RESUMEN
BACKGROUND: A major goal of asthma therapy is to achieve disease control, with maintenance of lung function, reduced need for rescue medication, and prevention of exacerbation. Despite current standard of care, up to 70% of patients with asthma remain poorly controlled. Analysis of serum and sputum biomarkers could offer insights into parameters associated with poor asthma control. OBJECTIVE: To identify signatures as determinants of asthma disease control, we performed proteomics using Olink proximity extension analysis. METHODS: Up to 3 longitudinal serum samples were collected from 23 controlled and 25 poorly controlled asthmatics. Nine of the controlled and 8 of the poorly controlled subjects also provided 2 longitudinal sputum samples. The study included an additional cohort of 9 subjects whose serum was collected within 48 hours of asthma exacerbation. Two separate pre-defined Proseek Multiplex panels (INF and CVDIII) were run to quantify 181 separate protein analytes in serum and sputum. RESULTS: Panels consisting of 9 markers in serum (CCL19, CCL25, CDCP1, CCL11, FGF21, FGF23, Flt3L, IL-10Rß, IL-6) and 16 markers in sputum (tPA, KLK6, RETN, ADA, MMP9, Chit1, GRN, PGLYRP1, MPO, HGF, PRTN3, DNER, PI3, Chi3L1, AZU1, and OPG) distinguished controlled and poorly controlled asthmatics. The sputum analytes were consistent with a pattern of neutrophil activation associated with poor asthma control. The serum analyte profile of the exacerbation cohort resembled that of the controlled group rather than that of the poorly controlled asthmatics, possibly reflecting a therapeutic response to systemic corticosteroids. CONCLUSIONS AND CLINICAL RELEVANCE: Proteomic profiles in serum and sputum distinguished controlled and poorly controlled asthmatics, and were maintained over time. Findings support a link between sputum neutrophil markers and loss of asthma control.
Asunto(s)
Asma/metabolismo , Biomarcadores , Proteoma , Proteómica , Esputo/metabolismo , Adulto , Asma/diagnóstico , Asma/inmunología , Asma/terapia , Citocinas , Femenino , Factor-23 de Crecimiento de Fibroblastos , Humanos , Masculino , Persona de Mediana Edad , Evaluación del Resultado de la Atención al Paciente , Proteómica/métodos , Pruebas de Función Respiratoria , Esputo/inmunología , Adulto JovenRESUMEN
BACKGROUND: Reperfusion injury with pulmonary edema continues to be a major complication after lung transplantation. Alveolar fluid homeostasis is regulated by Na+/K+-ATPase activity on the basolateral surface of alveolar epithelial cells. Intact Na+/K+-ATPase is essential to the resolution of pulmonary edema. We characterized the effects of cold ischemia and reperfusion on expression of Na+/K+-ATPase mRNA and protein. METHODS: Baseline values for Na+/K+-ATPase mRNA and protein were determined from freshly harvested lungs with no cold storage time or reperfusion (group I). Group II lungs were analyzed after cold storage times of 12 or 24 hr without subsequent reperfusion. Group III lungs were analyzed after cold storage times of 12 or 24 hr with subsequent reperfusion. Lungs were flushed with either Euro-Collins (EC) or University of Wisconsin (UW) solution in each group. All samples were quantified for Na+/K+-ATPase mRNA and Na+/K+-ATPase protein. Physiological parameters including oxygenation and compliance were also measured. RESULTS: There were no significant differences in the level of mRNA and protein for samples that were cold stored without reperfusion (group II). With reperfusion (group III) there was a significant increase in the level of the Na+/K+-ATPase mRNA after 12 hr of storage for both EC and UW. After 24 hr of storage and subsequent reperfusion, lungs flushed with EC had significantly decreased Na+/K+-ATPase protein and mRNA, although lungs preserved with UW maintained their increased levels of Na+/K+-ATPase protein and mRNA. CONCLUSIONS: Our data suggest that ischemia-reperfusion injury results in an initial up-regulation of Na+/K+-ATPase mRNA. With prolonged injury in lungs preserved with EC, the level of the mRNA decreased with a corresponding decrease in the Na+/K+-ATPase protein. The different response seen in EC versus UW may be explained by better preservation of pump function with UW than EC and correlates with improved physiological function in lungs preserved with UW solution.
