measured in epithelial and BAL cells and compared with asthma severity, control and eosinophilic inflammation. RESULTS: Eotaxin-2 and eotaxin-3 mRNA and eotaxin-2 protein were increased in airway epithelial brushings from patients with asthma and were highest in cases of severe asthma (p values 0.0155, 0.0033 and 0.0006, respectively), with eotaxin-2 protein increased with age at onset. BAL cells normally expressed high levels of eotaxin-2 mRNA/protein but BAL fluid levels of eotaxin-2 were lowest in severe asthma. Epithelial eotaxin-2 and eotaxin-3 mRNA/protein was associated with sputum eosinophilia, lower forced expiratory volume in 1 s and more asthma exacerbations. Airway epithelial cell eotaxin-2 protein differed by asthma severity only in those with late onset disease, and tended to be highest in those with late onset eosinophilic asthma. CONCLUSIONS: Epithelial eotaxin-2 and 3 are increased in asthma and severe asthma. Their expression may contribute to luminal migration of eosinophils, especially in later onset disease, asthma control and severity.


Assuntos
Asma/metabolismo , Quimiocina CCL24/metabolismo , Quimiocinas CC/metabolismo , Eosinofilia/metabolismo , Células Epiteliais/metabolismo , Corticosteroides/farmacologia , Adulto , Idade de Início , Lavagem Broncoalveolar , Broncoscopia , Quimiocina CCL11/metabolismo , Quimiocina CCL26 , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Estatísticas não Paramétricas
10.
J Immunol ; 188(12): 6046-54, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22573806

RESUMO

CCL11/eotaxin-1 is a potent eosinophilic CC chemokine expressed by primary human fibroblasts. The combination of TGF-ß1 and IL-13 synergistically increases CCL11 expression, but the mechanisms behind the synergy are unclear. To address this, human airway fibroblast cultures from normal and asthmatic subjects were exposed to IL-13 alone or TGF-ß1 plus IL-13. Transcriptional (nuclear run-on) and posttranscriptional (mRNA stability) assays confirmed that transcriptional regulation is critical for synergistic expression of CCL11. TGF-ß1 plus IL-13 synergistically increased STAT-6 phosphorylation, nuclear translocation, and binding to the CCL11 promoter as compared with IL-13 alone. STAT-6 small interfering RNA significantly knocked down both STAT-6 mRNA expression and phosphorylation and inhibited CCL11 mRNA and protein expression. Regulation of the IL-4Rα complex by TGF-ß1 augmented IL-13 signaling by dampening IL-13Rα2 expression, overcoming IL-13's autoregulation of its pathway and enhancing the expression of CCL11. Our data suggest that TGF-ß1 induced activation of the MEK/ERK pathway reduces IL-13Rα2 expression induced by IL-13. Thus, TGF-ß1, a pleiotropic cytokine upregulated in asthmatic airways, can augment eosinophilic inflammation by interfering with IL-13's negative feedback autoregulatory loop under MEK/ERK-dependent conditions.


Assuntos
Asma/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica/imunologia , Pulmão/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Asma/imunologia , Western Blotting , Quimiocina CCL11/imunologia , Quimiocina CCL11/metabolismo , Imunoprecipitação da Cromatina , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/imunologia , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-13/imunologia , Interleucina-13/metabolismo , Pulmão/imunologia , Receptores de Interleucina-13/imunologia , Receptores de Interleucina-13/metabolismo , Receptores de Interleucina-4/imunologia , Receptores de Interleucina-4/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT6/imunologia , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais/imunologia , Fator de Crescimento Transformador beta1/imunologia
11.
Proc Natl Acad Sci U S A ; 108(34): 14246-51, 2011 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-21831839

