RESUMEN
We previously developed transgenic mice expressing human platelet-derived growth factor B chain (PDGF-B) from the lung-specific surfactant protein C (SPC) promoter. These mice developed enlarged airspaces, inflammation, and fibrosis of varying severity. In the present study we examined potential causes of this phenotypic variation and tested whether constitutive PDGF-B expression exacerbated fibrosis induced by bleomycin and silica. The SPC-PDGFB transgene construct was modified by replacement of the PDGF-B 3' UTR, which contains motifs known to mediate instability in other cytokine genes, with SV40 sequences containing an intron and polyadenylation signal. This modification resulted in an increase in the efficiency with which the construct was expressed, but no difference in lung pathology was observed compared to the original construct. Backcrossing of mice carrying the original SPC-PDGFB construct to C57BL/6 and SJL inbred strains resulted in a more severe phenotype in SJL-bred mice compared to C57BL/6-bred mice after 4 generations. To determine whether SPC-PDGFB transgenic mice had increased susceptibility to fibrogenic agents, the mice were treated with bleomycin or silica. No significant differences were detected in lung weight, hydroxyproline content, or histopathologic changes between transgenic and wild-type mice after bleomycin or silica treatment. These results demonstrate that the amount of PDGF-BB produced in wild-type mice is not a limiting factor in the development of bleomycin- or silica-induced pulmonary fibrosis.
Asunto(s)
Enfermedades Pulmonares/inducido químicamente , Factor de Crecimiento Derivado de Plaquetas , Animales , Bleomicina , Sinergismo Farmacológico , Humanos , Pulmón/patología , Pulmón/fisiología , Enfermedades Pulmonares/genética , Enfermedades Pulmonares/patología , Ratones , Ratones Endogámicos/genética , Ratones Transgénicos/genética , Fenotipo , Factor de Crecimiento Derivado de Plaquetas/genética , Proteolípidos/genética , Proteínas Proto-Oncogénicas c-sis/genética , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Surfactantes Pulmonares/genética , Dióxido de Silicio , Virus 40 de los Simios/genéticaRESUMEN
Pulmonary surfactant dysfunction may significantly contribute to small airway obstruction during the asthmatic response, but neither its exact role nor its regulation is clear. Surfactant function and composition was studied in an Aspergillus fumigatus (Af)-induced late-phase allergic airway response in sensitized BALB/c mice. The peak of Af-induced airway hyperresponsiveness in sensitized and challenged mice 24 h after allergen provocation coincided with a significant fall in surface activity of the pulmonary surfactant. The underlying changes included time-dependent elaboration of eotaxin and IL-5 followed by eosinophil influx into the airways. The height of airway inflammation and hyperresponsiveness was preceded by release of IL-4 and marked reductions in surfactant protein (SP)-B, a hydrophobic surfactant protein responsible for maintaining low surface tension of the lining fluid of distal air spaces. Furthermore, intratracheal administration of IL-4 significantly inhibited SP-B, indicating a regulatory role of this cytokine in the surfactant biophysical changes. Thus surfactant dysfunction induced by an IL-4-driven SP-B deficiency after allergen provocation may be an important part of the late asthmatic airway response.
