Cultures of human tracheal gland cells of mucous or serous phenotype

Abstract

There are two main epithelial cell types in the secretory tubules of mammalian glands: serous and mucous. The former is believed to secrete predominantly water and antimicrobials, the latter mucins. Primary cultures of human airway gland epithelium have been available for almost 20 yr, but they are poorly differentiated and lack clear features of either serous or mucous cells. In this study, by varying growth supports and media, we have produced cultures from human airway glands that in terms of their ultrastructure and secretory products resemble either mucous or serous cells. Of four types of porous-bottomed insert tested, polycarbonate filters (Transwells) most strongly promoted the mucous phenotype. Coupled with the addition of epidermal growth factor (EGF), this growth support produced "mucous" cells that contained the large electron-lucent granules characteristic of native mucous cells, but lacked the small electron-dense granules characteristic of serous cells. Furthermore, they showed high levels of mucin secretion and low levels of release of lactoferrin and lysozyme (markers of native serous cells). By contrast, growth on polyethylene terephthalate filters (Cyclopore) in medium lacking EGF produced "serous" cells in which small electron-dense granules replaced the electron-lucent ones, and the cells had high levels of lactoferrin and lysozyme but low levels of mucins. Measurements of transepithelial resistance and short-circuit current showed that both "serous" and "mucous" cell cultures possessed tight junctions, had become polarized, and were actively secreting Cl.

Figure 1.

Release of lactoferrin into the mucosal medium. Cells were grown on four types of insert with (open columns) or without (hatched columns) EGF. Removal of EGF increased spontaneous secretion of lactoferrin, though maximal levels were dependent on type of insert. Values are means ± SE for 8 to 34 inserts from 3 to 11 different cultures. Asterisks indicate values significantly different from those obtained in the presence of EGF.

Figure 2.

Electron photomicrographs of serous and mucous gland cell cultures. (A) Serous cells. Note the apical electron-dense granules typical of native serous gland cells. (B) Mucous cells. Note the large electron-lucent secretory granules. Scale bar 5 µm.

Figure 3.

Immunocytochemical localization of lactoferrin in serous (A) or mucous cells (B). The signal (brown deposit) in mucous cells is no greater than in cells that did not receive primary antibody. Scale bar 10 µm.

Figure 4.

Short-circuit current records from mucous (upper trace) or serous cell cultures (lower trace) derived from the same trachea. Upward-pointing arrows methacholine (10⁻⁵ M, serosal side). Downward-pointing arrows CFTR_inh-172 (10⁻⁵ M, mucosal side). Cells had been pretreated with amiloride (10⁻⁵ M, mucosal side). The gap in the records was for 2 min, during which time ATP (10⁻⁵ M) was added. It caused small transient increases in I _sc ∼ 25% as large as the methacholine response.