RESUMO

Epithelial 15-lipoxygenase 1 (15LO1) and activated ERK are increased in asthma despite modest elevations in IL-13. MAPK kinase (MEK)/ERK activation is regulated by interactions of Raf-1 with phosphatidylethanolamine-binding protein 1 (PEBP1). Epithelial 15LO1 generates intracellular 15-hydroxyeicosatetraenoic acid (15HETE) conjugated to phosphatidylethanolamine (PE) (15HETE-PE). We hypothesized that (i) 15LO1 and its product 15HETE-PE serve as signaling molecules interacting with PEBP1 to activate Raf-1/MEK/ERK and that (ii) this 15LO1-15HETE-PE-regulated ERK activation amplifies IL-4Rα downstream pathways. Our results demonstrate that high epithelial 15LO1 levels correlate with ERK phosphorylation ex vivo. In vitro, IL-13 induces 15LO1, which preferentially binds to PEBP1, causing PEBP1 to dissociate from Raf-1 and activate ERK. Exogenous 15HETE-PE similarly induces dissociation of PEBP1 from Raf-1 independently of IL-13/15LO1. siRNA knockdown of 15LO1 decreases the dissociation of Raf-1 from PEBP1, and the resulting lower ERK activation leads to lower downstream IL-4Rα-related gene expression. Identical protein-protein interactions are observed in endobronchial biopsies and fresh epithelial cells from asthmatics ex vivo. Colocalization of Raf-1 to PEBP1 is low in asthmatic tissue and cells compared with normals, whereas there is striking colocalization of 15LO1 with PEBP1 in asthma. Low 15LO1 levels in normals limit its colocalization with PEBP1. The results confirm a previously unknown signaling role for 15LO1 and its PE-conjugated eicosanoid product in human airway epithelial cells. This pathway enhances critical inflammatory pathways integral to asthma pathogenesis.


Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Brônquios/patologia , Células Epiteliais/enzimologia , Sistema de Sinalização das MAP Quinases , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Asma/enzimologia , Asma/patologia , Ligação Competitiva/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Quimiocina CCL26 , Quimiocinas CC/metabolismo , Demografia , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Ácidos Hidroxieicosatetraenoicos/farmacologia , Interleucina-13/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mucina-5AC/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/metabolismo
12.
Am J Respir Crit Care Med ; 179(9): 782-90, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19218191

RESUMO

RATIONALE: 15-Lipoxygenase-1 (15LO1) and MUC5AC are highly expressed in asthmatic epithelial cells. IL-13 is known to induce 15LO1 and MUC5AC in human airway epithelial cells in vitro. Whether 15LO1 and/or its product 15-HETE modulate MUC5AC expression is unknown. OBJECTIVES: To determine the expression of 15LO1 in freshly harvested epithelial cells from subjects with asthma and normal control subjects and to determine whether IL-13-induced 15LO1 expression and activation regulate MUC5AC expression in human bronchial epithelial cells in vitro. METHODS: Human airway epithelial cells from subjects with asthma and normal subjects were evaluated ex vivo for 15LO1 and MUC5AC expression. The impact of 15LO1 on MUC5AC expression in vitro was analyzed by inhibiting 15LO1 through pharmacologic (PD146176) and siRNA approaches in human bronchial epithelial cells cultured under air-liquid interface. We analyzed 15 hydroxyeicosatetraenoic acid (15-HETE) by liquid chromatography/UV/mass spectrometry. MUC5AC and 15LO1 were analyzed by real-time RT-PCR, immunofluoresence, and Western blot. MEASUREMENTS AND MAIN RESULTS: Epithelial 15LO1 expression increased with asthma severity (P < 0.0001). 15LO1 significantly correlated with MUC5AC ex vivo and in vitro. IL-13 increased 15LO1 expression and stimulated formation of two molecular species of 15-HETE esterified to phosphotidylethanolamine (15-HETE-PE). Inhibition of 15LO1 suppressed 15-HETE-PE and decreased MUC5AC expression in the presence of IL-13 stimulation. The addition of exogenous 15-HETE partially restored MUC5AC expression. CONCLUSIONS: Epithelial 15LO1 expression increases with increasing asthma severity. IL-13 induction of 15-HETE-PE enhances MUC5AC expression in human airway epithelial cells. High levels of 15LO1 activity could contribute to the increases of MUC5AC observed in asthma.


Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Brônquios/citologia , Células Epiteliais/metabolismo , Interleucina-13/farmacologia , Mucina-5AC/metabolismo , Adulto , Araquidonato 15-Lipoxigenase/genética , Asma/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Cromatografia Líquida , Esterificação , Feminino , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacologia , Masculino , Espectrometria de Massas , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Índice de Gravidade de Doença , Transfecção
13.
J Allergy Clin Immunol ; 119(6): 1388-97, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17418380

RESUMO

BACKGROUND: TGF-beta induces expression of tissue inhibitor of metalloproteinase 1 (TIMP-1), a potent inhibitor of matrix metalloproteinases that controls extracellular matrix metabolism and deposition. IL-13 alone does not induce TIMP-1, but in combination with TGF-beta it augments TIMP-1 expression. Although these interactions have implications for remodeling in asthma, little is understood regarding the mechanisms controlling TIMP-1 product. OBJECTIVE: To explore the role of Smads and mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) in the TIMP-1 augmentation by IL-13+TGF-beta1 in primary human airway fibroblasts. METHODS: Real-time PCR, Western blot, ELISA, and transient transfection were used to evaluate the mechanisms of TIMP-1 augmentation. RESULTS: IL-13 enhanced TGF-beta1-induced Smad-2 and Smad-3 phosphorylation, transient transfection with dominant-negative Smad-2 or Smad-3 decreased TIMP-1 mRNA expression in the presence of TGF-beta1 and IL-13+TGF-beta1 through inhibition of Smad-2 or Smad-3 phosphorylation. ERK phosphorylation was increased by IL-13 and IL-13+TGF-beta1. MEK-ERK inhibition decreased TIMP-1 mRNA/protein to a greater degree after IL-13+TGF-beta1 stimulation versus TGF-beta1 alone. MEK-ERK inhibition also significantly increased Akt phosphorylation under all conditions and decreased Smad-3 phosphorylation in the presence of IL-13+TGF-beta1. In contrast, phosphoinositide-3 kinase-Akt inhibition increased phosphorylation of ERK and Smads, leading to increased TIMP-1. CONCLUSION: These results indicate that IL-13 augments TGF-beta1-induced TIMP-1 expression through increased Smad phosphorylation. These increases occur as TGF-beta1 downregulates IL-13-induced phosphoinositide-3 kinase activation while leaving the positive effect of IL-13-induced ERK on Smad signaling. CLINICAL IMPLICATIONS: This augmentation of TGF-beta1-induced TIMP-1 by IL-13 could contribute to the fibrosis and airway remodeling seen in the presence of T(H)2 inflammation in asthma.


Assuntos
Adjuvantes Imunológicos/fisiologia , Fibroblastos/enzimologia , Interleucina-13/fisiologia , Inibidor Tecidual de Metaloproteinase-1/fisiologia , Fator de Crescimento Transformador beta1/fisiologia , Adulto , Asma/enzimologia , Asma/imunologia , Asma/metabolismo , Asma/patologia , Brônquios/enzimologia , Brônquios/imunologia , Brônquios/patologia , Células Cultivadas , Regulação para Baixo/imunologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Feminino , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Humanos , Sistema de Sinalização das MAP Quinases/imunologia , Masculino , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Proteínas Smad/metabolismo , Proteínas Smad/fisiologia , Células Th2/imunologia , Células Th2/metabolismo , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Regulação para Cima/imunologia
14.
Am J Respir Crit Care Med ; 173(11): 1208-15, 2006 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-16543551

RESUMO

RATIONALE: Excessive deposition of extracellular matrix occurs in proximal airways of individuals with asthma, but fibrosis in distal lung has not been observed. Whether differing fibrotic capacities of fibroblasts from these two regions contribute to this variability is unknown. OBJECTIVES: We compared morphologic and functional characteristics of fibroblasts isolated from proximal airways and distal lung parenchyma to determine phenotypic differences. METHODS: Concurrent proximal airway and distal lung biopsies were obtained by bronchoscopy from subjects with asthma to isolate airway and distal lung fibroblasts, respectively. The following characteristics were compared: morphology, proliferation, alpha-smooth muscle actin expression, and synthesis of procollagen type I and eotaxin-1. RESULTS: Airway fibroblasts (AFs) are morphologically distinct from distal lung fibroblasts (DLFs): they are larger (2.3-fold greater surface area vs. matched DLFs; p = 0.02), stellate in appearance, and with more cytoplasmic projections compared with the spindle-shaped DLFs. AFs synthesized more procollagen type I than did DLFs at baseline (twofold higher; p = 0.003) and after transforming growth factor-beta stimulation (1.4-fold higher; p = 0.02). Similarly, AFs produced more eotaxin-1 than did DLFs at baseline (2.5-fold higher; p = 0.004) and after interleukin-13 stimulation (13-fold higher; p = 0.0001). In contrast, DLFs proliferate more than AFs with serum stimulation (about sixfold greater; p = 0.03). Unstimulated DLFs also expressed more alpha-smooth muscle actin than did corresponding AFs (p = 0.006). CONCLUSIONS: These studies suggest that at least two phenotypes of fibroblast exist in the lung. These phenotypic differences may partially explain the variable responses to injury and repair between proximal airways and distal lung/parenchyma in asthma and other respiratory diseases.


