RESUMEN
Epithelial-fibroblast interactions are thought to be very important in the adult lung in response to injury, but the specifics of these interactions are not well defined. We developed coculture systems to define the interactions of adult human alveolar epithelial cells with lung fibroblasts. Alveolar type II cells cultured on floating collagen gels reduced the expression of type 1 collagen (COL1A1) and α-smooth muscle actin (ACTA2) in fibroblasts. They also reduced fibroblast expression of hepatocyte growth factor (HGF), fibroblast growth factor 7 (FGF7, KGF), and FGF10. When type II cells were cultured at an air-liquid interface to maintain high levels of surfactant protein expression, this inhibitory activity was lost. When type II cells were cultured on collagen-coated tissue culture wells to reduce surfactant protein expression further and increase the expression of some type I cell markers, the epithelial cells suppressed transforming growth factor-ß (TGF-ß)-stimulated ACTA2 and connective tissue growth factor (CTGF) expression in lung fibroblasts. Our results suggest that transitional alveolar type II cells and likely type I cells but not fully differentiated type II cells inhibit matrix and growth factor expression in fibroblasts. These cells express markers of both type II cells and type I cells. This is probably a normal homeostatic mechanism to inhibit the fibrotic response in the resolution phase of wound healing. Defining how transitional type II cells convert activated fibroblasts into a quiescent state and inhibit the effects of TGF-ß may provide another approach to limiting the development of fibrosis after alveolar injury.
Asunto(s)
Células Epiteliales Alveolares/metabolismo , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Células Epiteliales Alveolares/efectos de los fármacos , Células Cultivadas , Colágeno/farmacología , Células Epiteliales/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Surfactantes Pulmonares/metabolismoRESUMEN
TGF beta is a multifunctional cytokine that is important in the pathogenesis of pulmonary fibrosis. The ability of TGF beta to stimulate smooth muscle actin and extracellular matrix gene expression in fibroblasts is well established. In this report, we evaluated the effect of TGF beta on the expression of HGF, FGF7 (KGF), and FGF10, important growth and survival factors for the alveolar epithelium. These growth factors are important for maintaining type II cells and for restoration of the epithelium after lung injury. Under conditions of normal serum supplementation or serum withdrawal TGF beta inhibited fibroblast expression of HGF, FGF7, and FGF10. We confirmed these observations with genome wide RNA sequencing of the response of control and IPF fibroblasts to TGF beta. In general, gene expression in IPF fibroblasts was similar to control fibroblasts. Reduced expression of HGF, FGF7, and FGF10 is another means whereby TGF beta impairs epithelial healing and promotes fibrosis after lung injury.
Asunto(s)
Fibroblastos/efectos de los fármacos , Fibrosis Pulmonar Idiopática/patología , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Factor de Crecimiento Transformador beta/farmacología , Anciano , Anciano de 80 o más Años , Células Cultivadas , Medio de Cultivo Libre de Suero , Femenino , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Factor 10 de Crecimiento de Fibroblastos/genética , Factor 7 de Crecimiento de Fibroblastos/biosíntesis , Factor 7 de Crecimiento de Fibroblastos/genética , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Factor de Crecimiento de Hepatocito/biosíntesis , Factor de Crecimiento de Hepatocito/genética , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Pulmón/metabolismo , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
TGF beta is a multifunctional cytokine that regulates alveolar epithelial cells as well as immune cells and fibroblasts. TGF beta inhibits surfactant protein A, B and C expression in fetal human lung and can inhibit type II cell proliferation induced by FGF7 (KGF). However, little is known about direct effects of TGF beta on adult human type II cells. We cultured alveolar type II cells under air/liquid interface conditions to maintain their state of differentiation with or without TGF beta. TGF beta markedly decreased expression of SP-A, SP-B, SP-C, fatty acid synthase, and the phospholipid transporter ABCA3. However, TGF beta increased protein levels of SP-D with little change in mRNA levels, indicating that it is regulated independently from other components of surfactant. TGF beta is a negative regulator of both the protein and the phospholipid components of surfactant. TGF beta did not induce EMT changes in highly differentiated human type II cells. SP-D is an important host defense molecule and regulated independently from the other surfactant proteins. Taken together these data are the first report of the effect of TGF beta on highly differentiated adult human type II cells. The effects on the surfactant system are likely important in the development of fibrotic lung diseases.