Asunto(s)
Asma/inmunología , Asma/metabolismo , Proteolípidos/metabolismo , Surfactantes Pulmonares/metabolismo , Animales , Aspergilosis Broncopulmonar Alérgica/inmunología , Aspergilosis Broncopulmonar Alérgica/metabolismo , Aspergillus fumigatus/inmunología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/metabolismo , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Quimiocina CCL11 , Quimiocinas CC/metabolismo , Eosinófilos/inmunología , Femenino , Humanos , Hipersensibilidad/inmunología , Hipersensibilidad/metabolismo , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Cinética , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Proteolípidos/genética , Surfactantes Pulmonares/genética , ARN Mensajero/análisis , Factor de Transcripción STAT6 , Tensión Superficial , Transactivadores/genética , Transcripción Genética/fisiologíaRESUMEN
Surfactant protein A (SP-A), a collectin associated with surfactant lipids, can have immune modulatory effects. We hypothesized that exogenous and basal endogenous SP-A can function to suppress donor T-cell-dependent inflammation that occurs during the generation of idiopathic pneumonia syndrome after bone marrow transplantation (BMT). Wild-type and SP-A-deficient mice were conditioned with cyclophosphamide and lethal irradiation and then given allogeneic donor bone marrow plus inflammation-inducing spleen T cells. On Day 7 after BMT, bronchoalveolar lavage fluids from SP-A-deficient mice contained increased numbers of inflammatory cells and higher levels of proinflammatory mediators tumor necrosis factor-alpha, interferon-gamma, and nitric oxide than wild-type mice. Exaggerated inflammation in SP-A-deficient mice was associated with decreased dynamic lung compliance and increased donor T-cell-dependent mortality (P = 0.0007, n = 10). Nitrative stress in alveolar macrophages from SP-A(-/-)-conditioned BMT recipients was higher than for SP-A(+/+) mice. Similarly, mice treated with transtracheal human SP-A (50 micro g), instilled on Day 4 after BMT during a time of in vivo donor T cell activation, exhibited decreased inflammation and improved early survival compared with buffer-instilled mice. We concluded that basal endogenous SP-A and enhanced alveolar SP-A level modulate donor T-cell-dependent immune responses and prolong survival after allogeneic BMT.
Asunto(s)
Trasplante de Médula Ósea/fisiología , Neumonía/prevención & control , Proteolípidos/metabolismo , Proteolípidos/farmacología , Surfactantes Pulmonares/metabolismo , Surfactantes Pulmonares/farmacología , Animales , Líquido del Lavado Bronquioalveolar , Humanos , Interferón gamma/metabolismo , Pulmón/metabolismo , Pulmón/patología , Macrófagos Alveolares/metabolismo , Ratones , Ratones Mutantes , Óxido Nítrico/metabolismo , Proteolípidos/genética , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , Surfactantes Pulmonares/genética , Tasa de Supervivencia , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiología , Trasplante Homólogo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Surfactant protein D (SP-D) is a molecule of the innate immune system that recognizes the patterns of surface carbohydrate on pathogens and targets them for phagocytosis and killing. SP-D-deficient mice show an increased number of macrophages in the alveolar space, excess surfactant phospholipid, overproduction of reactive oxygen species, and the development of emphysema. We report here that SP-D-deficient mice have a 5- to 10-fold increase in the number of apoptotic and necrotic alveolar macrophages, as defined by annexin V and propidium iodine staining, respectively. Intrapulmonary administration of a truncated 60-kDa fragment of human recombinant SP-D reduces the number of apoptotic and necrotic alveolar macrophages and partially corrects the lipid accumulation in SP-D-deficient mice. The same SP-D fragment binds preferentially to apoptotic and necrotic alveolar macrophages in vitro, suggesting that SP-D contributes to immune homeostasis in the lung by recognizing and promoting removal of necrotic and apoptotic cells.
Asunto(s)
Apoptosis , Glicoproteínas/administración & dosificación , Macrófagos Alveolares/citología , Surfactantes Pulmonares/administración & dosificación , Administración por Inhalación , Administración Intranasal , Animales , Anexina A5/análisis , Anexina A5/metabolismo , Apoptosis/genética , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Recuento de Células , Separación Celular , Sistema Libre de Células/química , Centrifugación , Citometría de Flujo , Fluoresceína-5-Isotiocianato/metabolismo , Glicoproteínas/análisis , Glicoproteínas/deficiencia , Glicoproteínas/genética , Humanos , Etiquetado Corte-Fin in Situ , Macrófagos Alveolares/química , Macrófagos Alveolares/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Necrosis , Fragmentos de Péptidos/administración & dosificación , Fosfolípidos/análisis , Propidio/análisis , Propidio/metabolismo , Proteínas/análisis , Proteína D Asociada a Surfactante Pulmonar , Surfactantes Pulmonares/análisis , Surfactantes Pulmonares/deficiencia , Surfactantes Pulmonares/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/metabolismo , Coloración y EtiquetadoRESUMEN
Pulmonary surfactant, a mixture of lipids and proteins, reduces the surface tension at the air-water interface of the lung alveoli by forming a surface active film. This way, it prevents alveoli from collapsing and facilitates the work of breathing. Surfactant protein C (SP-C) plays an important role in this surfactant function. SP-C is expressed as a proprotein (proSP-C), which becomes posttranslationally modified with palmitate and undergoes several rounds of proteolytical cleavage. This results in the formation of mature SP-C, which is stored in the lamellar bodies (LB) and finally secreted into the alveolar space. Recently, new insights into the sorting, processing and palmitoylation of proSP-C have been obtained by mutagenesis studies. Moreover, reports on the association of development of lung disease with SP-C deficiency have led to new insights into the importance of SP-C for proper surfactant homeostasis. In addition, new information has become available on the role of the palmitoyl chains of SP-C in surface activity. This review summarizes these recent developments in the processing and function of SP-C, with particular emphasis on the signals for and role of palmitoylation of SP-C.