Asunto(s)
Criopreservación , Pulmón/enzimología , Daño por Reperfusión/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Conservación de Tejido , Adenosina/farmacología , Alopurinol/farmacología , Animales , Western Blotting , Expresión Génica , Glutatión/farmacología , Insulina/farmacología , Pulmón/irrigación sanguínea , Rendimiento Pulmonar , Masculino , Soluciones Preservantes de Órganos/farmacología , ARN Mensajero/metabolismo , Rafinosa/farmacología , Ratas , Ratas Sprague-Dawley , Ribonucleasas/análisisRESUMEN
We investigated expression of the alpha(3)-integrin subunit by rat alveolar epithelial cells (AECs) grown in primary culture as well as the effects of monoclonal antibodies with blocking activity against the alpha(3)-integrin subunit on AEC monolayer formation. alpha(3)-Integrin subunit mRNA and protein were detectable in AECs on day 1 and increased with time in culture. alpha(3)- and beta(1)-integrin subunits coprecipitated in immunoprecipitation experiments with alpha(3)- and beta(1)-subunit-specific antibodies, consistent with their association as the alpha(3)beta(1)-integrin receptor at the cell membrane. Treatment with blocking anti-alpha(3) monoclonal antibody from day 0 delayed development of transepithelial resistance, reduced transepithelial resistance through day 5 compared with that in untreated AECs, and resulted in large subconfluent patches in monolayers viewed by scanning electron microscopy on day 3. These data indicate that alpha(3)- and beta(1)-integrin subunits are expressed in AEC monolayers where they form the heterodimeric alpha(3)beta(1)-integrin receptor at the cell membrane. Blockade of the alpha(3)-integrin subunit inhibits formation of confluent AEC monolayers. We conclude that the alpha(3)-integrin subunit modulates formation of AEC monolayers by virtue of the key role of the alpha(3)beta(1)-integrin receptor in AEC adhesion.
Asunto(s)
Antígenos CD/fisiología , Integrinas/fisiología , Alveolos Pulmonares/citología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Antígenos CD/metabolismo , Northern Blotting , Western Blotting , Adhesión Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Células Cultivadas , Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Técnica del Anticuerpo Fluorescente , Integrina alfa3 , Integrinas/inmunología , Integrinas/metabolismo , Masculino , Microscopía Electrónica de Rastreo , Pruebas de Precipitina , Alveolos Pulmonares/fisiología , Alveolos Pulmonares/ultraestructura , Ratas , Ratas Sprague-DawleyRESUMEN
Using the patch-clamp technique, we studied the effects of epidermal growth factor (EGF) on whole cell and single channel currents in adult rat alveolar epithelial type II cells in primary culture in the presence or absence of EGF for 48 h. In symmetrical sodium isethionate solutions, EGF exposure caused a significant increase in the type II cell whole cell conductance. Amiloride (10 microM) produced approximately 20-30% inhibition of the whole cell conductance in both the presence and absence of EGF, such that EGF caused the magnitude of the amiloride-sensitive component to more than double. Northern analysis showed that alpha-, beta- and gamma-subunits of rat epithelial Na(+) channel (rENaC) steady-state mRNA levels were all significantly decreased by EGF. At the single channel level, all active inside-out patches demonstrated only 25-pS channels that were amiloride sensitive and relatively nonselective for cations (P(Na(+))/P(K(+)) approximately 1.0:0.48). Although the biophysical characteristics (conductance, open-state probability, and selectivity) of the channels from EGF-treated and untreated cells were essentially identical, channel density was increased by EGF; the modal channel per patch was increased from 1 to 2. These findings indicate that EGF increases expression of nonselective, amiloride-sensitive cation channels in adult alveolar epithelial type II cells. The contribution of rENaC to the total EGF-dependent cation current under these conditions is quantitatively less important than that of the nonselective cation channels in these cells.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Alveolos Pulmonares/citología , Canales de Sodio/metabolismo , Factores de Edad , Amilorida/farmacología , Animales , Northern Blotting , Separación Celular , Supervivencia Celular , Células Cultivadas , Diuréticos/farmacología , Conductividad Eléctrica , Canales Epiteliales de Sodio , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Alveolos Pulmonares/química , Alveolos Pulmonares/efectos de los fármacos , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Sodio/metabolismo , Canales de Sodio/genética , Regulación hacia Arriba/fisiologíaRESUMEN
