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The extracellular matrix has been demonstrated to affect the differentiation of epithelial cells. We present evidence that in a three-dimensional (3-D) type I collagen gel matrix, isolated human adult tracheal gland (HTG) cells are capable of reconstructing new functional gland-like tubules in vitro. During the first two weeks in culture, HTG cells developed globular epithelial cell aggregates in which lumina is absent. By the third week in culture, the tubulogenesis and the formation of branching structures became evident with a polarized morphology, which in many aspects resembles the in vivo morphology. A central lumen was lined by polarized secretory epithelial cells exhibiting well-developed microvilli and apical secretory granules. Furthermore, we showed that the capacity of in vitro tracheal gland differentiation was associated with the basal deposition of laminin and type IV collagen around the gland-like tubules. A cell-associated 72 kDa type IV collagenase was expressed in developing tubule cells, as shown by immunocytochemistry. The secretion of the antileucoprotease (ALP), a protein marker of tracheal gland serous cells, was bidirectional in gland-like tubules, since up to 65% of released ALP was in the basolateral direction. Taken together, these observations indicate that isolated HTG cells in a 3-D collagen matrix form functional tracheal gland-like tubules and suggest that similar new tracheobronchial gland formations may occur during the human normal gland development and remodeling. © 1994 Wiley-Liss, Inc.

During airway inflammation, proteinases such as human leukocyte elastase are actively secreted. Secretory leukocyte protease inhibitor is a major serine proteinase inhibitor, secreted by bronchial, bronchiolar and lung epithelial cells. We recently identified secretory leukocyte protease inhibitor in human nasal epithelium, exclusively in remodelled areas of the surface epithelium. We now investigated the influence of remodelling and inflammation of the nasal tissue on the in vitro capacity of these cells to respond to human leukocyte elastase. Primary cultures of surface epithelial cells were established from various nasal polyp samples. At confluency, cell cultures were exposed to different human leukocyte elastase concentrations. The secretory leukocyte protease inhibitor immunocytolocalisation, expression and secretion were then investigated. Immunocytochemistry, showed a human leukocyte elastase dose-dependent increase of secretory leukocyte protease inhibitor containing cells and a basal extracellular localization of secretory leukocyte protease inhibitor after incubation with 100 microg/ml human leukocyte elastase. The relative amount of secretory leukocyte protease inhibitor mRNA transcripts increased with respect to the human leukocyte elastase concentration. Nevertheless, the potential stimulation of secretory leukocyte protease inhibitor secretion by human leukocyte elastase was lower in the more remodelled and inflamed tissue. Our results suggest that the contribution of the surface epithelial cells of poorly remodelled tissues to the protection against the deleterious effect of neutrophil proteinases is severely decreased in highly remodelled and inflamed tissues.

We describe a method for establishing the culture of bovine tracheal submucosal gland (BTG) cells, in which we have also examined the influence of a reconstituted basement membrane matrix derived from the Engelbreth-Holm-Swarm tumor (EHS) on the growth and morphological differentiation of these cells. BTG cells have been isolated by tissue enzymatic digestion using trypsin, deoxyribonuclease I, elastase, hyaluronidase and EGTA for 1 hr at 37 degrees C. Afterwards, cells and tissue were collected by centrifugation and were incubated for 15 min with 15% newborn calf serum to inactivate the proteolytic enzymes. Enzymatic digestion using only trypsin, centrifugation and inactivation steps were repeated three times. Using this protocol, we obtained 15 +/- 4 (X 10(6] cells per g of tracheal submucosa with 72 +/- 2% (n = 5) cell viability. On microscopic observation, isolated cells were mainly composed of serous type glandular cells. Cells were cultured in a 1:1 medium of Dulbecco's Modified Eagle's/Ham's F12 supplemented with 10% fetal calf serum and subcultured in either plastic flasks or flasks coated with EHS matrix. On the plastic, the BTG cells exhibited at confluency an epithelioid appearance. They stained positively with the immunofluorescent anticytokeratin antibody and contained PAS-staining granules. By electron microscopy, lactoferrin, a protein marker specific to the serous cells, was demonstrated immunocytochemically in small secretory vesicles. BTG cells cultured on EHS matrix revealed a significantly increased growth in comparison to those cultured on plastic. In post-confluent culture of BTG cells on EHS matrix, we observed numerous dome-like structures formed by differentiated cells which were joined together around luminal spaces.