Asunto(s)
Péptidos/metabolismo , Proteolípidos/química , Proteolípidos/metabolismo , Surfactantes Pulmonares/química , Surfactantes Pulmonares/metabolismo , Secuencia de Aminoácidos , Animales , Homeostasis , Humanos , Pulmón/fisiología , Enfermedades Pulmonares/fisiopatología , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteolípidos/genética , Proteína C Asociada a Surfactante Pulmonar , Surfactantes Pulmonares/genética , Transducción de SeñalRESUMEN
Surfactant protein D (SP-D) appears to play an important role in regulating local pulmonary inflammatory responses to pathogens. There is also in vitro evidence that SP-D may suppress local T cell responses. However, the role of SP-D in regulating T cell responses directly in the lung has not been previously evaluated in vivo. SP-D(-)(/-) mice demonstrate peribronchial and perivascular accumulations of lymphocytes. Therefore, we investigated the functional status and abundance of intrapulmonary lymphocytes in SP-D(-)(/-) mice. By morphometric analysis, SP-D(-)(/-) mice demonstrated increased numbers of airway- and vessel-associated lymphocytes without increases in interstitial lymphocytes. There was increased proliferative activity of lymphocytes isolated by enzymatic disassociation of minced lung. Flow cytometry was used to determine the number and functional activation status of intrapulmonary CD4(+) and CD8(+) T cells, as well as B cells and NK cells. Cytokine expression patterns in lung tissues were evaluated using RNase protection assays, reverse transcriptase/polymerase chain reaction, and enzyme-linked immunosorbent assay. There was marked T cell activation in the lungs of SP-D(-)(/-) mice, as reflected by an increased percentage of both CD4(+) and CD8(+) T cells expressing CD69 and CD25. BAL CD4 lymphocytes were increased and the fraction expressing CD69 was also increased. Although there were increases in BAL CD8 lymphocytes, apparent increases in CD69-positive CD8 lymphocytes did not reach statistical significance. In contrast, splenic T cells were not activated in SPD(-)(/-) mice. Of the proinflammatory cytokines evaluated, only interleukin (IL)-12 and IL-6 expression were consistently upregulated in the lungs of SPD(-)(/-) mice. Increased IL-2 expression was apparent but did not reach statistical significance. We conclude that the lack of local pulmonary production of SP-D leads to a state of persistent T cell activation, possibly in response to exogenous antigens. This study therefore provides further evidence of the important local immunoregulatory role of SP-D in vivo.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Glicoproteínas/fisiología , Pulmón/inmunología , Activación de Linfocitos/inmunología , Surfactantes Pulmonares/fisiología , Animales , Antígenos CD/biosíntesis , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , División Celular , Glicoproteínas/deficiencia , Glicoproteínas/genética , Interleucina-2/biosíntesis , Interleucina-2/inmunología , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Lectinas Tipo C , Pulmón/fisiología , Activación de Linfocitos/fisiología , Ratones , Ratones Transgénicos , Proteína D Asociada a Surfactante Pulmonar , Surfactantes Pulmonares/deficiencia , Surfactantes Pulmonares/genética , Receptores de Interleucina-2/biosíntesis , Receptores de Interleucina-2/inmunologíaRESUMEN
Genetic factors are thought to influence the risk for lung cancer. Since pulmonary surfactant mediates the response to inhaled carcinogenic substances, candidate genes may be among those coding for pulmonary surfactant proteins. In the present matched case-control study a polymorphism within intron 4 of the gene coding for surfactant specific protein B was analysed in 357 individuals. They were divided into 117 patients with lung cancer (40 patients with small cell lung cancer, 77 patients with non small cell lung cancer), matched controls and 123 healthy individuals. Surfactant protein B gene variants were analysed using specific PCR and cloned surfactant protein B sequences as controls. The frequency of the intron 4 variation was similar in both control groups (13.0% and 9.4%), whereas it was increased in the small cell lung cancer group (17.5%) and the non small cell lung cancer group (16.9%). The gene variation was found significantly more frequently in patients with squamous cell carcinoma (25.0%, P=0.016, odds ratio=3.2, 95%CI=1.24-8.28) than in the controls. These results indicate an association of the surfactant protein B intron 4 variants and/or its flanking loci with mechanisms that may enhance lung cancer susceptibility, especially to squamous cell carcinoma of the lung.