We investigated the effects of epidermal growth factor (EGF) on active Na+ absorption by alveolar epithelium. Rat alveolar epithelial cells (AEC) were isolated and cultivated in serum-free medium on tissue culture-treated polycarbonate filters. mRNA for rat epithelial Na+ channel (rENaC) alpha-, beta-, and gamma-subunits and Na+ pump alpha1- and beta1-subunits were detected in day 4 monolayers by Northern analysis and were unchanged in abundance in day 5 monolayers in the absence of EGF. Monolayers cultivated in the presence of EGF (20 ng/ml) for 24 h from day 4 to day 5 showed an increase in both alpha1 and beta1 Na+ pump subunit mRNA but no increase in rENaC subunit mRNA. EGF-treated monolayers showed parallel increases in Na+ pump alpha1- and beta1-subunit protein by immunoblot relative to untreated monolayers. Fixed AEC monolayers demonstrated predominantly membrane-associated immunofluorescent labeling with anti-Na+ pump alpha1- and beta1-subunit antibodies, with increased intensity of cell labeling for both subunits seen at 24 h following exposure to EGF. These changes in Na+ pump mRNA and protein preceded a delayed (>12 h) increase in short-current circuit (measure of active transepithelial Na+ transport) across monolayers treated with EGF compared with untreated monolayers. We conclude that EGF increases active Na+ resorption across AEC monolayers primarily via direct effects on Na+ pump subunit mRNA expression and protein synthesis, leading to increased numbers of functional Na+ pumps in the basolateral membranes.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Alveolos Pulmonares/fisiología , Canales de Sodio/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Sodio/metabolismo , Animales , Células Cultivadas , Medio de Cultivo Libre de Suero , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Canales Epiteliales de Sodio , Cinética , Sustancias Macromoleculares , Masculino , Alveolos Pulmonares/citología , Alveolos Pulmonares/efectos de los fármacos , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Transcripción GenéticaRESUMEN
T1alpha is a recently identified gene expressed in the adult rat lung by alveolar type I (AT1) epithelial cells but not by alveolar type II (AT2) epithelial cells. We evaluated the effects of modulating alveolar epithelial cell (AEC) phenotype in vitro on T1alpha expression using either soluble factors or changes in cell shape to influence phenotype. For studies on the effects of soluble factors on T1alpha expression, rat AT2 cells were grown on polycarbonate filters in serum-free medium (MDSF) or in MDSF supplemented with either bovine serum (BS, 10%), rat serum (RS, 5%), or keratinocyte growth factor (KGF, 10 ng/ml) from either day 0 or day 4 through day 8 in culture. For studies on the effects of cell shape on T1alpha expression, AT2 cells were plated on thick collagen gels in MDSF supplemented with BS. Gels were detached on either day 1 (DG1) or day 4 (DG4) or were left attached until day 8. RNA and protein were harvested at intervals between days 1 and 8 in culture, and T1alpha expression was quantified by Northern and Western blotting, respectively. Expression of T1alpha progressively increases in AEC grown in MDSF +/- BS between day 1 and day 8 in culture, consistent with transition toward an AT1 cell phenotype. Exposure to RS or KGF from day 0 prevents the increase in T1alpha expression on day 8, whereas addition of either factor from day 4 through day 8 reverses the increase. AEC cultured on attached gels express high levels of T1alpha on days 4 and 8. T1alpha expression is markedly inhibited in both DG1 and DG4 cultures, consistent with both inhibition and reversal of the transition toward the AT1 cell phenotype. These results demonstrate that both soluble factors and alterations in cell shape modulate T1alpha expression in parallel with AEC phenotype and provide further support for the concept that transdifferentiation between AT2 and AT1 cell phenotypes is at least partially reversible.
Asunto(s)
Células Epiteliales/citología , Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento/farmacología , Proteínas de la Membrana/biosíntesis , Alveolos Pulmonares/citología , Animales , Sangre , Bovinos , Adhesión Celular , Células Cultivadas , Colágeno , Medios de Cultivo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Geles , Regulación de la Expresión Génica/efectos de los fármacos , Cinética , Masculino , Glicoproteínas de Membrana , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
We investigated the role of keratinocyte growth factor (KGF) in regulation of alveolar epithelial cell (AEC) phenotype in vitro. Effects of KGF on cell morphology, expression of surfactant apoproteins A, B, and C (SP-A, -B, and -C), and expression of aquaporin 5 (AQP5), a water channel present in situ on the apical surface of alveolar type I (AT1) cells but not expressed in alveolar type II (AT2) cells, were evaluated in AECs grown in primary culture. Observations were made on AEC monolayers grown in serum-free medium without KGF (control) or grown continuously in the presence of KGF (10 ng/ml) from either Day 0 (i.e., the time of plating) or Day 4 or 6 through Day 8 in culture. AECs monolayers express AQP5 only on their apical surfaces as determined by cell surface biotinylation studies. Control AECs grown in the absence of KGF through Day 8 express increasing levels of AQP5, consistent with transition toward the AT1 cell phenotype. Exposure of AECs to KGF from Day 0 results in decreased AQP5 expression, retention of a cuboidal morphology, and greater numbers of lamellar bodies relative to control on Day 8 in culture. AECs treated with KGF from Day 4 or 6 exhibit a decrease in AQP5 expression through subsequent days in culture, as well as an increase in expression of surfactant apoproteins. These data, showing that KGF both prevents and reverses the increase in AQP5 (and decrease in surfactant apoprotein) expression that accompanies progression of the AT2 toward the AT1 cell phenotype, support the concepts that transdifferentiation between AT2 and AT1 cell phenotypes is at least partially reversible and that KGF may play a major role in modulating AEC phenotype.
