Mucous Cell Differentiation Research Articles

Culture of conducting airway epithelial cells in serum-free medium

Reproducible techniques for the isolation and culture of conducting airway epithelial cells from various species including human are described. Tissues recovered after necropsy or surgery are treated with 0.1% protease at 4°C overnight. The epithelial lining cells are liberated from adventitia by flushing with ice-cold minimal essential medium containing 10% fetal bovine serum. The resulting suspension of single cells and cell clusters is centrifuged and then plated on type 1 collagen gel substratum in a serum-free F12 medium supplemented with insulin, transferrin, epidermal growth factor, cholera toxin, hydrocortisone (or dexamethasone), bovine hypothalamus extract, and retinol. Increased calcium concentration in the medium to 1 to 3 mM augments in vitro mucous cell differentiation. Both a squamouslike and mucociliary differentiation of the cultured epithelium can be identified by biochemical, immunohistologic, and morphologic means. The polarity of epithelial cell cultures is promoted by use of biphasic culture methods, in which epithelial cells are fed basally and are in direct contact with the air phase.

Growth and differentiation of primary tracheal epithelial cells in culture: Regulation by extracellular calcium

Growth and differentiation of primary monkey tracheal epithelial (MTE) cells maintained on collagen gel substrata were studied in a defined serum-free culture medium containing 0.03 to 3.0 mM extracellular calcium. Cell attachment efficiency (40-60%) was not altered by different calcium levels. Growth of primary MTE cells on collagen gel substrata, which was vitamin A dependent, was enhanced 50% in the medium supplemented with high calcium (greater than 0.3 mM). High calcium medium also increased cell-cell interactions, formation of desmosomes, and multi-cell layering. The relative content of mucous cells, which were identified by a mucin-specific monoclonal antibody and the presence of mucus-secreting granules at the ultrastructural level, was greater in the high-calcium medium. Furthermore, the secretion of mucin into the medium, determined either by an ELISA or by the incorporation of 3H-glucosamine into mucous glycoprotein fractions, was also increased more than 5-fold in media containing high calcium content (greater than 0.6 mM). In contrast, MTE cells cultured in low calcium medium (less than 0.15 mM) were squamous-like with prominent tonofilaments, and their secretory product was mainly hyaluronate. These results demonstrate that media containing a high calcium content promote conducting airway epithelium to express mucous cell differentiation, while media with low calcium content promote squamous cell differentiation.

Expression of Mucin Synthesis and Secretion in Human Tracheobronchial Epithelial Cells Grown in Culture

The effects of culture conditions on growth and differentiation of human tracheobronchial epithelial (HTBE) cells have been defined. Epithelial cells were dissociated from tissues by protease treatment and were plated on tissue culture dishes in F12 medium supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, cholera toxin, bovine hypothalamus extract, and retinol. HTBE cells did not express any mucociliary function (ciliogenesis or mucin secretion) on tissue culture plastic, but they could be passaged 3 to 5 times with a total of 10 to 25 population doublings. Cells from early passages re-express both these functions when transplanted to tracheal grafts. When tissue culture plates were coated with collagen film or collagen gel substrata, cell attachment and proliferation were stimulated. However, the expression of mucous cell function in culture occurred only when cells were plated on collagen gel substrata and vitamin A (retinol) was present in the medium. Mucous cell differentiation under optimal conditions was defined by ultrastructural studies, by immunologic studies with mucin-specific monoclonal antibodies, and by carbohydrate and amino acid compositional analyses of mucin-like glycoproteins purified from culture medium. These results demonstrate for the first time that HTBE cells can express mucin synthesis and secretion under appropriate culture conditions.

A biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells.

A simple, disposable, biphasic cultivation chamber has been developed for respiratory tract epithelial cells. This chamber, the Whitcutt chamber, contains a movable, transparent, permeable gelatin membrane that can be employed either submerged in the culture medium, thereby feeding the cells by the traditional immersion method, or raised to the surface of the culture medium, to bring the apical surfaces of the cells into contact with air and provide nutrients only from below (basal feeding). The effects of biphasic cultivation on the growth and differentiation of respiratory tract epithelial cells from different sources have been studied in Whitcutt chambers. Primary hamster tracheal epithelial (HTE) cells grown to confluence with basal feeding developed a ciliated columnar morphology, with differentiated features (cilia and mucous granules) located in the apical region of the epithelial layer. These cells secreted mucinlike molecules from the apical surface (i.e. the surface in contact with air). Although the apical localization of differentiation features was greater, mucous cell differentiation achieved by basal feeding was quantitatively not greater than that achieved by continuous immersion feeding. Similarly, basal feeding did not alter the degree of epithelial cell differentiation in cultures derived from rat, rabbit, and monkey tracheas or from human bronchial and nasal tissues. In contrast, the differentiation of guinea pig tracheal epithelial cells in culture was significantly influenced by the feeding method employed. When fed basally, guinea pig tracheal epithelial cell cultures expressed various mucociliary functions with resemblance to mucociliary layers in vivo, whereas constantly immersed cultures seemed stratified and squamous. These results suggest that, at least for guinea pigs, the combination of feeding methods provided by the Whitcutt chamber can be used to achieve differentiated cultures of tracheal epithelial cells with a polarity of differentiation that is similar to that observed in intact airways in vivo.

Cyclophosphamide-mediated enhancement of delayed hypersensitivity reactions in the lung.