Asunto(s)
Carcinoma de Células Escamosas/genética , Neoplasias Pulmonares/genética , Proteolípidos/genética , Surfactantes Pulmonares/genética , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Carcinoma de Pulmón de Células no Pequeñas/epidemiología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Pequeñas/epidemiología , Carcinoma de Células Pequeñas/genética , Carcinoma de Células Escamosas/epidemiología , Estudios de Casos y Controles , Cromosomas Humanos Par 2/genética , Análisis Mutacional de ADN , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Variación Genética , Genotipo , Alemania/epidemiología , Humanos , Intrones/genética , Neoplasias Pulmonares/epidemiología , Masculino , Persona de Mediana Edad , Mutagénesis Insercional , Reacción en Cadena de la Polimerasa , Proteolípidos/fisiología , Surfactantes Pulmonares/fisiología , Factores de Riesgo , Eliminación de Secuencia , Fumar/epidemiologíaRESUMEN
Surfactant protein A (SP-A), the major lung surfactant-associated protein, mediates local defense against pathogens and modulates inflammation in the alveolus. Tumor necrosis factor (TNF)-alpha, a proinflammatory cytokine, inhibits SP-A gene expression in lung epithelial cells. Inhibitors of the phosphatidylinositol 3-kinase pathway, i.e., wortmannin, LY-294002, and rapamycin, did not block the inhibitory effects of TNF-alpha on SP-A mRNA levels. An inhibitor of the p44/42 mitogen-activated protein kinase (MAPK) pathway, PD-98059, was also ineffective. PD-169316 and SB-203580, inhibitors of p38 MAPK, blocked the TNF-alpha-mediated inhibition of SP-A mRNA levels. TNF-alpha increased the phosphorylation of p38 MAPK within 15 min. Anisomycin, an activator of p38 MAPK, increased p38 MAPK phosphorylation and decreased SP-A mRNA levels in a dose-dependent manner. Finally, TNF-alpha increased the phosphorylation of ATF-2, a transcription factor that is a p38 MAPK substrate. We conclude that TNF-alpha downregulates SP-A gene expression in lung epithelial cells via the p38 MAPK signal transduction pathway.
Asunto(s)
Expresión Génica/efectos de los fármacos , Pulmón/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Proteolípidos/genética , Surfactantes Pulmonares/genética , Factor de Necrosis Tumoral alfa/farmacología , Factor de Transcripción Activador 2 , Anisomicina/farmacología , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Células Epiteliales/fisiología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Pulmón/citología , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , Transducción de Señal/fisiología , Factores de Tiempo , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The collectins mannan-binding lectin (MBL) and lung surfactant protein D (SP-D) play a significant role in innate immunity. Structural as wells as promoter variants are known for MBL and different alleles correlate with low MBL concentrations in serum and predispose to infectious diseases. Structural variants are also known for SP-D but these have not been linked to disease states. The aim of the present study was to provide heritability estimates for the constitutional levels of MBL and SP-D in children. A population of 26 monozygotic (MZ) and 36 dizygotic (DZ) like-sexed twin pairs aged 6-9 years was studied. Intraclass correlations were significantly higher in MZ than in DZ twins, indicating substantial genetic influence on both MBL and SP-D levels. Biometric model fitting showed that the estimated heritability was 0.96 (95% CI 0.92-0.97) for MBL with the presence of non-additive genetic factors and non-shared environmental factors and 0.91 (95% CI 0.83-0.95) for SP-D with additive genetic and non-shared environmental factors. The data indicate quantitatively very strong genetic dependence for the serum levels of both MBL and SP-D.