Asunto(s)
Acuaporinas , Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento/farmacología , Proteínas de la Membrana , Alveolos Pulmonares/citología , Proteínas Asociadas a Surfactante Pulmonar , Animales , Apoproteínas/efectos de los fármacos , Apoproteínas/genética , Acuaporina 5 , Polaridad Celular , Células Cultivadas , Células Epiteliales/clasificación , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Canales Iónicos/análisis , Canales Iónicos/efectos de los fármacos , Canales Iónicos/genética , Masculino , Fenotipo , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Alveolos Pulmonares/efectos de los fármacos , Surfactantes Pulmonares/efectos de los fármacos , Surfactantes Pulmonares/genética , ARN Mensajero/análisis , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
We evaluated the effects of keratinocyte growth factor (KGF) on alveolar epithelial cell (AEC) active ion transport and on rat epithelial Na channel (rENaC) subunit and Na(+)-K(+)-adenosinetriphosphatase (ATPase) subunit isoform expression using monolayers of AEC grown in primary culture. Rat alveolar type II cells were plated on polycarbonate filters in serum-free medium, and KGF (10 ng/ml) was added to confluent AEC monolayers on day 4 in culture. Exposure of AEC monolayers to KGF on day 4 resulted in dose-dependent increases in short-circuit current (Isc) compared with controls by day 5, with further increases occurring through day 8. Relative Na(+)-K(+)-ATPase alpha 1-subunit mRNA abundance was increased by 41% on days 6 and 8 after exposure to KGF, whereas alpha 2-subunit mRNA remained only marginally detectable in both the absence and presence of KGF. Levels of mRNA for the beta 1-subunit of Na(+)-K(+)-ATPase did not increase, whereas cellular alpha 1- and beta 1-subunit protein increased 70 and 31%, respectively, on day 6. mRNA for alpha-, beta-, and gamma-rENaC all decreased in abundance after treatment with KGF. These results indicate that KGF upregulates active ion transport across AEC monolayers via a KGF-induced increase in Na pumps, primarily due to increased Na(+)-K(+)-ATPase alpha 1-subunit mRNA expression. We conclude that KGF may enhance alveolar fluid clearance after acute lung injury by upregulating Na pump expression and transepithelial Na transport across the alveolar epithelium.
Asunto(s)
Factores de Crecimiento de Fibroblastos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Sustancias de Crecimiento/farmacología , Alveolos Pulmonares/fisiología , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Cinética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Alveolos Pulmonares/citología , Alveolos Pulmonares/efectos de los fármacos , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Transcripción Genética/efectos de los fármacosRESUMEN
The Na(+)-K(+)-ATPase is a heterodimeric plasma membrane protein that consists of a catalytic alpha-subunit and a smaller glycosylated beta-subunit that has not been fully characterized in alveolar epithelial cells (AEC) to date. In this study, we identified the Na(+)-K(+)-ATPase beta-subunit protein in rat AEC and lung membranes using immunochemical techniques. Rat AEC grown in primary culture and rat lung, brain, and kidney membranes were solubilized in either 2% sodium dodecyl sulfate (SDS) sample buffer for SDS-polyacrylamide gel electrophoresis or in 1% Nonidet P-40 lysis buffer for immunoprecipitation studies. Na(+)-K(+)-ATPase beta-subunit was not detected in either AEC or lung membranes on Western blots when probed with a panel of antibodies (Ab) against beta-subunit isoforms, whereas brain and kidney beta-subunit were recognized as broad approximately 50-kDa bands. AEC, lung, and kidney membranes were immunoprecipitated with anti-beta Ab IEC 1/48, a monoclonal Ab that recognizes beta-subunit protein only in its undenatured state. The beta-subunit was detected in the immunoprecipitate (IP) from kidney membranes by several different anti-beta-subunit Ab. The beta-subunit was faintly detectable from AEC and lung IP as a broad approximately 50-kDa band when blotted with the polyclonal anti-beta 1-subunit Ab SpET but could not be detected by blotting with other anti-beta Ab. Treatment of the IP from kidney, lung, and AEC with N-glycosidase F for 2 h at 37 degrees C resulted in immunodetection of identical approximately 35 kDa bands when probed with all anti-beta 1 Ab on Western blots. From these results, we conclude that rat lung and AEC possess immunoreactive beta-subunit protein that is only readily detectable after deglycosylation. Because anti-beta Ab fail to detect the Na(+)-K(+)-ATPase beta-subunit in rat lung or AEC by standard Western blotting techniques under the conditions of these experiments, our results suggest that lung beta-subunit may be glycosylated differently from kidney and other tissues. These differences appear to be due to organ- or cell-specific posttranslational processing of the beta 1-subunit and may result in altered regulation of sodium pumps in lung compared with other epithelia.