Asunto(s)
Células Epiteliales Alveolares/metabolismo , Proteínas Asociadas a Surfactante Pulmonar/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Células Epiteliales Alveolares/efectos de los fármacos , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Lipogénesis/efectos de los fármacos , Proteínas Asociadas a Surfactante Pulmonar/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Severe acute respiratory syndrome (SARS)-coronavirus (CoV) produces a devastating primary viral pneumonia with diffuse alveolar damage and a marked increase in circulating cytokines. One of the major cell types to be infected is the alveolar type II cell. However, the innate immune response of primary human alveolar epithelial cells infected with SARS-CoV has not been defined. Our objectives included developing a culture system permissive for SARS-CoV infection in primary human type II cells and defining their innate immune response. Culturing primary human alveolar type II cells at an air-liquid interface (A/L) improved their differentiation and greatly increased their susceptibility to infection, allowing us to define their primary interferon and chemokine responses. Viral antigens were detected in the cytoplasm of infected type II cells, electron micrographs demonstrated secretory vesicles filled with virions, virus RNA concentrations increased with time, and infectious virions were released by exocytosis from the apical surface of polarized type II cells. A marked increase was evident in the mRNA concentrations of interferon-ß and interferon-λ (IL-29) and in a large number of proinflammatory cytokines and chemokines. A surprising finding involved the variability of expression of angiotensin-converting enzyme-2, the SARS-CoV receptor, in type II cells from different donors. In conclusion, the cultivation of alveolar type II cells at an air-liquid interface provides primary cultures in which to study the pulmonary innate immune responses to infection with SARS-CoV, and to explore possible therapeutic approaches to modulating these innate immune responses.
Asunto(s)
Células Epiteliales/inmunología , Inmunidad Innata , Síndrome Respiratorio Agudo Grave/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Enzima Convertidora de Angiotensina 2 , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Diferenciación Celular , Citoplasma/inmunología , Citoplasma/ultraestructura , Citoplasma/virología , Células Epiteliales/virología , Humanos , Interferón beta/inmunología , Interferón beta/metabolismo , Interferones , Interleucinas/inmunología , Interleucinas/metabolismo , Peptidil-Dipeptidasa A/inmunología , Peptidil-Dipeptidasa A/metabolismo , Cultivo Primario de Células , Alveolos Pulmonares/citología , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/virología , ARN Mensajero/metabolismo , Receptores Virales/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/virología , Factores de Tiempo , Liberación del VirusRESUMEN
Alveolar Type II (ATII) cells are important targets for seasonal and pandemic influenza. To investigate the influenza-induced innate immune response in those cells, we measured the global gene expression profile of highly differentiated ATII cells infected with the influenza A virus at a multiplicity of infection of 0.5 at 4 hours and 24 hours after inoculation. Infection with influenza stimulated a significant increase in the mRNA concentrations of many host defense-related genes, including pattern/pathogen recognition receptors, IFN, and IFN-induced genes, chemokines, and suppressors of cytokine signaling. We verified these changes by quantitative real-time RT-PCR. At the protein level, we detected a robust virus-induced secretion of the three glutamic acid-leucine-arginine (ELR)-negative chemokines CXCL9, CXCL10, and CXCL11, according to ELISA. The ultraviolet inactivation of virus abolished the chemokine and cytokine response. Viral infection did not appear to alter the differentiation of ATII cells, as measured by cellular mRNA and concentrations of surfactant proteins. However, viral infection significantly reduced the secretion of surfactant protein (SP)-A and SP-D. In addition, influenza A virus triggered a time-dependent activation of phosphatidylinositol 3-kinase signaling in ATII cells. The inhibition of this pathway significantly decreased the release of infectious virus and the chemokine response, but did not alter virus-induced cell death. This study provides insights into influenza-induced innate immunity in differentiated human ATII cells, and demonstrates that the alveolar epithelium is a critical part of the initial innate immune response to influenza.
Asunto(s)
Inmunidad Innata , Virus de la Influenza A/metabolismo , Alveolos Pulmonares/citología , Adulto , Anciano , Anciano de 80 o más Años , Diferenciación Celular , Quimiocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fosfatidilinositol 3-Quinasas/metabolismo , Fumar , Tensoactivos/metabolismoRESUMEN
Severe acute respiratory syndrome (SARS) is a disease characterized by diffuse alveolar damage. We isolated human alveolar type II cells and maintained them in a highly differentiated state. Type II cell cultures supported SARS-CoV replication as evidenced by RT-PCR detection of viral subgenomic RNA and an increase in virus titer. Virus titers were maximal by 24 h and peaked at approximately 10(5) pfu/mL. Two cell types within the cultures were infected. One cell type was type II cells, which were positive for SP-A, SP-C, cytokeratin, a type II cell-specific monoclonal antibody, and Ep-CAM. The other cell type was composed of spindle-shaped cells that were positive for vimentin and collagen III and likely fibroblasts. Viral replication was not detected in type I-like cells or macrophages. Hence, differentiated adult human alveolar type II cells were infectible but alveolar type I-like cells and alveolar macrophages did not support productive infection.
