RESUMEN
The Non-Ciliated Bronchiolar Cell (NCBC) is responsible for the defense and maintenance of the bronchiolar epithelium. Several cellular defense mechanisms have been associated with an increase in the secretion of CC16 and changes in the phenotype of the cell; these mechanisms could be linked to tolerance to the damage due to exposure to inhaled Particulate Matter (PM) of the epithelium. These defense mechanisms have not been sufficiently explored. In this article, we studied the response of the NCBC to inhaled vanadium, an element which adheres to PM. This response was measured by the changes in the phenotype of the NCBC and the secretion of CC16 in a mouse model. Mice were exposed in two phases to different vanadium concentrations; 1.27 mg/m³ in the first phase and 2.56 mg/m³ in the second phase. Mice were sacrificed on the 2nd, 4th, 5th, 6th and 8th weeks. In the second phase, we observed the following: sloughing of the NCBC, hyperplasia and small inflammatory foci remained without changes and that the expression of CC16 was higher in this phase than in phase I. We also observed a change in the phenotype with a slow decrease in both phases. The increase in the secretion of CC16 and the phenotype reversion could be due to the anti-inflammatory activity of CC16. The changes observed in the second phase could be attributed to the tolerance to inhaled vanadium.
Asunto(s)
Bronquiolos , Células Epiteliales , Uteroglobina/metabolismo , Vanadio/toxicidad , Contaminantes Atmosféricos/toxicidad , Animales , Antiinflamatorios/metabolismo , Bronquiolos/citología , Bronquiolos/metabolismo , Bronquiolos/patología , Tolerancia a Medicamentos/fisiología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Epitelio/metabolismo , Epitelio/patología , Inflamación , Inhalación , Pulmón/metabolismo , Ratones , Material Particulado/toxicidadRESUMEN
Clara cells are the main airway secretory cells able to regenerate epithelium in the distal airways through transdifferentiating into goblet cells, a process under negative regulation of the Notch pathway. Pneumocystis is a highly prevalent fungus in humans occurring between 2 and 5 months of age, a period when airways are still developing and respiratory morbidity typically increases. Pneumocystis induces mucus hyperproduction in immunocompetent host airways and whether it can stimulate Clara cells is unknown. Markers of Clara cell secretion and Notch1 activation were investigated in lungs of immunocompetent rats at 40, 60, and 80 days of age during Pneumocystis primary infection with and without Valproic acid (VPA), a Notch inducer. The proportion of rats expressing mucin increased in Pneumocystis-infected rats respect to controls at 60 and 80 days of age. Frequency of distal airways Clara cells was maintained while mRNA levels for the mucin-encoding genes Muc5B and Muc5ac in lung homogenates increased 1.9 and 3.9 times at 60 days of infection (P. = 0.1609 and P. = 0.0001, respectively) and protein levels of the Clara cell marker CC10 decreased in the Pneumocystis-infected rats at 60 and 80 days of age (P. = 0.0118 & P. = 0.0388). CC10 and Muc5b co-localized in distal airway epithelium of Pneumocystis-infected rats at day 60. Co-localization of Muc5b and Ki67 as marker of mitosis in distal airways was not observed suggesting that Muc5b production by Clara cells was independent of mitosis. Notch levels remained similar and no transnucleation of activated Notch associated to Pneumocystis infection was detected. Unexpectedly, mucus was greatly increased at day 80 in Pneumocystis-infected rats receiving VPA suggesting that a Notch-independent mechanism was triggered. Overall, data suggests a Clara to goblet cell transdifferentiation mechanism induced by Pneumocystis and independent of Notch.
Asunto(s)
Pulmón/metabolismo , Pulmón/microbiología , Mucina 5AC/biosíntesis , Mucina 5B/biosíntesis , Infecciones por Pneumocystis/metabolismo , Infecciones por Pneumocystis/microbiología , Pneumocystis/patogenicidad , Receptores Notch/metabolismo , Animales , Transdiferenciación Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Antígeno Ki-67/metabolismo , Mitosis/efectos de los fármacos , Mucina 5AC/genética , Mucina 5AC/metabolismo , Mucina 5B/genética , Mucina 5B/metabolismo , Pneumocystis/efectos de los fármacos , Infecciones por Pneumocystis/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Transducción de Señal , Uteroglobina/metabolismo , Ácido Valproico/farmacologíaRESUMEN
The aim of this pilot study was to determine Clara cell protein (CC16) concentration in bronchoalveolar lavages (BAL) fluid from full-term and preterm (<37 weeks' gestational age) neonates requiring respiratory support, having symptoms of neonatal respiratory distress syndrome, and at risk of bronchopulmonary dysplasia (BPD). We hypothesized that CC16 may be predictive of BPD diagnosis regardless of gestational age. BAL fluid CC16 was measured by ELISA at birth and at day 7 of life. Both groups that developed BPD showed significantly decreased BAL fluid CC16 levels compared to those infants that did not develop the disease. CC16 positively correlated with diagnosis of BPD and negatively with the severity of the disease. These results suggest that BAL fluid CC16 levels may have a diagnostic value at day 7 for BPD in both term and preterm infants. This study demonstrates the potential utility of BAL fluid CC16 levels as a biomarker for BPD in term infants.