Asunto(s)
Isoenzimas/metabolismo , Alveolos Pulmonares/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Western Blotting , Células Epiteliales , Epitelio/enzimología , Fucosa , Glicosilación , Immunoblotting , Masculino , Manosa , Pruebas de Precipitina , Alveolos Pulmonares/citología , Ratas , Ratas Sprague-DawleyRESUMEN
We evaluated the effects of epidermal growth factor (EGF) on transepithelial resistance (Rt) and active ion transport by alveolar epithelial cell (AEC) monolayers on tissue culture-treated polycarbonate filters. Rat type II cells were cultured in completely defined serum-free medium (MDSF) or MDSF supplemented with EGF. The addition of EGF from either day 0 (chronic) or day 4 (subacute) resulted in significant increases in Rt and short-circuit current (ISC) on day 5. After subacute exposure, these effects were delayed in onset by 6-12 h and sustained for > 24 h. Basolateral (but not apical) EGF was responsible for these effects, which were prevented by preincubation with tyrphostin RG-50864, a reversible specific inhibitor of the EGF receptor tyrosine kinase. ISC decreased, with a sensitivity to apical inhibitors of sodium transport in the order benzamil > amiloride > 5-(N-ethyl-N-isopropyl) amiloride in MDSF +/- EGF, and was completely inhibited by the addition of basolateral ouabain. Net sodium flux and Na+, K+ -ATPase activity both increased approximately 50% in the presence of EGF. These results indicate that 1) EGF decreases tight junctional permeability and increases active sodium transport by AEC monolayers via basolaterally located EGF receptors, and 2) the pathways for AEC sodium entry and exit (+/- EGF) are apical high amiloride affinity sodium channels and basolateral sodium pumps.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Alveolos Pulmonares/metabolismo , Sodio/metabolismo , Amilorida/farmacología , Animales , Transporte Biológico Activo/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero , Células Epiteliales , Epitelio/metabolismo , Masculino , Ouabaína/farmacología , Permeabilidad/efectos de los fármacos , Alveolos Pulmonares/citología , Ratas , Ratas Sprague-DawleyRESUMEN
Alveolar epithelial type II (AT2) cells have been thought to be the progenitors of terminally differentiated type I (AT1) cells in the adult animal in vivo. In this study, we used an AT1 cell-specific monoclonal antibody (mAb VIII B2) to investigate expression of the AT1 cell phenotype accompanying reversible changes in expression of the AT2 cell phenotype. AT2 cells were isolated and cultured either on attached collagen gels or on gels detached 1 or 4 days after plating and maintained thereafter as floating gels. Monolayers on both attached and floating gels were harvested on days 4 and 8 and analyzed by electron microscopy for changes in morphology and binding of mAb VIII B2. Results indicate that: (1) alveolar epithelial cells (AEC) on attached gels develop characteristics of the AT1 cell phenotype, (2) AEC on gels detached on day 1 maintain features of the AT2 cell phenotype (and do not react with mAb VIII B2), and (3) the expression of AT1 cell phenotypic traits seen by day 4 on attached gels is reversed after detachment. We conclude that commitment to the AT1 and AT2 cell lineages requires continuous regulatory input to maintain the differentiated states, and that transdifferentiation between AT2 and AT1 cells may be reversible.
Asunto(s)
Alveolos Pulmonares/citología , Animales , Antígenos de Superficie/análisis , Diferenciación Celular , Células Cultivadas , Colágeno , Células Epiteliales , Epitelio/inmunología , Epitelio/metabolismo , Geles , Expresión Génica , Masculino , Proteolípidos/genética , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/metabolismo , Proteínas Asociadas a Surfactante Pulmonar , Surfactantes Pulmonares/genética , ARN Mensajero/genética , Ratas , Ratas Sprague-DawleyRESUMEN
We investigated the polarized distribution and isoform specificity of anion exchange (Cl(-)-HCO3- exchange) in alveolar epithelial cell monolayers. Rat alveolar type II epithelial cell monolayers were grown in primary culture on detachable tissue culture-treated nuclepore filters. Each filter was mounted in a cuvette containing two fluid compartments (apical and basolateral) separated by the monolayer, the cells loaded with pH-sensitive dye, and intracellular pH (pHi) measured spectrofluorometrically. To assay for Cl(-)-HCO3- exchange, monolayers were incubated in medium containing 24 mM HCO3-/5% CO2 and 140 mM NaCl at pH 7.4 and acutely alkalinized by replacement of the fluid by HCO3(-)-free buffer containing Hepes (6 mM) at pH 7.4. Monolayers exhibited basolateral (but not apical) Cl(-)-dependent, Na(+)-independent recovery from an alkaline load that was abolished when Cl- was substituted by equimolar gluconate in the basolateral fluid, or if DIDS (500 microM) was present basolaterally. Substitution of gluconate for Cl- in the basolateral fluid, but not the apical fluid, resulted in a rise in steady-state pHi that was reversible on replacement of the basolateral fluid with Cl(-)-containing buffer, which occurred in HCO3(-)- but not Hepes-buffered medium. These data indicate that alveolar epithelial cells express basolateral membrane domain of these cells. Northern analysis of alveolar epithelial cell mRNA using anion exchanger (AE) isoform-specific cDNA probes indicates that alveolar epithelial cells express the AE2 isoform predominantly, if not exclusively, and do not express detectable AE1 (i.e., band-3 protein) or AE3.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas de Transporte de Anión , Antiportadores/metabolismo , Bicarbonatos/metabolismo , Cloruros/metabolismo , Proteínas de la Membrana/metabolismo , Alveolos Pulmonares/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Antiportadores/genética , Polaridad Celular , Células Cultivadas , Antiportadores de Cloruro-Bicarbonato , ADN Complementario , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/genética , Alveolos Pulmonares/citología , Alveolos Pulmonares/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Proteínas SLC4A , Sodio/metabolismoRESUMEN
