RESUMEN
Measurement of the magnitude and kinetics of exocytosis from intact epithelia has historically been difficult. Using well-differentiated cultures of human bronchial epithelial cells, we describe the use of transepithelial impedance analysis to enable the real-time quantification of mucin secretagogue-induced changes in membrane capacitance (surface area) and conductance. ATPgammaS, UTP, ionomycin, and PMA induced robust increases in total cellular capacitance that were demonstrated to be dominated by a specific increase in apical membrane surface area. The UTP-induced increase in capacitance occurred in parallel with goblet cell emptying and the secretion of mucin and was associated with decreases in apical and basolateral membrane resistances. The magnitude and kinetics of the capacitance increases were dependent on the agonist and the sidedness of the stimulation. The peak increase in capacitance induced by UTP was approximately 30 mucin granule fusions per goblet cell. Secretagogue-induced decreases in apical membrane resistance were independent of exocytosis, although each of the secretagogues induced profound reductions in basolateral membrane resistance. Transepithelial impedance analysis offers the potential to study morphological and conductance changes in cultured human bronchial epithelial cells.
Asunto(s)
Bronquios/metabolismo , Capacidad Eléctrica , Conductividad Eléctrica , Mucinas/metabolismo , Mucosa Respiratoria/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Bronquios/efectos de los fármacos , Membrana Celular , Células Cultivadas , Exocitosis , Humanos , Ionomicina/farmacología , Mucosa Respiratoria/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Uridina Trifosfato/farmacologíaRESUMEN
Repetitive, acute inflammatory insults elicited by cigarette smoke (CS) contribute to the development of chronic obstructive pulmonary disease (COPD), a disorder associated with lung inflammation and mucus hypersecretion. Presently, there is a poor understanding of the acute inflammatory mechanisms involved in this process. The aims of this study were to develop an acute model to investigate temporal inflammatory changes occurring after CS exposure. Rats were exposed to whole body CS (once daily) generated from filtered research cigarettes. Initial studies indicated the generation of a neutrophilic/mucus hypersecreting lung phenotype in <4 days. Subsequent studies demonstrated that just two exposures to CS (15 h apart) elicited a robust inflammatory/mucus hypersecretory phenotype that was used to investigate mechanisms driving this response. Cytokine-induced neutrophil chemoattractants (CINCs) 1-3, the rat growth-related oncogene-alpha family homologs, and IL-1beta demonstrated time-dependent increases in lung tissue or lavage fluid over the 24-h period following CS exposure. The temporal changes in the neutrophil chemokines, CINCs 1-3, mirrored increases in neutrophil infiltration, indicative of a role in neutrophil migration. In addition, a specific CXCR2 antagonist, SB-332235, effectively inhibited CS-induced neutrophilia in a dose-dependent manner, supporting this conclusion. This modeling of the response of the rat airways to acute CS exposure indicates 1) as few as two exposures to CS will induce a phenotype with similarities to COPD and 2) a novel role for CINCs in the generation of this response. These observations represent a paradigm for the study of acute, repetitive lung insults that contribute to the development of chronic disease.
Asunto(s)
Pulmón/metabolismo , Moco/metabolismo , Nicotiana/efectos adversos , Neumonía/etiología , Receptores de Interleucina-8B/metabolismo , Humo/efectos adversos , Animales , Ligandos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
In addition to a direct proinflammatory role, IL-13 has been demonstrated to induce a goblet cell metaplastic phenotype in the airway epithelium in vivo. We have studied the direct effects of IL-13 (and IL-4) on well-differentiated, air-liquid interface cultures of human bronchial epithelial cells (HBEs) and provide a quantitative assessment of the development of a mucus hypersecretory phenotype induced by these cytokines. Using Alcian blue staining of goblet cells and immunohistochemical detection of MUC5AC, we found that IL-13 (and IL-4) induced increases in the goblet cell density (GCD) of the HBE cultures. The effects of these cytokines were critically dependent on concentration: 1 ng/ml routinely induced a 5- to 10-fold increase in GCD that was associated with a hypersecretory ion transport phenotype. Paradoxically, 10 ng/ml of either cytokine induced a profound reduction in GCD. Removal of EGF from the culture media or treatment of the cells with AG-1478 [a potent inhibitor of EGF receptor tyrosine kinase (EGFR-TK)] demonstrated that the EGFR-TK pathway was key to the regulation of the basal GCD but that it was not involved in the IL-13-driven increase. The IL-13-driven increase in GCD was, however, sensitive to inhibition of MEK (PD-98059, U-0126), p38 MAPK (SB-202190), and phosphatidylinositol (PtdIns) 3-kinase (LY-294002). These data support the concept that IL-13 is in part able to induce a mucus hypersecretory phenotype through a direct interaction with the airway epithelium and that MAP kinase and PtdIns 3-kinase signaling pathways are involved.
Asunto(s)
Células Caliciformes/enzimología , Interleucina-13/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Bronquios/citología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/metabolismo , Butadienos/farmacología , Recuento de Células , Células Cultivadas , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Células Epiteliales/enzimología , Células Epiteliales/inmunología , Receptores ErbB/metabolismo , Flavonoides/farmacología , Células Caliciformes/citología , Células Caliciformes/inmunología , Humanos , Imidazoles/farmacología , Interleucina-4/farmacología , Canales Iónicos/metabolismo , Sistema de Señalización de MAP Quinasas/inmunología , Morfolinas/farmacología , Nitrilos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piridinas/farmacologíaRESUMEN
In this study, the authors describe a new technique enabling the rapid assessment of mucociliary clearance (MCC) in rats and characterize this aspect of innate host defense in 2 animal models of bronchitis. Following instillation into the airways, fluorescent microspheres were rapidly cleared over 24 hours, with 60% to 80% of clearance occurring within 4 hours. On a background of airway neutrophilia and mucus hypersecretion, induced by either lipopolysaccharide or cigarette smoke, MCC was significantly enhanced. This reserve capacity in the MCC system will need to become overwhelmed in order to model the clinically observed impairment of lung mucus clearance in an animal system.