RESUMEN
Overproduction of mucus is a central feature of asthma. The cytokine, IL-13, epidermal growth factor receptor (EGFR), and transcription factor, FOXA2, have each been implicated in mucus production, but the mechanistic relationships between these molecules are not yet well understood. To address this, we established a primary normal human bronchial epithelial cell culture system with IL-13-induced mucus production and gene transcript expression changes similar to those seen in vivo in mice. IL-13 did not stimulate release of the EGFR ligand, transforming growth factor (TGF)-alpha. However, there was constitutive release of TGF-alpha from normal human bronchial epithelial cells, and inhibition of TGF-alpha or EGFR reduced both constitutive and IL-13-induced mucin production. Microarray analysis revealed that IL-13 and the EGFR pathway appear to have almost completely independent effects on transcript expression. IL-13 induced a relatively small set of transcripts, including several novel transcripts that might play a role in pathogenesis of allergic airway disease. In contrast, EGFR activity had extensive effects, including altered expression of many transcripts associated with cell metabolism, survival, transcription, and differentiation. One of the few common effects of IL-13 and EGFR signaling was decreased expression of FOXA2, which is known to prevent mucus production. We conclude that the IL-13 and EGFR pathways make critical but quite distinct contributions to gene regulation in airway epithelial cells, and that both pathways affect expression of the key transcription factor, FOXA2, a known regulator of mucus production.
Asunto(s)
Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Receptores ErbB/metabolismo , Interleucina-13/farmacología , Mucinas/biosíntesis , Mucinas/efectos de los fármacos , Administración Intranasal , Animales , Bovinos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Factor Nuclear 3-beta del Hepatocito/metabolismo , Humanos , Interleucina-13/administración & dosificación , Masculino , Metaplasia , Ratones , Ratones Endogámicos BALB C , Mucina 5AC , Mucinas/genética , Mucinas/metabolismo , Moco/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sistema Respiratorio/citología , Sistema Respiratorio/efectos de los fármacosRESUMEN
RATIONALE: Macrophages are believed to play a central role in emphysema based largely on data from mouse models. However, the relevance of these models to smoking-related lung disease in humans is uncertain. OBJECTIVES: We sought to comprehensively characterize the effects of smoking on gene expression in human alveolar macrophages and to compare these with effects seen in transgenic mouse models of emphysema. METHODS: We used DNA microarrays with genomewide coverage to analyze alveolar macrophages from 15 smokers, 15 nonsmokers, and 15 subjects with asthma (disease control). Selected gene expression changes were validated by polymerase chain reaction and ELISA. Expression changes were compared with those identified by microarray analysis of interleukin-13-overexpressing and integrin-beta6-deficient mice, which both develop emphysema. MEASUREMENTS AND MAIN RESULTS: All 15 smokers shared a common pattern of macrophage gene expression that distinguished them from nonsmokers, a finding not observed in subjects with asthma. We identified 110 genes as differentially expressed in smokers despite using conservative statistical methods. Matrix metalloproteinase 12, a proteinase that plays a critical role in mouse models, was the third most highly induced gene in smokers (ninefold, p < 0.0001). However, most changes in smokers were not reflected in mouse models. One such finding was increased osteopontin expression in smokers (fourfold, p = 0.006), which was confirmed at the protein level and correlated with the degree of airway obstruction. CONCLUSIONS: Smoking induces a remarkably consistent and distinctive pattern of alveolar macrophage activation. These studies identify aspects of mouse models that are directly relevant to human smokers and also reveal novel potential mediators of smoking-related diseases.
Asunto(s)
Expresión Génica , Activación de Macrófagos/genética , Macrófagos Alveolares/metabolismo , Metaloendopeptidasas/genética , ARN/genética , Fumar/metabolismo , Adulto , Animales , Líquido del Lavado Bronquioalveolar , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Metaloproteinasa 12 de la Matriz , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Enfisema Pulmonar/etiología , Enfisema Pulmonar/genética , Enfisema Pulmonar/metabolismo , Fumar/efectos adversos , Fumar/genéticaRESUMEN
BACKGROUND: Asthma functional genomics studies are challenging because it is difficult to relate gene expression changes to specific disease mechanisms or pathophysiologic features. Use of simplified model systems might help to address this problem. One such model is the IL-13/Epi (IL-13-overexpressing transgenic mice with STAT6 expression limited to epithelial cells) focused transgenic mouse, which isolates the effects of a single mediator, IL-13, on a single cell type, the airway epithelial cell. These mice develop airway hyperreactivity and mucus overproduction but not airway inflammation. OBJECTIVE: To identify how effects of IL-13 on airway epithelial cells contribute to gene expression changes in murine asthma models and determine whether similar changes are seen in people with asthma. METHODS: We analyzed gene expression in ovalbumin allergic mice, IL-13-overexpressing mice, and IL-13/Epi mice with microarrays. We analyzed the expression of human orthologues of genes identified in the mouse studies in airway epithelial cells from subjects with asthma and control subjects. RESULTS: In comparison with the other 2 models, IL-13/Epi mice had a remarkably small subset of gene expression changes. Human orthologues of some genes identified as increased in the mouse models were more highly expressed in airway epithelial cells from subjects with asthma than in controls. These included calcium-activated chloride channel 1, 15-lipoxygenase, trefoil factor 2, and intelectin. CONCLUSION: The combination of focused transgenic models, DNA microarray analyses, and translational studies provides a powerful approach for analyzing the contributions of specific mediators and cell types and for focusing attention on a limited number of genes associated with specific pathophysiologic aspects of asthma.