Asunto(s)
Displasia Broncopulmonar/metabolismo , Respiración Artificial/efectos adversos , Síndrome de Dificultad Respiratoria del Recién Nacido/metabolismo , Uteroglobina/metabolismo , Líquido del Lavado Bronquioalveolar/química , Displasia Broncopulmonar/etiología , Femenino , Humanos , Recién Nacido , Recien Nacido Prematuro , Masculino , Proyectos Piloto , Síndrome de Dificultad Respiratoria del Recién Nacido/complicaciones , Síndrome de Dificultad Respiratoria del Recién Nacido/terapia , Uteroglobina/análisisRESUMEN
Atopic asthma is a chronic allergic disease that involves T-helper type 2 (Th2)-inflammation and airway remodeling. Bronchiolar club cells (CC) and alveolar macrophages (AM) are sentinel cells of airway barrier against inhaled injuries, where allergy induces mucous metaplasia of CC and the alternative activation of AM, which compromise host defense mechanisms and amplify Th2-inflammation. As there is evidence that high levels of environmental endotoxin modulates asthma, the goal of this study was to evaluate if the activation of local host defenses by Lipopolysaccharide (LPS) previous to allergy development can contribute to preserving CC and AM protective phenotypes. Endotoxin stimulus before allergen exposition reduced hallmarks of allergic inflammation including eosinophil influx, Interleukin-4 and airway hyperreactivity, while the T-helper type 1 related cytokines IL-12 and Interferon-γ were enhanced. This response was accompanied by the preservation of the normal CC phenotype and the anti-allergic proteins Club Cell Secretory Protein (CCSP) and Surfactant-D, thereby leading to lower levels of CC metaplasia and preventing the increase of the pro-Th2 cytokine Thymic stromal lymphopoietin. In addition, classically activated alveolar macrophages expressing nitric oxide were promoted over the alternatively activated ones that expressed arginase-1. We verified that LPS induced a long-term overexpression of CCSP and the innate immune markers Toll-like receptor 4, and Tumor Necrosis Factor-α, changes that were preserved in spite of the allergen challenge. These results demonstrate that LPS pre-exposition modifies the local bronchioalveolar microenvironment by inducing natural anti-allergic mechanisms while reducing local factors that drive Th2 type responses, thus modulating allergic inflammation.
Asunto(s)
Asma/inmunología , Macrófagos Alveolares/inmunología , Mucosa Respiratoria/citología , Mucosa Respiratoria/inmunología , Animales , Western Blotting , Modelos Animales de Enfermedad , Endotoxinas/inmunología , Endotoxinas/toxicidad , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Fenotipo , Uteroglobina/metabolismoRESUMEN
To get further insights on the estrogen regulation of the uteroglobin (UG) gene, the 5'-flanking region of the UG gene from the brown hare (Lepus capensis) (Lc) was cloned and compared with those from two phylogenetically related species: the rabbit (Orictolagus cuniculus) (Oc) and the volcano rabbit (Romerolagus diazi) (Rd). The Lc-UG gene is very similar to those from rabbits (94%) and volcano rabbits (95%), and shares a number of genetic elements, including an estrogen response element (ERE). The estrogen-regulated transcription of a series of progressive 5'-deletion mutants of the Lc-UG gene, identified a functional ERE in the promoter region exhibiting the same orientation and relative position than that previously described in rabbits. The Lc-ERE is identical to the Oc-ERE, but different from both the Rd-ERE and the consensus ERE (c-ERE) by one nucleotide. We also detected important species-specific differences in the estrogen-regulated transcription of the UG gene. A luciferase reporter driven by 333 base pairs (bp) of the Lc-UG promoter elicited a higher response to estradiol than its related counterparts when expressed in estrogen-sensitive MCF-7 cells. Several ERE-like motifs which failed to act as functional EREs were also identified; one of them exhibited two mismatches in its palindromic sequence, a characteristic exhibited in many other natural occurring EREs, including the Rd-ERE.