Asunto(s)
Proteínas Portadoras/genética , Glicoproteínas/genética , Surfactantes Pulmonares/genética , Biometría , Proteínas Portadoras/sangre , Niño , Colectinas , Femenino , Glicoproteínas/sangre , Humanos , Masculino , Proteína D Asociada a Surfactante Pulmonar , Surfactantes Pulmonares/sangre , Gemelos Dicigóticos , Gemelos MonocigóticosRESUMEN
Interleukin (IL)-13, a key mediator of Th2-mediated immunity, contributes to the pathogenesis of asthma and other pulmonary diseases via its ability to generate fibrosis, mucus metaplasia, eosinophilic inflammation, and airway hyperresponsiveness. In these studies, we compared surfactant accumulation in wild-type mice and mice in which IL-13 was overexpressed in the lung. When compared with littermate controls, transgenic animals showed alveolar type II cell hypertrophy under light and electron microscopy. Over time, their alveoli also filled with surfactant in a pulmonary alveolar proteinosis pattern. At the same time, prominent interstitial fibrosis occurs. Bronchoalveolar lavage fluid from these mice had a three- to sixfold increase in surfactant phospholipids. Surfactant proteins (SP)-A, -B, and -C showed two- to threefold increases, whereas SP-D increased 70-fold. These results indicate that IL-13 is a potent stimulator of surfactant phospholipid and surfactant accumulation in the lung. IL-13 may therefore play a central role in the broad range of chronic pulmonary conditions in which fibrosis, type II cell hypertrophy, and surfactant accumulation occur.
Asunto(s)
Interleucina-13/genética , Proteolípidos/genética , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/patología , Surfactantes Pulmonares/genética , Animales , Asma/inmunología , Asma/patología , Líquido del Lavado Bronquioalveolar/inmunología , Expresión Génica/inmunología , Hipertrofia , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Proteolípidos/análisis , Alveolos Pulmonares/química , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/patología , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , Surfactantes Pulmonares/análisis , ARN Mensajero/análisisRESUMEN
Previous in vitro studies have suggested that surfactant protein A (SP-A) may play a role in pulmonary surfactant homeostasis by mediating surfactant secretion and clearance. However, mice made deficient in SP-A [SP-A (-/-) animals] have relatively normal levels of surfactant compared with wild-type SP-A (+/+) animals. We hypothesize that SP-A may play a role in surfactant homeostasis after acute lung injury. Bacterial lipopolysaccharide was instilled into the lungs of SP-A (-/-) mice and SP-A (+/+) mice to induce injury. Surfactant phospholipid levels were increased 1.6-fold in injured SP-A (-/-) animals, although injury did not alter [3H]choline or [14C]palmitate incorporation into dipalmitoylphosphatidylcholine (DPPC), suggesting no change in surfactant synthesis/secretion 12 h after injury. Clearance of [3H]DPPC from the lungs of injured SP-A (-/-) animals was decreased by approximately 40%. Instillation of 50 microg of exogenous SP-A rescued both the clearance defect and the increased phospholipid defect in injured SP-A (-/-) animals, suggesting that SP-A may play a role in regulating clearance of surfactant phospholipids after acute lung injury.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/metabolismo , Homeostasis/fisiología , Enfermedades Pulmonares/metabolismo , Proteolípidos/genética , Proteolípidos/metabolismo , Surfactantes Pulmonares/genética , Surfactantes Pulmonares/metabolismo , Animales , Colina/farmacocinética , Lipopolisacáridos , Liposomas/metabolismo , Enfermedades Pulmonares/inducido químicamente , Enfermedades Pulmonares/patología , Ratones , Ratones Noqueados , Palmitatos/farmacocinética , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , TritioRESUMEN
