Cultures Of Human Bronchial Epithelial Cells Research Articles

Ciliogenesis in human bronchial epithelial cells cultured at the air-liquid interface.

We present a study on modification of culture conditions in serially cultured human bronchial epithelial cells (HBEC), necessary to achieve bronchial epithelial cells similar to the native epithelium. Cells were obtained from bronchial biopsies and serially cultured using a previously described method (In Vitro Cell. Dev. Biol. 1993; 29A:379-387). At the air-liquid interface, the second and the subsequent passages of HBEC cultures were grown 7 to 31 days, in medium containing fetal calf serum, using de-epidermized dermis or collagen discs as substratum. Scanning and transmission electron microscopy revealed ciliogenesis after 7 days and maturation of the cilia up to 31 days, irrespective of whether de-epidermized dermis or collagen membrane was used. The transmission electron microscopy of the developing cilia showed fibrogranular masses, procentrioles, basal bodies, and in the mature cilia a normal ultrastructure of the axoneme, the nine doublets, the central pair, radial spokes, and dynein arms in the ciliary shaft. In contrast, the submerged cultures showed no signs of ciliogenesis in the same time course. Results of experiments, in which cell seeding density, the substrate used, and the manner of nutrient supplementation were modulated, revealed that the air-exposure of the cultured HBEC is a necessary requirement for the ciliogenesis. The development pathway of ciliated cells in air-exposed HBEC cultures was similar to the differentiation and maturation pattern in human fetal tracheal cells. The in vitro model of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy offers an attractive model for future studies on the function of human bronchial epithelial cells under normal and pathologic conditions.

Serial culturing of human bronchial epithelial cells derived from biopsies.

In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy. The cell cultures were initiated from small amounts of material (2 mm forceps biopsies) using either explants or epithelial cell suspensions in combination with a feeder-layer technique. The rate of cell proliferation and the number of passages (up to 8 passages) achieved were similar, irrespective of whether the explants or dissociated cells were used. To modulate the extent of differentiation, the bronchial epithelial cells were cultured either under submerged, low calcium (0.06 mM) (proliferating), normal calcium (1.6 mM) (differentiation enhancing) conditions, or at the air-liquid interface. Characterization of the bronchial epithelial cell cultures was assessed on the basis of cell morphology, cytokeratin expression, and ciliary activity. The cells cultured under submerged conditions formed a multilayer consisting of maximally three layers of polygonal-shaped, small cuboidal cells, an appearance resembling the basal cells in vivo. In the air-exposed cultures, the formed multilayer consisted of three to six layers exhibiting squamous metaplasia. The cytokeratin profile in cultured bronchial epithelial cells was similar in submerged and air-exposed cultures and comparable with the profile found in vivo. In addition to cytokeratins, vimentin was co-expressed in a fraction of the subcultured cells. The ciliary activity was observed in primary culture, irrespective of whether the culture had been established from explants or from dissociated cells. This activity was lost upon subculturing and it was not regained by prolongation of the culture period. In contrast to submerged cultures and despite the squamous metaplasia appearance, the cells showed a reappearance of cilia when cultured at the air-liquid interface. Human bronchial epithelial cell cultures can be a representative model for controlling the mechanisms of regulation of bronchial epithelial cell function.

Histamine (H) does not Affect Permeability of Human Bronchial Epithelial Cell Cultures (HBEC), in vitro

Histamine (H) does not Affect Permeability of Human Bronchial Epithelial Cell Cultures (HBEC), <i>in vitro</i>

Nitrogen Dioxide (NO2)-Induced Increase in Permeability and Synthesis of GM-CSF and IL-8, in Human Bronchial Epithelial Cell Cultures (HBEC)

Nitrogen Dioxide (NO2)-Induced Increase in Permeability and Synthesis of GM-CSF and IL-8, in Human Bronchial Epithelial Cell Cultures (HBEC)

Respiratory syncytial virus infection of human primary nasal and bronchial epithelial cell cultures and bronchoalveolar macrophages.