Asunto(s)
Epitelio/virología , Alveolos Pulmonares/citología , Alveolos Pulmonares/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Replicación Viral , Diferenciación Celular , Células Cultivadas , Efecto Citopatogénico Viral , Humanos , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Virología/métodosRESUMEN
Cultures of differentiating fetal human type II cells have been available for many years. However, studies with differentiated adult human type II cells are limited. We used a published method for type II cell isolation and developed primary culture systems for maintenance of differentiated adult human alveolar epithelial cells for in vitro studies. Human type II cells cultured on Matrigel (basolateral access) or a mixture of Matrigel and rat tail collagen (apical access) in the presence of keratinocyte growth factor, isobutylmethylxanthine, 8-bromo-cyclicAMP, and dexamethasone (KIAD) expressed the differentiated type II cell phenotype as measured by the expression of surfactant protein (SP)-A, SP-B, SP-C, and fatty acid synthase and their morphologic appearance. These cells contain lamellar inclusion bodies and have apical microvilli. In both systems the cells appear well differentiated. In the apical access system, type II cell differentiation markers initially decreased and then recovered over 6 d in culture. Lipid synthesis was also increased by the addition of KIAD. In contrast, type II cells cultured on rat tail collagen (or tissue culture plastic) slowly lose their lamellar inclusions and expression of the surfactant proteins and increase the expression of type I cell markers. The expression of the phenotypes is regulated by the culture conditions and is, in part, reversible in vitro.
Asunto(s)
Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Células Epiteliales , Alveolos Pulmonares/citología , Animales , Biomarcadores/metabolismo , Polaridad Celular , Forma de la Célula , Células Cultivadas , Colágeno/metabolismo , Combinación de Medicamentos , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Laminina/metabolismo , Lípidos/biosíntesis , Fenotipo , Proteoglicanos/metabolismo , RatasRESUMEN
Keratinocyte growth factor (KGF) stimulates fatty acid and phospholipid synthesis in alveolar type II cells in vitro. KGF stimulates lipogenic enzymes, including fatty acid synthase and stearyl-CoA desaturase-1, and transcription factors involved in lipogenesis, such as sterol regulatory element binding protein (SREBP)-1c and CCAAT/enhancer binding protein (C/EBP)alpha and C/EBPdelta. To define the role of SREBP-1c on the induction of lipogenic genes and lipogenesis by KGF in primary cultures of rat type II cells, we used adenoviral vectors to alter levels of SREBP-1c. Overexpression of a dominant-negative form of SREBP-1 decreased lipogenesis and decreased the induction of fatty acid synthase and stearyl coenzyme A desaturase-1 by KGF. Conversely, adenovirus-mediated overexpression of a constitutively active form of SREBP-1c mimicked the effect of KGF on lipogenic enzymes and lipogenesis. These data indicate that SREBP-1c is required for the stimulation of lipogenesis by KGF in the alveolar type II cells and is a key regulator of lung lipid metabolism and that expression of SREBP-1c is sufficient to induce lipogenesis in rat type II cells.
Asunto(s)
Ácidos Grasos/biosíntesis , Factor 7 de Crecimiento de Fibroblastos/farmacología , Lipogénesis/efectos de los fármacos , Alveolos Pulmonares/efectos de los fármacos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Adenoviridae/genética , Animales , Técnicas de Cultivo de Célula , Células Cultivadas , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genéticaRESUMEN
Alveolar epithelial cells are among the first cells to encounter inhaled particles or organisms. These cells likely participate in the initiation and modulation of the inflammatory response by production of chemokines. However, there is little information on the extent or regulation of chemokine production by these cells. Rat type II cells were studied under differentiated and dedifferentiated conditions to determine their ability to express and secrete CXC chemokines. Both differentiated and dedifferentiated type II cells secreted MIP-2, MCP-1, and CINC-2 in response to a cytokine mixture of IL-1beta, TNF-alpha, and IFN-gamma or to IL-1beta alone. The cytokine mixture also induced iNOS expression and nitrite secretion. Both differentiated and dedifferentiated type II cells expressed CINC-1 (GRO), CINC-2alpha, CINC-3 (MIP-2), and MCP-1 mRNA, and their expression was increased by the cytokine mixture or by IL-1beta alone. However, CINC-2beta, a splice variant of CINC-2, was only expressed under differentiated conditions stimulated by KGF and was not increased by the cytokine mixture or by IL-1beta. In situ hybridization of normal lung and lung instilled with Ad-KGF demonstrated that CINC-2beta was expressed by alveolar and bronchiolar epithelial cells in vivo. We conclude that CINC-2beta is regulated differently from most other chemokines and that its expression is related to the state of alveolar type II cell differentiation.