CTP:phosphocholine cytidylyltransferase (CT) is the rate-limiting enzyme in the biosynthesis by type II pneumocytes of phosphatidylcholine (PC), the predominant phospholipid in lung surfactant. Augmentation of endogenous CT activity might therefore result in enhanced surfactant PC production. To test this hypothesis, transgenic mice were created in which rat CT (rCT) was expressed under control of the human surfactant protein C (SP-C) promoter. Transgenic mice were identified by tail-clip PCR analysis and studies of four founder lines were initiated. Lung CT gene expression was enhanced in two transgenic founder lines relative to wild-type controls. These two transgenic lines also exhibited significantly higher levels of immunoreactive CT protein and CT activity in whole-lung homogenates and in cultured type II cell extracts. Disaturated PC (DSPC) content in whole-lung homogenates and the rate of DSPC synthesis in cultured type II cells were significantly increased in one transgenic line. However, neither the incorporation of radiolabeled precursors (choline and palmitate) into DSPC in vivo nor the cellular metabolism of DSPC differed significantly between transgenic and control mice. This transgenic model provides opportunity for further study of factors controlling surfactant phospholipid production in vivo.
Asunto(s)
Citidililtransferasa de Colina-Fosfato/metabolismo , Pulmón/metabolismo , Proteolípidos/genética , Surfactantes Pulmonares/genética , Animales , Secuencia de Bases , Cartilla de ADN , Pulmón/citología , Ratones , Ratones Transgénicos , RatasRESUMEN
Familial pulmonary fibrosis is a heterogeneous group of interstitial lung diseases of unknown cause that is associated with multiple pathologic subsets. Mutations in the surfactant protein C (SP-C) gene (SFTPC) are associated with familial desquamative and nonspecific interstitial pneumonitis. Genetic studies in familial usual interstitial pneumonitis have been inconclusive. Using a candidate gene approach, we found a heterozygous exon 5 + 128 T-->A transversion of SFTPC in a large familial pulmonary fibrosis kindred, including adults with usual interstitial pneumonitis and children with cellular nonspecific interstitial pneumonitis. The mutation is predicted to substitute a glutamine for a conserved leucine residue and may hinder processing of SP-C precursor protein. SP-C precursor protein displayed aberrant subcellular localization by immunostaining. Electron microscopy of affected lung revealed alveolar type II cell atypia, with numerous abnormal lamellar bodies. Mouse lung epithelial cells transfected with the SFTPC mutation were notable for similar electron microscopy findings and for exaggerated cellular toxicity. We show that an SFTPC mutation segregates with the pulmonary fibrosis phenotype in this kindred and may cause type II cellular injury. The presence of two different pathologic diagnoses in affected relatives sharing this mutation indicates that in this kindred, these diseases may represent pleiotropic manifestations of the same central pathogenesis.
