Human surface respiratory epithelial (HSRE) cells from nasal polyps have been cultured within collagen lattices in a serum-free defined medium. Cell growth observed over a period of 12 days showed a population doubling time of 36 h. Under these culture conditions, we observed a contraction of the lattices. Phase-contrast light microscopy and transmission electron microscopy demonstrated that the HSRE cells formed tubular ductlike structures. Lumens formed by HSRE cells were surrounded by cuboidal-shaped polarized cells with numerous ciliated cells, secretory cells, and undifferentiated cells. Epidermal growth factor (EGF) was observed to stimulate the tubule formation and the contraction of the lattices. Videomicroscopic observations and analysis of the ciliary beating frequency (CBF) demonstrated that the cilia were homogeneously distributed on the whole apical surface of the ciliated cells and that their movement was well coordinated, with a CBF similar to that observed in outgrowth cells from cultured human nasal and tracheal epithelia. Immunofluorescent staining of basement membrane components synthesized and secreted by cells revealed the presence of type III collagen around the tubules. Type IV collagen and laminin were present in the cytoplasm and at the periphery of the cells. The biotin-streptavidin-gold immunocytochemical technique with monoclonal anti-mucin antibody showed intracellular localization of mucins in secretory granules of the secretory cells. With the use of substrate gel electrophoresis polyacrylamide gels impregnated with gelatin, collagenase activity was detected in the conditioned medium of the cultured HSRE cells. These results suggest that both three-dimensional collagen gel and soluble factors such as EGF regulate tubule formation by HSRE cells. Moreover, the capacity of the epithelial cells to contract the gel suggests they may be involved in the wound healing process.

We have investigated the effect of both adenosine triphosphate (ATP) and uridine triphosphate (UTP) on the fluid transport, transepithelial electric potential difference (PD), and unidirectional chloride flux when applied apically to cultured human surface respiratory epithelial (HSRE) cells in a double-compartment chamber. The effects of ATP and UTP (both 100 microM) were examined in cells either untreated or pretreated with 100 microM amiloride in lactated Ringer's solution. ATP or UTP was added to the apical solution in a 100 microliters final volume. After a 2-h incubation period, the change in fluid transport was measured by weighing the apical fluid. Compared with control, amiloride blocked the fluid absorption by HSRE cells. The addition of ATP or UTP, either alone or after pretreatment with amiloride, induced a similar and significant increase in the apical fluid and chloride flux (P < 0.001 and P < 0.005, respectively). The changes in both fluid transport and chloride flux were accompanied by changes in PD. A blocker for chloride transport, 4,4'-diisothiocyano-2,2'-stilbene disulfonate, at 500 microM significantly blocked the ATP-stimulated fluid transport (P < 0.05) and chloride flux (P < 0.01). These results support the hypothesis that extracellular ATP and UTP increase the fluid transport by respiratory epithelial cells and may be useful in the hydration of mucus and respiratory mucosa.

Inflammation and epithelial damage of the bronchial mucosa are frequently identified in children with bronchial diseases. Nevertheless, until now the quantitative assessment of the epithelial damage has never been studied in relation to clinical or respiratory function or mucus abnormalities. Bronchial biopsies and brushings were performed in 31 children with recurrent bronchitis and without atopia. The quantitative histologic data were compared with clinical results, the endoscopic appearance of the mucosa, ciliary beating frequency, mucus transport capacity, leukocyte count, and protein concentration in mucus samples. Most of the biopsies (87%) collected in this group of children without recent acute infections showed extensive epithelial damage. A significant correlation was observed between the degree of shedding and edema (p < 0.01). Bronchial epithelial edema was associated with a significantly decreased (p < 0.01) mucus transport rate. Inflammation of the submucosa was significantly correlated with lymphocyte epithelial infiltration (p < 0.01), total mucus protein content (p < 0.01), and local airway inflammation estimated by bronchoscopy. These results demonstrate that children with recurrent bronchitis develop a severe bronchial inflammation associated with an increased mucus protein content and a reduction in the mucociliary function.

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