Asunto(s)
Quimiocinas CXC/metabolismo , Células Epiteliales/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Alveolos Pulmonares/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CXCL1 , Quimiocina CXCL2 , Quimiocinas CXC/genética , Citocinas/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Alveolos Pulmonares/citología , Alveolos Pulmonares/efectos de los fármacos , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Strategies to stimulate endogenous surfactant production require a detailed understanding of the regulation of lipogenesis in alveolar type II cells. We developed culture conditions in which keratinocyte growth factor (KGF) stimulates fatty acid and phospholipid synthesis. KGF stimulated acetate incorporation into phosphatidylcholine, disaturated phosphatidylcholine, and phosphatidylglycerol more than 5% rat serum alone. To determine the mRNA levels of lipogenic enzymes and transport proteins, we analyzed gene expression by oligonucleotide microarrays. KGF increased the mRNA levels for fatty acid synthase, stearoyl-CoA desaturase-1 (SCD-1), and epidermal fatty acid-binding protein more than rat serum alone. In addition, KGF increased the mRNA levels of the transcription factors CCAAT/enhancer-binding protein alpha (C/EBPalpha) and C/EBPdelta as well as SREBP-1c (ADD-1), but not PPARgamma. These changes in C/EBPalpha and C/EBPdelta were confirmed by in situ hybridization. SCD-1 was also found to be highly expressed in alveolar type II cells in vivo. Furthermore, KGF increased protein levels of fatty acid synthase, C/EBPalpha, C/EBPdelta, SREBP-1, epidermal fatty acid-binding protein, and SCD. Finally, the liver X receptor agonist T0901317 increased acetate incorporation and SREBP-1 but not SREBP-2 protein levels. In summary, KGF stimulates lipogenesis in type II cells by a coordinated expression of lipogenic enzymes and transport proteins regulated by C/EBP isoforms and SREBP-1c.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/metabolismo , Ácidos Grasos/metabolismo , Factores de Crecimiento de Fibroblastos/fisiología , Animales , Western Blotting , Proteína delta de Unión al Potenciador CCAAT , Colágeno/farmacología , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Factor 7 de Crecimiento de Fibroblastos , Immunoblotting , Hibridación in Situ , Laminina/farmacología , Metabolismo de los Lípidos , Receptores X del Hígado , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos/química , Receptores Nucleares Huérfanos , Fosfolípidos/metabolismo , Isoformas de Proteínas , Proteoglicanos/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Proteína 2 de Unión a Elementos Reguladores de Esteroles , Factores de Transcripción/metabolismoRESUMEN
Secretion of surfactant proteins A and D (SP-A and SP-D) has been difficult to study in vitro because a culture system for maintaining surfactant secretion has been difficult to establish. We evaluated several growth factors, corticosteroids, rat serum, and a fibroblast feeder layer for the ability to produce and maintain a polarized epithelium of type II cells that secretes SP-A and SP-D into the apical medium. Type II cells were plated on a filter insert coated with an extracellular matrix and were cultured at an air-liquid interface. Keratinocyte growth factor (KGF) stimulated type II cell proliferation and secretion of SP-A and SP-D more than fibroblast growth factor-10 (FGF-10), hepatocyte growth factor (HGF), or heparin-binding epidermal-like growth factor (HB-EGF). Cells cultured in the presence of KGF and rat serum with or without fibroblasts had high surfactant protein mRNA levels and exhibited a high level of SP-A and SP-D secretion. Dexamethasone inhibited type II cell proliferation but increased expression of SP-B. In the presence of KGF, rat serum, and dexamethasone, the mRNAs for the surfactant proteins were maintained at high levels. Secretion of SP-A and SP-D was found to be independent of phospholipid secretion.