Asunto(s)
Enfermedades Pulmonares Intersticiales/genética , Mutación , Proteolípidos/genética , Fibrosis Pulmonar/genética , Surfactantes Pulmonares/genética , Adolescente , Adulto , Secuencia de Bases , Femenino , Heterocigoto , Humanos , Lactante , Enfermedades Pulmonares Intersticiales/patología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Linaje , Fibrosis Pulmonar/patologíaAsunto(s)
Enfermedades Pulmonares/metabolismo , Pulmón/metabolismo , Surfactantes Pulmonares/fisiología , Amiloide/química , Amiloide/metabolismo , Animales , Humanos , Pulmón/diagnóstico por imagen , Pulmón/inmunología , Enfermedades Pulmonares/inmunología , Enfermedades Pulmonares/microbiología , Enfermedades Pulmonares/patología , Ratones , Ratones Noqueados , Vaina de Mielina/metabolismo , Fosfolípidos/química , Fosfolípidos/metabolismo , Alveolos Pulmonares/diagnóstico por imagen , Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares/química , Surfactantes Pulmonares/genética , Surfactantes Pulmonares/metabolismo , UltrasonografíaRESUMEN
The lung of the preterm infant is easily injured and an initial indication of the injury is an inflammatory response. Surfactant treatment and gentle ventilation will minimize the initiation and progression of injury. We asked if the initial lung injury response differed when preterm ventilated lambs were treated with complete natural sheep surfactant, a lipid extract of sheep surfactant, a surfactant used to treat RDS (Survanta), or a synthetic surfactant containing recombinant SP-C (Venticute). We used a gentle style of ventilation and a positive end expiratory pressure of 4 cmH(2)0 to minimize injury. The surfactants were not distinguishable based on gas exchange, compliance or lung gas volumes over the 6h ventilation period. When compared with unventilated controls the ventilated lambs had increased protein and inflammatory cells in alveolar lavages. The cells from the alveolar lavages produced more H(2)0(2), expressed more surface adhesion antigens and CD-14 receptors, and expressed more mRNA for the pro-inflammatory cytokines IL-1 beta and IL-8 than did cells from unventilated lungs. Lung tissue expressed primarily increased IL-6 mRNA relative to unventilated controls. However, there were no consistent differences in any of the inflammatory indicators between the different surfactant treated groups. Because endotoxin free natural surfactant containing SP-A was not superior to three other surfactants containing differing amounts of the surfactant proteins, additions of these proteins to clinical surfactants may not decrease the indicators of lung inflammation that accompany the initiation of ventilation of the preterm lung.
Asunto(s)
Lesión Pulmonar , Neumonía/prevención & control , Proteolípidos/genética , Proteolípidos/farmacología , Surfactantes Pulmonares/genética , Surfactantes Pulmonares/farmacología , Respiración Artificial/métodos , Animales , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Femenino , Citometría de Flujo , Expresión Génica , Edad Gestacional , Interleucina-6/análisis , Interleucina-8/análisis , Pulmón/química , Pulmón/inmunología , Neumonía/etiología , Neumonía/inmunología , Embarazo , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , ARN Mensajero/análisis , Respiración Artificial/efectos adversos , Ovinos , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Members of the CCAAT enhancer binding protein (C/EBP) transcription factor family were detected in fetal lung of both human and rat. In rat lung, the level of C/EBPs increased with time of gestation, peaking around birth. In adult rat lung, C/EBPs were localized to the alveolar type II cells. The effect of C/EBPs on pulmonary surfactant protein A (SP-A), which is also expressed late in gestation, was investigated. In contrast to control plasmids, C/EBP delta expressing plasmids reversed the action of a transcriptional silencer just upstream of the rat SP-A promoter. In order to test the effect of C/EBPs on endogenous SP-A gene expression, cells that express SP-A were exposed to a phosphorothioate-substituted, double-stranded oligonucleotide matching the consensus C/EBP binding site (decoy oligonucleotide) at concentrations from 0.5 to 10 microM for 72 h. A mutant oligonucleotide with an 8-base pair (bp) substitution served as a control. The decoy oligonucleotide reduced SP-A mRNA as much as 75% compared to a mutant oligonucleotide both in the human lung cell line, NCI-H441, and in primary human fetal alveolar type II cells. The data indicate that C/EBPs facilitate SP-A gene expression, possibly by overcoming transcriptional silencing.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Proteolípidos/genética , Surfactantes Pulmonares/genética , Animales , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células Cultivadas , Regulación de la Expresión Génica , Silenciador del Gen , Humanos , Pulmón/fisiología , Proteolípidos/biosíntesis , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , Surfactantes Pulmonares/biosíntesis , Ratas , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