Assuntos
Asma/patologia , Brônquios/citologia , Fibroblastos/citologia , Pulmão/citologia , Actinas/efeitos dos fármacos , Actinas/metabolismo , Adulto , Proliferação de Células , Quimiocina CCL11 , Quimiocinas CC/biossíntese , Colágeno Tipo I/biossíntese , Matriz Extracelular/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Fator de Crescimento Transformador beta/farmacologia
15.
J Allergy Clin Immunol ; 115(1): 110-7, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15637555

RESUMO

BACKGROUND: Airway eosinophilia and thickened subepithelial basement membrane have previously been reported to increase with increases in TGF-beta expression. However, little is known regarding the expression of specific TGF-beta isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) in asthma, despite recent evidence suggesting that isoforms may have differing biologic activities. OBJECTIVE: This study examined airway tissue expression of the 3 TGF-beta isoforms and several downstream pathway elements in 48 patients with severe asthma with or without persistent eosinophilia, 14 patients with mild asthma, and 21 normal subjects. METHODS: Immunochemistry/immunofluorescence, quantitative real-time PCR and enzyme immunoassay were used to evaluate the 3 TGF-beta isoforms, their receptors, collagen I deposition, connective tissue growth factor expression, and tissue inhibitor of metalloproteinases 1 levels. RESULTS: Of the isoforms, only TGF-beta2 was different among the groups and increased in severe asthma (overall P < .0001). The increase was due to severe asthma tissue eosinophils which, unlike eosinophils in other groups, expressed high amounts of TGF-beta2. Subjects with severe asthma also had the thickest subbasement membrane and highest tissue inhibitor of metalloproteinases 1 levels. In contrast, TGF-beta receptor 1 and connective tissue growth factor were both consistently downregulated in asthma, regardless of severity. CONCLUSION: TGF-beta2, expressed mainly by eosinophils, is the predominant isoform expressed in severe asthma, and is associated with increased profibrotic responses. Decreased expression of TGF-beta receptor 1 and connective tissue growth factor in all asthma severity groups suggests a degree of activation of the TGF-beta pathway in airway tissue of all asthmatic compared with normal airways.


Assuntos
Asma/imunologia , Eosinofilia/imunologia , Fator de Crescimento Transformador beta/biossíntese , Adulto , Asma/metabolismo , Brônquios/imunologia , Tecido Conjuntivo/metabolismo , Eosinofilia/metabolismo , Eosinófilos/metabolismo , Feminino , Substâncias de Crescimento/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Músculo Liso/imunologia , Isoformas de Proteínas/análise , Isoformas de Proteínas/biossíntese , RNA Mensageiro/análise , Receptores de Fatores de Crescimento Transformadores beta/análise , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta2
16.
Am J Physiol Cell Physiol ; 288(2): C435-42, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15456694