In adults, clinical symptoms caused by respiratory syncytial virus (RSV) are usually confined to the upper respiratory tract, whereas RSV infection in infants frequently causes bronchiolitis and pneumonia. The preferential localization of RSV infection to the upper airways may partially be due to protective immunity, but may also depend on a difference in susceptibility of epithelial cells from upper and lower airways, or on antiviral activities of bronchoalveolar macrophages (AM). In this study, we have compared the susceptibility of primary adult human nasal epithelium, primary adult human bronchial epithelium, a human bronchial epithelial cell line (BEAS-2B), and adult human AM to infection with RSV. The cell cultures were infected with multiplicities of infection (moi) of 1 and 0.1. Virus release into the supernatants was assayed at days 1, 2, 4, and 7, and the percentage of virus-positive cells determined by immunofluorescence at the same time points. Similar proportions of nasal epithelial cells (NE) and bronchial epithelial cells (BE) were infected with RSV. Approximately 50 to 75% (with moi 1) and 2 to 10% (with moi 0.1) of the cells were infected by 24 h; almost all the cells were RSV positive by day 4. However, BE released less infectious RSV than do NE. With moi 0.1, 10-fold less virus was released over 4 days of culture. By days 4 to 7, cytopathic effects (CPE) were maximal in all epithelial cell cultures, but CPE developed latest in BE infected with moi 0.1. AM were also productively infected with RSV, with peak virus production at day 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Time course of IL1 and IL6 synthesis and release in human bronchial epithelial cell cultures exposed to toluene diisocyanate.

We have previously demonstrated that human bronchial epithelial cells release appreciable amounts of interleukin 1 (IL1) and interleukin 6 (IL6) when exposed to toluene diisocyanate (TDI) in vitro. TDI is an inflammatory and asthmogenic stimulus presumed to act at least in part through immunological mechanisms. The epithelial cell-derived IL1 and IL6 can promote T cell activation and proliferation in culture, and if this also happens in vivo they may contribute to the persistence of the inflammatory response of the bronchial mucosa observed in TDI-sensitive asthmatics. In this study, we confirmed the release of biologically active IL1 beta and IL6-like substances from bronchial epithelial cells exposed to isocyanates in vitro, and related the rate and the magnitude of the cytokine secretion with the pattern of IL1 beta and IL6 gene expression and the extent of epithelial cell injury. In the epithelial cell cultures exposed to TDI, there was a parallel, progressive increase in the expression of IL6 mRNA and in the secretion of IL6 protein between 48 hours and 6 days after exposure. By contrast, although increasing amounts of biologically active IL1 beta were detected in the supernatants of TDI-exposed epithelial cells throughout the 6-day period following exposure, augmented levels of IL1 beta mRNA were only evident 6 days after exposure, suggesting that TDI exposure might have initially affected the enzymatic cleavage of the intracellular IL1 beta precursor and the mechanisms which regulate the secretion of mature IL1 beta.

Histamine Receptors (HR) and the Effect of Histamine (H) on Permeability in Human Bronchial Epithelial Cell Cultures, in Vitro

Histamine Receptors (HR) and the Effect of Histamine (H) on Permeability in Human Bronchial Epithelial Cell Cultures, in Vitro

Acute effects of 12-O-tetradecanoylphorbol-13-acetate, teleocidin B, or 2,3,7,8-tetrachlorodibenzo-p-dioxin on cultured normal human bronchial epithelial cells.

Effects of teleocidin B, 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,7-dichlorodibenzo-p-dioxin (DCDD) on normal human bronchial epithelial cell cultures were assessed by quantitation of cellular morphology, clonal growth (population doublings per day), cross-linked envelope (CLE) formation and the enzymatic activities of aryl hydrocarbon hydroxylase (AHH), ornithine decarboxylase (ODC) and plasminogen activator (PA). Toxicity was assessed by clonal growth assays. Teleocidin B and TPA had similar effects on growth, morphology and enzyme activities. When the cells were incubated with TPA or teleocidin B at concentrations of 1-100 nM for 6 h, RNA synthesis was unaffected, but DNA synthesis decreased and squamous differentiation, marked by an increase in cell surface area and cross-linked envelope formation, was increased. TPA and teleocidin B also increased ODC activity in LHC-0 medium (a maintenance medium without epidermal growth factor) but caused a decrease of ODC activity in LHC-4 (a growth medium containing epidermal growth factor). Finally, TPA and teleocidin B each caused an increase of PA and a decrease of AHH activities in both media. Phorbol, a non-promoting analogue of TPA, had no effect on growth, morphology or biochemical assays. TCDD (100 nM) caused a 15% decrease in cell growth when cells were incubated in LHC-4, and this was accompanied by an increase in cell surface area, PA activity, and CLE formation. TCDD caused an increase in AHH and ODC activities when the cells were incubated in either LHC-0 or LHC-4 medium. DCDD did not alter cell growth, and its morphological and biochemical effects were similar to those of TCDD although less marked. In conclusion, results reported here are consistent with the hypothesis that an important property of some tumor promoters is their ability to induce terminal differentiation in normal, non-initiated epithelial cells.