Asunto(s)
Estrógenos/farmacología , Liebres/genética , Transcripción Genética/efectos de los fármacos , Uteroglobina/genética , Región de Flanqueo 5'/genética , Animales , Secuencia de Bases , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Datos de Secuencia Molecular , Motivos de Nucleótidos/genética , Conejos , Elementos de Respuesta/genética , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Uteroglobina/metabolismoRESUMEN
Bronchiolar Clara cells play a critical role in lung homoeostasis. The main goal of this study was to evaluate the effects of chronic allergy on these cells and the efficacy of budesonide (BUD) and montelukast (MK) in restoring their typical phenotypes after ovalbumin-induced chronic allergy in mice. Chronic allergy induced extensive bronchiolar Alcian blue-periodic acid-Schiff (AB/PAS)-positive metaplasia. In addition, cells accumulated numerous big electron-lucent granules negative for Clara cell main secretory protein (CC16), and consequently, CC16 was significantly reduced in bronchoalveolar lavage. A concomitant reduction in SP-D and CYP2E1 content was observed. The phenotypic changes induced by allergy were pharmacologically reversed by both treatments; MK was more efficient than BUD in doing so. MK decreased AB/PAS reactivity to control levels whereas they remained persistently elevated after BUD. Moreover, most non-ciliated cells recovered their normal morphology after MK, whereas for BUD normal cells coexisted with 'transitional' cells that contained remnant mucous granules and stained strongly for CC16 and SP-D. Glucocorticoids were also less able to reduce inflammatory infiltration and maintained higher percentage of neutrophils, which may have contributed to prolonged mucin expression. These results show that chronic allergy-induced mucous metaplasia of Clara cells affects their defensive mechanisms. However, anti-inflammatory treatments were able to re-establish the normal phenotype of Clara cell, with MK being more efficient at restoring a normal profile than BUD. This study highlights the role of epithelial cells in lung injuries and their contribution to anti-inflammatory therapies.
Asunto(s)
Acetatos/uso terapéutico , Asma/tratamiento farmacológico , Asma/patología , Bronquios/patología , Budesonida/uso terapéutico , Fenotipo , Quinolinas/uso terapéutico , Acetatos/farmacología , Animales , Antiasmáticos/farmacología , Antiasmáticos/uso terapéutico , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Asma/inducido químicamente , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Budesonida/farmacología , Enfermedad Crónica , Ciclopropanos , Citocromo P-450 CYP2E1/metabolismo , Modelos Animales de Enfermedad , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Epitelio/patología , Femenino , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/efectos adversos , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Quinolinas/farmacología , Sulfuros , Uteroglobina/metabolismoRESUMEN
BACKGROUND/AIMS: Clara cell protein (cc-10) has been shown to negatively regulate inflammation, protect pulmonary surfactant from degradation in the lung, and administration of this recombinant protein improves the condition of infant respiratory distress syndrome (iRDS), a disease that occurred mainly in preterm infants. In view of the possibility that altered expression of cc-10 might regulate its protective function, we attempted to characterize this protein in infants with iRDS. METHODS: Using bronchotraqueal aspirates from human infants, we analyzed cc-10 in two-dimensional gel electrophoresis (2-DE) by combining immunoprecipitation, carbonyl groups and total protein immunoblotting. RESULTS: Seven forms of cc-10 were detected with western immunoblots in infants with iRDS while only four forms were present in neonates who needed mechanical ventilation for other reasons without any lung disease (control group). The overall levels of cc-10 in iRDS were lower and differences were seen in isoform pattern and distribution. CONCLUSION: Our demonstration that cc-10 is differentially expressed in infants with iRDS may point the way towards one possible mechanism that potentially involves modifications of the protein structure with its anti-inflammatory and surfactant protective function and could be detrimental for this airway disorder.