RESUMO

Tissue inhibitor of metalloproteinase (TIMP)-1 is a potent inhibitor of activated matrix metalloproteinases (MMPs) such as gelatinases and collagenases. TIMP-1 is induced by transforming growth factor-beta1 (TGF-beta1), but details regarding signaling pathways remain unclear. T-helper-2 cytokines also have profibrotic properties and can interact with TGF-beta. In the present study, we examined the effects of interleukin (IL)-13 (2,500 pM) on TGF-beta1 (200 pM)-induced expression of TIMP-1 mRNA and protein in primary human airway fibroblasts obtained from 57 human subjects. IL-13 alone had no effect on TIMP-1 mRNA or protein expression. However, IL-13 synergistically augmented TGF-beta1-induced TIMP-1 mRNA and protein expression (P < 0.001 vs. TGF-beta1 alone). The upregulation of TIMP-1 by the combination of TGF-beta1 and IL-13 involved increased transcription, with little effect on mRNA stabilization. Initial exploration of the pathways leading to the synergy determined that activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway by IL-13 may have a negative effect on TIMP-1 production. The specific PI3K inhibitor LY-294002 in the presence of TGF-beta1, IL-13, or the combination of the two caused significant increases in TIMP-1 mRNA expression, while LY-294002 increased TIMP-1 protein levels in the presence of IL-13 alone. These results suggest that IL-13 augments TGF-beta1-induced profibrotic responses at both the mRNA and protein levels. Although IL-13 induced activation of PI3K-Akt, the activation did not contribute to the synergy observed with TGF-beta1 plus IL-13 in TIMP-1 expression and in fact may dampen it. The mechanisms behind the synergy remain to be determined.


Assuntos
Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Interleucina-13/farmacologia , Transdução de Sinais/fisiologia , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Fator de Crescimento Transformador beta/farmacologia , 1-Fosfatidilinositol 4-Quinase/metabolismo , Células Cultivadas , Sinergismo Farmacológico , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-13/metabolismo , Pulmão/fisiologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo
17.
Am J Pathol ; 165(4): 1097-106, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15466377

RESUMO

The transforming growth factor (TGF)-beta family is important for tissue repair in pathological conditions including asthma. However, little is known about the impact of either TGF-beta1 or TGF-beta2 on asthmatic airway epithelial mucin expression. We evaluated bronchial epithelial TGF-beta1 and TGF-beta2 expression and their effects on mucin expression, and the role of TGF-beta1 or TGF-beta2 in interleukin (IL)-13-induced mucin expression. Epithelial TGF-beta1, TGF-beta2, and mucin expression were evaluated in endobronchial biopsies from asthmatics and normal subjects. The effects of TGF-beta1 or TGF-beta2 on mucin MUC5AC protein and mRNA expression, and the impact of IL-13 on epithelial TGF-beta1, TGF-beta2, and MUC5AC were determined in cultured bronchial epithelial cells from endobronchial brushings of both subject groups. In biopsy tissue, epithelial TGF-beta2 expression levels were higher than TGF-beta1 in both asthmatics and normals. TGF-beta2, but not TGF-beta1, was increased in asthmatics compared with normals, and significantly correlated with mucin expression. TGF-beta2, but not TGF-beta1, increased mucin expression in cultured epithelial cells from both subject groups. IL-13 increased the release of TGF-beta2, but not TGF-beta1, from epithelial cells. A neutralizing TGF-beta2 antibody partially inhibited IL-13-induced mucin expression. These data suggest that TGF-beta2 production by asthmatic bronchial epithelial cells may increase airway mucin expression. IL-13-induced mucin expression may occur in part through TGF-beta2 up-regulation.


Assuntos
Asma/metabolismo , Mucinas/biossíntese , Mucosa Respiratória/metabolismo , Fator de Crescimento Transformador beta/biossíntese , Adulto , Biópsia , Células Cultivadas , Feminino , Humanos , Interleucina-13/farmacologia , Masculino , Pessoa de Meia-Idade , Mucinas/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Fator de Crescimento Transformador beta/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia
18.
J Allergy Clin Immunol ; 112(6): 1064-71, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14657859