Asunto(s)
Asma/genética , Perfilación de la Expresión Génica , Animales , Bronquios/metabolismo , Células Cultivadas , Citocinas , Proteínas Ligadas a GPI , Humanos , Interleucina-13/farmacología , Lectinas/genética , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Factor Trefoil-2RESUMEN
BACKGROUND: Asthma is characterized by type 2 T-helper cell (Th2) inflammation, goblet cell hyperplasia, airway hyperreactivity, and airway fibrosis. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and its receptor, CCR2, have been shown to play important roles in the development of Th2 inflammation. CCR2-deficient mice have been found to have altered inflammatory and physiologic responses in some models of experimental allergic asthma, but the role of CCR2 in contributing to inflammation and airway hyperreactivity appears to vary considerably between models. Furthermore, MCP-1-deficient mice have not previously been studied in models of experimental allergic asthma. METHODS: To test whether MCP-1 and CCR2 are each required for the development of experimental allergic asthma, we applied an Aspergillus antigen-induced model of Th2 cytokine-driven allergic asthma associated with airway fibrosis to mice deficient in either MCP-1 or CCR2. Previous studies with live Aspergillus conidia instilled into the lung revealed that MCP-1 and CCR2 play a role in anti-fungal responses; in contrast, we used a non-viable Aspergillus antigen preparation known to induce a robust eosinophilic inflammatory response. RESULTS: We found that wild-type C57BL/6 mice developed eosinophilic airway inflammation, goblet cell hyperplasia, airway hyperreactivity, elevations in serum IgE, and airway fibrosis in response to airway challenge with Aspergillus antigen. Surprisingly, mice deficient in either MCP-1 or CCR2 had responses to Aspergillus antigen similar to those seen in wild-type mice, including production of Th2 cytokines. CONCLUSION: We conclude that robust Th2-mediated lung pathology can occur even in the complete absence of MCP-1 or CCR2.
Asunto(s)
Aspergilosis Broncopulmonar Alérgica/inmunología , Hiperreactividad Bronquial/inmunología , Quimiocina CCL2/deficiencia , Eosinofilia/inmunología , Fibrosis/inmunología , Receptores de Quimiocina/deficiencia , Células Th2/inmunología , Animales , Antígenos Fúngicos/inmunología , Aspergilosis Broncopulmonar Alérgica/complicaciones , Aspergilosis Broncopulmonar Alérgica/microbiología , Aspergilosis Broncopulmonar Alérgica/patología , Aspergillus fumigatus/inmunología , Hiperreactividad Bronquial/complicaciones , Hiperreactividad Bronquial/microbiología , Hiperreactividad Bronquial/patología , Quimiocina CCL2/inmunología , Citocinas/inmunología , Eosinofilia/complicaciones , Eosinofilia/microbiología , Eosinofilia/patología , Fibrosis/complicaciones , Fibrosis/microbiología , Fibrosis/patología , Ratones , Ratones Noqueados , Receptores CCR2 , Receptores de Quimiocina/inmunologíaRESUMEN
BACKGROUND: The cell adhesion molecule integrin alpha 4 beta 7 helps direct the migration of blood lymphocytes to the intestine and associated lymphoid tissues. We hypothesized that beta 7+ and beta 7- blood memory T helper cells differ in their expression of genes that play a role in the adhesion or migration of T cells. RESULTS: RNA was prepared from beta 7+ and beta 7- CD4+ CD45RA- blood T cells from nine normal human subjects and analyzed using oligonucleotide microarrays. Of 21357 genes represented on the arrays, 16 were more highly expressed in beta 7+ cells and 18 were more highly expressed in beta 7- cells (>/=1.5 fold difference and adjusted P < 0.05). Several of the differentially expressed transcripts encode proteins with established or putative roles in lymphocyte adhesion and chemotaxis, including the chemokine receptors CCR9 and CCR10, the integrin alpha 4 subunit, L-selectin, KLRB1 (CD161), NT5E (CD73), LGALS1 and LGALS2 (galectin-1 and -2), and RGS1. Flow cytometry was used to determine whether differences in levels of transcripts encoding cell surface proteins were associated with differential expression of those proteins. Using this approach, we found that surface expression of KLRB1, LAIR1, and NT5E proteins was higher on beta 7+ memory/effector T cells than on beta 7- cells. CONCLUSIONS: Memory/effector T cells that express integrin beta 7 have a distinct pattern of expression of a set of gene transcripts. Several of these molecules can affect cell adhesion or chemotaxis and are therefore likely to modulate the complex multistep process that regulates trafficking of CD4+ memory T cell subsets with different homing behaviors.