Asunto(s)
Isoformas de Proteínas/metabolismo , Síndrome de Dificultad Respiratoria del Recién Nacido/patología , Uteroglobina/metabolismo , Western Blotting , Estudios de Casos y Controles , Electroforesis en Gel Bidimensional , Humanos , Recién Nacido , Recien Nacido Prematuro , Síndrome de Dificultad Respiratoria del Recién Nacido/metabolismoRESUMEN
OBJECTIVE: Evaluation of uteroglobin (UG) expression in the fallopian tube in different tubal diseases. DESIGN: The UG was screened and quantified in samples of fallopian tubes from patients with salpingitis, hydrosalpinx, and ectopic pregnancy by exposing the UG with immunohistochemical techniques. SETTING: University hospital and electron microscopy center. PATIENT(S): Women with pelvic inflammatory disease (PID) and complicated tubal ectopic pregnancy consulting for medical care. INTERVENTION(S): Salpingectomy. MAIN OUTCOME MEASURE(S): Tubal tissues were collected and examined using regular pathologic techniques. The UG immunoreactivity in the tubal epithelium was also assessed. RESULT(S): Fallopian tube epithelium displayed an increased UG expression in patients with PID and complicated tubal pregnancy compared with control patients. CONCLUSION(S): Uteroglobin is present in the human fallopian tube as a secretory protein and appears to be involved in immunosuppressive responses in the fallopian tube.
Asunto(s)
Trompas Uterinas/metabolismo , Inflamación/etiología , Embarazo Ectópico/genética , Uteroglobina/metabolismo , Adulto , Trompas Uterinas/inmunología , Trompas Uterinas/cirugía , Femenino , Humanos , Inmunohistoquímica , Embarazo , Salpingitis/cirugíaRESUMEN
Analysis of the transcriptional regulation of the Clara cell secretory protein (CCSP) gene has resulted in the characterization of several trans-acting factors that regulate the activity of this gene. However, little is known about negative regulatory elements involved in CCSP gene transcription. Using transient transfections of luciferase reporter constructs driven by various fragments of the Neotomodon CCSP (nCCSP) promoter, we identified an inhibitory region that contains an inverted CCAAT box located -225 to -221 bp upstream of the transcriptional start site. Sequence analysis in a broad region of the nCCSP promoter (-744/+33) identified another potentially important CCAAT motif (-459/-455). Gel shift and supershift assays indicated that the transcription factor NF-Y binds to both CCAAT boxes. Mutation of the CCAAT motif prevented the in vitro binding of NF-Y and led to a significant increase of CCSP promoter activity in both pulmonary (H441) and non-pulmonary (HeLa and MCF-7) cells, suggesting that NF-Y is involved in a negative transcriptional regulation that may potentially contribute to the highly cell-specific expression of the anti-inflammatory CCSP gene.
Asunto(s)
Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/metabolismo , Regulación de la Expresión Génica/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Uteroglobina/metabolismo , Animales , Ratones , Regiones Promotoras Genéticas/genética , Uteroglobina/genéticaRESUMEN
Uteroglobin (UG) or Clara cell protein (CC16), the main secretory product of bronchiolar Clara cells, plays an important protective role in the respiratory tract against inflammatory processes. In the lung, protein secretion is regulated by glucocorticoids, but also proinflammatory cytokines, such as interferon-gamma (IFN-gamma) and TNF-alpha, have been found to modulate the expression of this peptide. We have previously demonstrated that the acute exposure to an organophosphoreted insecticide induces an enhanced production of UG/CC16 by Clara cells. In the present report, we worked with intact and adrenalectomised (ADX) animals to study the mechanism involved in the UG/CC16 increase caused by the insecticide and the role played by a glucocorticoid (dexamethasone; DEX). In intact rats we found that DEX treatment could not reproduce such an increase of UG/CC16 synthesis with pharmacological doses. In ADX rats, even though glucocorticoid deprivation provoked a strong inhibition of UG/CC16 synthesis, the exposure to the organophosphoreted insecticide stimulated the synthesis of the protein, shown by the great accumulation of secretory granules in the cytoplasm of Clara cells and the increase of UG/CC16 detected by immunocytochemistry and western blot. These results imply that glucocorticoids are not essential to trigger the increase of UG/CC16 in response to an injury, and they also suggest an involvement of other molecules associated with inflammation. In coincidence with these observations, we have found that IFN-gamma, a proinflammatory cytokine, increased after insecticide exposition in both groups, intact and ADX, mainly in ADX rats. The stimulation of UG/CC16 synthesis occurring during inflammatory processes of the respiratory tract caused by acute inhalation of a toxicant appears to be functional without the intervention of glucocorticoids and mediated by IFN-gamma as a mechanism for local control of the inflammatory response.