RESUMO

BACKGROUND: Matrix metalloproteinase (MMP)-9 levels are increased in bronchoalveolar lavage (BAL) fluid from patients with severe asthma on high doses of glucocorticoids (GCs). OBJECTIVE: We sought to identify neutrophils as the source of increased BAL fluid MMP-9 in severe asthma and to evaluate the effects of GCs on this MMP-9. METHODS: MMP-9 protein, activity, and mRNA were measured in BAL fluid and cells at baseline, and after in vitro GCs in patients with severe asthma and controls using enzyme immunoassays, zymography, Western blotting, and real-time PCR. RESULTS: The high molecular weight (HMW) form of MMP-9 was significantly increased in severe asthma (P =.02). Western blotting confirmed a heterodimer of MMP-9 and neutrophil gelatinase-associated lipocalin. The HMW MMP-9 correlated with BAL neutrophils (r =.65, P <.0001). BAL cell supernatant MMP-9 protein levels also tended to be higher in patients with severe asthma (overall, P =.09), whereas the HMW activity form was increased (P =.03). MMP-9 protein (and HMW activity) correlated with neutrophils in the cell pellet (r =.75, P <.0001). In contrast to protein and activity, BAL cell mRNA levels were marginally lower in patients with severe asthma than in control subjects (overall, P =.06). Although GCs decreased BAL cell MMP-9 protein and mRNA in vitro, the effect was significantly smaller in severe asthma (P <.01 for both). GCs decreased the pro-MMP-9 activity in patients with severe asthma and normal control subjects, while having no effects on the HMW form (P =.22). Peripheral blood neutrophil MMP-9 protein was not affected by GCs. CONCLUSIONS: BAL neutrophils contribute to BAL fluid MMP-9 protein and activity and are poorly inhibited by GCs.


Assuntos
Asma/enzimologia , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Neutrófilos/enzimologia , Proteínas da Fase Aguda/metabolismo , Adulto , Asma/sangue , Western Blotting , Lavagem Broncoalveolar , Líquido da Lavagem Broncoalveolar/citologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Lipocalina-2 , Lipocalinas , Masculino , Metaloproteinase 9 da Matriz/genética , Neutrófilos/efeitos dos fármacos , Proteínas Oncogênicas/metabolismo , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas , RNA Mensageiro/metabolismo
20.
J Immunol ; 169(8): 4613-9, 2002 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-12370400

RESUMO

Chronic diseases may involve an "innate" response followed by an adaptive immune response, of a Th1 or Th2 variety. Little is known regarding the interactions of these responses. We hypothesized that TGF-beta1 (innate response factor associated with wound repair) in combination with IL-13 (Th2 factor) might augment inflammatory processes associated with asthma. Airway fibroblasts were cultured from asthmatic subjects and normal controls. These fibroblasts were exposed to TGF-beta1 and IL-13 alone or in combination, and eotaxin-1 expression and production were evaluated. At 48 h, eotaxin-1 production was markedly increased with the combination of TGF-beta1 and IL-13 (p < 0.0001) compared with either stimulus alone. mRNA increased slightly at 1 h with IL-13 or TGF-beta1 plus IL13, peaked, and became significantly increased over IL-13 alone at 24 h. Protein was measurable from 6 h with IL-13 and TGF-beta1 plus IL-13, but greater levels were measured over time with the combination. Actinomycin ablated the increase in mRNA and protein seen with IL-13 alone and with TGF-beta1 plus IL-13. Cycloheximide blocked the increase in mRNA at 6 h in both conditions, but also blocked the increase at 24 h with TGF-beta1 plus IL-13. STAT-6 was rapidly activated with both IL-13 and the combination, without difference. Finally, eotaxin-1-positive fibroblasts were identified in severe asthma biopsies in greater numbers than in normals. These results support the concept that interactions of innate and adaptive immune systems may be important in promoting the tissue eosinophilia of asthma, particularly in those with more severe disease.


Assuntos
Adjuvantes Imunológicos/farmacologia , Quimiocinas CC/biossíntese , Fatores Quimiotáticos de Eosinófilos/biossíntese , Fibroblastos/metabolismo , Interleucina-13/farmacologia , Pulmão/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Regulação para Cima/imunologia , Asma/imunologia , Asma/metabolismo , Asma/patologia , Northern Blotting , Brônquios/imunologia , Brônquios/metabolismo , Células Cultivadas , Quimiocina CCL11 , Quimiocinas CC/antagonistas & inibidores , Quimiocinas CC/genética , Fatores Quimiotáticos de Eosinófilos/antagonistas & inibidores , Fatores Quimiotáticos de Eosinófilos/genética , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Relação Dose-Resposta Imunológica , Sinergismo Farmacológico , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Humanos , Interleucina-8/biossíntese , Pulmão/imunologia , Pulmão/patologia , Reação em Cadeia da Polimerase , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Fator de Transcrição STAT6 , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transativadores/metabolismo , Fator de Crescimento Transformador beta1 , Regulação para Cima/genética
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