Serum contains a number of polypeptide growth factors, hormones, and soluble matrix components and may influence the state of differentiation of epithelial cells in general and of alveolar epithelial cells (AEC) in particular. To evaluate the influence of sera on the transition from the type II toward the type I cell phenotype, we compared the effects of newborn bovine serum (NBS) and rat serum (RS) on morphologic changes and expression of a type I cell-specific epitope in AEC monolayers with time in primary culture. Rat type II AEC were harvested and cultured in defined serum-free medium (MDSF), MDSF + RS (5%), or MDSF + NBS (10%). Monolayer integrity was monitored by measuring transepithelial resistance (approximately 2,000 omega.cm2) and short-circuit current (approximately 4 microA/cm2). Binding of the type I cell-specific monoclonal antibody VIIIB2 was assessed between day 1 and day 11 by cell-based enzyme-linked immunosorbent assay (ELISA) and by immunoelectron microscopy (IEM). By ELISA, in MDSF and MDSF + NBS, VIIIB2 binding increased markedly after day 2, rising approximately 4-fold by day 8 (compared with day 1). In dramatic contrast, there was essentially no increase in VIIIB2 binding through day 11 in MDSF + RS. Results from IEM for apical surface binding of VIIIB2 were similar to those obtained by ELISA. Some morphologic differences were also noted, with cells in MDSF + RS being somewhat less spread at later times than those in MDSF or MDSF + NBS. These data indicate that the rate of rat type II AEC differentiation toward the type I cell phenotype is significantly modulated by soluble factor(s) present in rat serum.
Asunto(s)
Sangre , Alveolos Pulmonares/citología , Animales , Animales Recién Nacidos , Antígenos de Diferenciación/análisis , Antígenos de Diferenciación/metabolismo , Biomarcadores/análisis , Bovinos , Diferenciación Celular , Membrana Celular/química , Medio de Cultivo Libre de Suero , Electrofisiología , Células Epiteliales , Masculino , Fenotipo , Alveolos Pulmonares/fisiología , Ratas , Ratas Sprague-Dawley , Organismos Libres de Patógenos EspecíficosRESUMEN
Recent studies in fetal lung using immunological and molecular probes have revealed type I and type II cell phenotypic markers in primordial lung epithelial cells prior to the morphogenesis of these cell types. We have recently developed monoclonal antibodies specific for adult type I cells. To evaluate further the temporal appearance of the type I cell phenotype during alveolar epithelial cell ontogeny, we analyzed fetal lung development using one of our monoclonal antibodies (mAb VIII B2). The epitope recognized by mAb VIII B2 first appears in the canalicular stage of fetal lung development, at approx. embryonic day 19 (E19), in occasional, faintly stained tubules. Staining with this type I cell probe becomes more intense and more widespread with increasing gestational age, during which time the pattern of staining changes. Initially, all cells of the distal epithelial tubules are uniformly labelled along their apical and basolateral surfaces. As morphological differentiation of the alveolar epithelium proceeds, type I cell immunoreactivity appears to become restricted to the apical surface of the primitive type I cells in a pattern approaching that seen in the mature lung. We concurrently analyzed developing fetal lung with an antiserum to surfactant apoprotein-A (alpha-SP-A). Consistent with the findings of others, labeling of SP-A was first detectable in scattered cuboidal cells at E18. Careful examination of the double-labeled specimens suggested that some cells were reactive with both the VIII B2 and SP-A antibodies, particularly at E20. Confocal microscopic analysis of such sections from E20 lung confirmed this impression. Three populations of cells were detected: cells labeled only with alpha-SP-A, cells labeled only with mAb VIII B2, and a smaller subset of cells labeled by both.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Epítopos/análisis , Pulmón/embriología , Alveolos Pulmonares/citología , Alveolos Pulmonares/inmunología , Animales , Anticuerpos Monoclonales , Antígenos de Diferenciación/análisis , Desarrollo Embrionario y Fetal , Células Epiteliales , Epitelio/inmunología , Microscopía Confocal , Proteolípidos/análisis , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , Surfactantes Pulmonares/análisis , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Isolated type II pneumocytes grown in serum on tissue culture-treated polycarbonate filters form monolayers with characteristic bioelectric properties, and change morphologically with time in culture to resemble type I cells. Concurrently, the cells express type I cell surface epitopes, making this a potentially useful in vitro model with which to study regulation of alveolar epithelial cell function and differentiation. To define specific soluble growth factors and matrix substances that may regulate these processes, it would be preferable to culture isolated pneumocytes de novo under completely defined, serum-free conditions. In this study, we developed a completely defined serum-free medium that is capable of supporting alveolar epithelial cells in primary culture, allowing the formation of monolayers with characteristic bioelectric and phenotypic properties. Freshly isolated rat type II cells were resuspended in completely defined serum-free medium and plated de novo on polycarbonate filters. Plating efficiency, bioelectric properties, morphology, and binding of a type I cell-specific monoclonal antibody were determined as functions of time. Plating efficiency plateaus at about 14% by Day 3 in culture. Transepithelial resistance rises to high levels, peaking at 1.76 +/- 0.14 K omega-cm2 by Day 5 in culture. Short-circuit current peaks on Day 3 in culture at 2.71 +/- 0.35 microA/cm2. With time, the cells gradually become flattened with protuberant nuclei and long cytoplasmic extensions, more closely resembling type I cells, and begin to express a type I cell surface epitope.