C/EBP delta, a member of the leucine zipper transcription factor family, is expressed at higher levels in the lung than in any other tissue. We detected C/EBP delta mRNA and protein in NCI-H441 cells, a cell line derived from a human adenocarcinoma that produces surfactant protein A (SP-A). NCI-H441 cells were exposed to phosphorothioate-substituted antisense oligonucleotides directed against C/EBP delta. After exposure to the oligonucleotides, cells were harvested, total RNA prepared, and levels of mRNA for C/EBP delta, SP-A and a control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were quantified from Northern blots. An oligonucleotide that overlapped the translational start was effective in reducing C/EBP delta mRNA. Oligonucleotides that corresponded to regions upstream and downstream from the translational start were not as effective. The loss of C/EBP delta was accompanied by a decrease in the level of SP-A mRNA. The overlapping oligonucleotide was tested more extensively. After 72 h, antisense oligonucleotide at 3 and 5 microM reduced the level of C/EBP delta mRNA and protein by 50% or more as compared with sense and scrambled controls. The SP-A mRNA level was reduced even more, by about 75%. GAPDH mRNA was not affected. We conclude that C/EBP delta plays a role in the regulation of SP-A gene expression.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Regulación de la Expresión Génica , Proteolípidos/genética , Surfactantes Pulmonares/genética , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína delta de Unión al Potenciador CCAAT , Humanos , Ratones , Ratones Noqueados , Oligonucleótidos Antisentido/genética , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , Factores de Transcripción/genéticaRESUMEN
We report the use of HOPE-fixation (HOPE = Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) for specimens utilized for in situ hybridization targeting mRNA. For this purpose, an optimized protocol was developed and repeatedly tested on HOPE-fixed lung specimens. We observed that neither pretreatment, permeabilizing the cells, nor prehybridization is necessary to generate signals. After deparaffinizing, the random primed digoxigenin-labeled probes are directly hybridized together with yeast tRNA for blocking unspecific signals. Detection was performed using anti digoxigenin antibodies conjugated with alkaline phosphatase and new-fuchsine or NBT/BCIP as substrates. The results were verified by RT-PCR and adequate negative controls. Signals for human surfactant protein-A and interferon-gamma-inducible protein-10 developed rapidly within 10 min, accompanied by high signal intensities comparable to those observed in immunohistochemistry. Signal enhancement by biotinyl-tyramide, although giving suitable results as well, did not lead to higher signal intensities, and thus was not necessary in conjunction with the probes tested so far. These experiments were performed with material stored under appropriate conditions (at +4 degrees C) up to five years. To sum up, these initial results, obtained with the novel HOPE-fixative, are promising as regards the enhancement of the capabilities of in situ hybridization in the future.
Asunto(s)
Hibridación in Situ/métodos , Pulmón/metabolismo , Proteolípidos/metabolismo , Surfactantes Pulmonares/metabolismo , Fijación del Tejido/métodos , Células Cultivadas , Reactivos de Enlaces Cruzados/química , Humanos , Adhesión en Parafina , Proteolípidos/genética , Proteínas Asociadas a Surfactante Pulmonar , Surfactantes Pulmonares/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
Surfactant protein D (SP-D) and serum conglutinin are closely related members of the collectin family of host defense lectins. Although normally synthesized at different anatomic sites, both proteins participate in the innate immune response to microbial challenge. To discern the roles of specific domains in the function of SP-D in vivo, a fusion protein (SP-D/Cong(neck+CRD)) consisting of the NH(2)-terminal and collagenous domains of rat SP-D (rSP-D) and the neck and carbohydrate recognition domains (CRDs) of bovine conglutinin (Cong) was expressed in the respiratory epithelium of SP-D gene-targeted (SP-D(-/-)) mice. While SP-D/Cong(neck+CRD) fusion protein did not affect lung morphology and surfactant phospholipid levels in the lungs of wild type mice, the chimeric protein substantially corrected the increased lung phospholipids in SP-D(-/-) mice. The SP-D/Cong(neck+CRD) fusion protein also completely corrected defects in influenza A clearance and inhibited the exaggerated inflammatory response that occurs following viral infection. However, the chimeric protein did not ameliorate the ongoing lung inflammation, enhanced metalloproteinase expression, and alveolar destruction that characterize this model of SP-D deficiency. By contrast, a single arm mutant (RrSP-D(Ser15,20)) partially restored antiviral activity but otherwise failed to rescue the deficient phenotype. Our findings directly implicate the CRDs of both SP-D and conglutinin in host defense in vivo. Our findings also strongly suggest that the molecular mechanisms underlying impaired pulmonary host defense and abnormal lipid metabolism are distinct from those that promote ongoing inflammation and the development of emphysema.