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Medios de Cultivo , Alveolos Pulmonares/citología , Animales , Anticuerpos Monoclonales , Conductividad Eléctrica , Electrofisiología , Filtración/instrumentación , Sustancias de Crecimiento , Masculino , Microscopía Electrónica , Modelos Biológicos , Fenotipo , Cemento de Policarboxilato , Alveolos Pulmonares/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
An understanding of the process of alveolar epithelial cell growth and differentiation requires the ability to trace and analyze the phenotypic transitions that the cells undergo. This analysis demands specific phenotypic probes to type II and, especially, type I pneumocytes. To this end, monoclonal antibodies have been generated to type I alveolar epithelial cells using an approach designed to enhance production of lung-specific clones from a crude lung membrane preparation. The monoclonal antibodies were screened by a combination of enzyme-linked immunosorbent assay and immunohistochemical techniques, with the determination of type I cell specificity resting primarily on immunoelectron microscopic localization. Two of these new markers of the type I pneumocyte phenotype (II F1 and VIII B2) were used to analyze primary cultures of type II cells growing on standard tissue culture plastic and on a variety of substrata reported to affect the morphology of these cells in culture. On tissue culture plastic, the antibodies fail to react with early (days 1 to 3) type II cell cultures. The cells become progressively more reactive with time in culture to a plateau of approximately 6 times background by day 8, with a maximum rate of increase between days 3 and 5. This finding is consistent with the hypothesis that type II cells in primary culture undergo at least partial differentiation into type I cells. Type II cells grown on laminin, which reportedly delays the loss of type II cell appearance, and on fibronectin, which has been reported to facilitate cell spreading and loss of type II cell features, develop the type I cell markers during cultivation in vitro with kinetics similar to those on uncoated tissue culture plastic. Cells on type I collagen and on tissue culture-treated Nuclepore filters, which have been reported to support monolayers with type I cell-like morphology, also increase their expression of the II F1 and VIII B2 epitopes around days 3 to 5. Taken together with available morphologic information, these data suggest that expression of different alveolar epithelial cell phenotypic markers by type II cells in primary culture may be independently regulated. The monoclonal antibody probes described in this report should prove useful in the continued investigation of the mechanisms and regulation of alveolar epithelial cell differentiation.
Asunto(s)
Anticuerpos Monoclonales , Alveolos Pulmonares/metabolismo , Animales , Anticuerpos Monoclonales/ultraestructura , Diferenciación Celular , Células Cultivadas , Medios de Cultivo , Conductividad Eléctrica , Células Epiteliales , Epitelio/crecimiento & desarrollo , Epitelio/metabolismo , Técnica del Anticuerpo Fluorescente , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Inmunoelectrónica , Alveolos Pulmonares/citología , Alveolos Pulmonares/crecimiento & desarrollo , Ratas , Ratas EndogámicasRESUMEN
A plasma membrane proton-translocating adenosinetriphosphatase (ATPase) has been identified in rat alveolar pneumocytes in primary culture using the pH-sensitive fluorescent probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Intracellular pH (pHi) was acutely lowered by NH3 prepulse in HCO3(-)-free medium buffered with 6 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and its recovery was measured thereafter under control conditions, in the presence of amiloride to inhibit Na(+)-H+ antiport, and in the presence of N-ethylmaleimide (NEM), a plasma membrane H(+)-ATPase inhibitor. Initial rate of pHi recovery was reduced by 67% in the presence of amiloride, 52% in the presence of NEM, and 96% in the presence of both. Recovery was decreased but not abolished in Na(+)-free buffer, was essentially abolished when NEM was present in the absence of Na+, and was also abolished by addition of the metabolic inhibitor KCN in glucose- and Na(+)-free medium. These data suggest that alveolar epithelial cells possess a plasma membrane H(+)-ATPase. In Na(+)-containing buffer at pH 7.4, steady-state pHi was 7.50. This value was unaffected by amiloride but decreased to 7.01 in the presence of NEM, suggesting active H(+)-ATPase and inactive Na(+)-H+ antiport at steady-state pHi. We conclude that this plasma membrane proton-translocating ATPase in alveolar pneumocytes may be an important mechanism contributing to regulation of steady-state pHi, recovery from acute intracellular acidification, and modulation of extracellular alveolar fluid pH.
Asunto(s)
Alveolos Pulmonares/fisiología , Amilorida/farmacología , Animales , Células Cultivadas , Epitelio/efectos de los fármacos , Epitelio/fisiología , Etilmaleimida/farmacología , Fluoresceínas , Colorantes Fluorescentes , Concentración de Iones de Hidrógeno , Cinética , Masculino , Cianuro de Potasio/farmacología , Ratas , Ratas Endogámicas , Sodio/farmacología , Espectrometría de FluorescenciaRESUMEN
Monoclonal antibodies reactive with desmin (D3 and D76) have been generated and their specificities validated by immunoblots, RIAs, and immunocytochemistry. No cross-reaction with other IFPs has been observed. The McAbs recognized different epitopes but both reside in the amino-terminal rod domain of desmin. Whereas McAb D3 produces a staining pattern characteristic of desmin throughout the development of cardiac and skeletal muscles, McAb D76 was selectively unreactive with certain regions of early (three days in ovo) embryonic cardiac anlage, with cultured cardiac myocytes derived from 7-day-old embryos, and with skeletal myotubes in early stages of myogenesis in vitro. Positive reactivity of D76 was seen at stages of myofibrillogenesis when the sarcomeres assume lateral alignment. Evidence was presented that differential reactivity of D76 did not result from the biosynthesis of a new desmin isoform or the post-translational modification of an existing protein. We suggest that the appearance of D76 immunoreactivity during striated muscle development represents an unmasking of the epitope by some IF-associated protein. Since this transition during skeletal muscle differentiation occurs during lateral alignment of the myofibrils, this antibody may serve as a useful probe for exploring this reorganization of the contractile apparatus during myogenesis and muscle regeneration.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Citoesqueleto/análisis , Desmina/análisis , Filamentos Intermedios/análisis , Músculos/análisis , Miocardio/análisis , Animales , Embrión de Pollo , Desmina/inmunología , Técnica del Anticuerpo Fluorescente , Ratones , Músculos/embriología , Miofibrillas/fisiología , ConejosRESUMEN
Monoclonal antibodies ( McAbs ) have been generated against a preparation of intermediate filament proteins (IFP) from adult chicken gizzard. Two antibodies, D3 and D76 , have been characterized in detail. They bind specifically to desmin but recognize different epitopes. In the adult chicken, both McAbs produced equivalent immunofluorescent staining patterns, reacting in frozen sections with all forms of muscle tissue, including vascular smooth muscle, but with no other tissue types. In isolated skeletal myofibrils and in longitudinal frozen sections of cardiac and skeletal muscle, desmin was detected with both McAbs at the Z-band and in longitudinally-oriented filament bundles between myofibrils. In contrast to these results in the adult, the intermediate filaments (IF) of embryonic cardiac myocytes in primary cultures were decorated only with McAb D3, whereas McAb D76 was completely unreactive with these cells. Similarly, frozen sections through the heart at early stages of embryonic chick development (Hamburger-Hamilton stages 17-18) revealed regions of myocytes, identified by double immunofluorescence with myosin-specific McAbs , that were unstained with McAb D76 even though similar regions were stained by McAb D3. That McAb D76 reacted with desmin in all adult cardiac myocytes but not with all embryonic heart cells indicates that embryonic and adult cardiac IF are immunologically distinct and implies a conversion in IF immunoreactivity during cardiac development.
Asunto(s)
Citoesqueleto/ultraestructura , Corazón/embriología , Proteínas de Filamentos Intermediarios/análisis , Miofibrillas/ultraestructura , Animales , Anticuerpos Monoclonales , Células Cultivadas , Embrión de Pollo , Pollos , Desmina , Técnica del Anticuerpo Fluorescente , Proteínas de Filamentos Intermediarios/inmunología , Músculos/ultraestructura , Miocardio/ultraestructura , RadioinmunoensayoRESUMEN
Antibodies raised against chicken gizzard smooth muscle desmin were shown to be specific by immunofluorescence cytochemistry and immunoautoradiography after two-dimensional polyacrylamide gel electrophoresis. Embryonic chick heart cell cultures (permeabilized with Triton X-100) and enucleated adult chicken erythrocyte ghosts (Granger, B. L., E. A. Rapasky, and E. Lazarides, 1982, J. Cell Biol. 92:299-312) were then used for immunoelectronmicroscopic localization of desmin. As expected, all intermediate filaments (IF) of the cardiac myocytes were labeled heavily and uniformly with the desmin antibodies. No periodicity or helicity was detectable along the labeled IF. Of interest was the intermittent but clear labeling of the IF of the nonmuscle, fibroblastic cells in the identical cultures. These antibodies did not bind vimentin from embryonic chick heart homogenates; furthermore, they did not label IF of avian erythrocytes known to contain vimentin but not desmin. We conclude that IF of cardiac fibroblastic cells contain low, but significant, concentrations of desmin and that this protein probably forms a copolymer with vimentin in these cells.