The appearance of mononuclear cells, mast cells and mucous cells in the airways was studied in Balb/c mice in relation to cell-mediated immunity reflected as delayed hypersensitivity (DH). The animals were pretreated with cyclophosphamide (Cy) to increase cell-mediated immunity and to decrease the influence of antibodies. Mice pretreated with Cy and epicutaneously sensitized with picrylchloride (PiCl) had stronger DH reactions as compared with sensitized mice not pretreated with Cy. The Cy treatment decreased the IgG antibody formation after sensitization. The Cy-pretreated, sensitized and challenged mice had increased numbers of mucus-producing cells and to some extent also mononuclear cells in their lungs compared with sensitized and challenged non-Cy-treated animals. However, the mucous cell numbers were also increased in Cy-treated, but nonsensitized mice in which most of the airway epithelium had differentiated into mucus-containing cells. The present results indicate that local cell-mediated immunity involves the appearance of mononuclear cells and mucus-producing cells in the lung. This inflammatory reaction is enhanced by Cy treatment, although mucous cell differentiation seems to be induced by exposure to Cy alone.

Effects of elevated levels of cyclic AMP on the embryonic chick mandible in vitro

Mandibles were cultured in the presence of dibutyryl cyclic AMP (1 mM) and theophylline (1 mM) and in control medium. Controls differentiated normally but the timing of events differed from that in ovo and feathers did not form. With elevated intracellular cyclic-AMP levels, membrane bone did not form in the earlier stages tested and was inhibited or reduced in older ones; chondrogenesis was inhibited only in young explants (HH stage 20) and, in certain instances, it was enhanced. There was precocious and hyperplastic differentiation of mucous cells within oral epithelium but the aboral epithelium was unchanged.

Mechanisms for rapid re-epithelialization of the gastric mucosal surface.

Defects in the mucosal surface of the stomach are generally thought to be re-epithelialized by cells generated from proliferating stem cells in the neck region of the gastric glands. Regeneration by mitotic proliferation, however, would require at least 1-2 days for the migration and differentiation of the surface mucous cells (13, 21). It seems reasonable that there be a more rapid process of re-epithelialization to repair superficial defects incurred in daily living, especially because the gastric mucosal surface can be and is damaged by many common foods and drugs. The early recognition that the stomach could repair itself rapidly was by Grant (6). who in 1945 reported that the damage of the superficial third of the mucosa of cat stomachs exposed to 60% ethanol was repaired within 4 hr. In this early study the process was interpreted to be regeneration, but the cellular mechanism was not otherwise clarified. Morris & Wallace ( 15) recently proposed t�at the reestablishment of rat gastric mucosal epithelial continuity after ethanol injury occurred by migration of underlying epithelial cells from the gastric pits. In 1982, we described the cellular process of reconstitution in in vitro amphibian gastric mucosa mounted in an Ussing chamber, a process in which reestablishment of epithelial continuity occurred following destruction of the surface layer with hypertonic NaCl (22). More recently, gastric mucosal restitution has been identified in the in vitro guinea

Relation between components of the immune response in rats sensitized by aerosol and subcutaneous injections.

Relations between the appearance of various components of the immune response were analyzed in two groups of rats sensitized by aerosol and subcutaneous injections in the neck region, respectively. The relations were expressed by Spearman rank correlation coefficient and studied by cluster analysis. In the aerosol-sensitized animals, there was a close association between IgA and IgG antibody levels in bronchial fluid and these in turn were related to IgG levels in serum and more loosely to IgE levels in bronchial fluid. There was an apparent association between IgE antibody formation and mast cell maturation in cultures of regional lymph node cells and the appearance of mucous cells in the lungs. These variables seemed associated with spontaneous cell proliferation in vitro and the numbers of mast cells in the lungs. This indicates that local stimulation with antigen induces local immune responses and immune-mediated migration of cells. In subcutaneously sensitized animals, formation of IgG antibodies in vitro seemed related to the stimulated proliferation of regional lymph node cells. The levels of IgG and IgE antibodies in bronchial fluid and in serum also appeared to be related. Unlike the findings in aerosol-sensitized animals there was no apparent relation between the differentiation of mast cells and mucous cells. This was possibly due to lack of immune-mediated antigen-induced cell migration. The different immune response patterns in aerosol and subcutaneously sensitized rats should be considered when studies are designed aiming to explore the pathogenesis of allergic inflammatory diseases. The findings also indicate that the various parameters of immunity are more closely related in aerosol than in subcutaneously immunized animals.

Ultrastructure of well-differentiated adenocarcinomas of the lung with special reference to bronchioloalveolar carcinoma.

The cytologic phenotypes of 20 well-differentiated pulmonary adenocarcinomas were determined by electron microscopy. On examination of more than 100 cells in each case, the tumors were classified according to the predominant cell types. Nine cases (45%) were of mucous cell type, further divided into 7 cases of bronchial surface epithelial cell type, 1 case of bronchial gland cell type, and 1 case of metaplastic bronchiolar goblet cell type. The remainder included 5 cases (25%) of Clara cell type, 2 cases (10%) of type II cell type, and 4 cases (20%) of mixed cell type. The predominant histologic pattern by light microscopy was "typically" bronchioloalveolar (Manning et al.'s type 1) in the metaplastic goblet cell tumor and papillary in most Clara cell-type tumors, while it was glandular in bronchial surface and bronchial gland cell types, although variable in type II cell or mixed cell type. Therefore, bronchioloalveolar carcinomas, when histologically defined inclusive of papillary tumors, present cytologic phenotypes also related to the bronchioloalveolar epithelium, i.e., metaplastic goblet or Clara or type II cell subtypes, which is in accordance with some previous reports. These tumors could be distinguished from the other (glandular) adenocarcinomas that show primarily bronchial mucous cell differentiation.

Relation between mucous cells and lymphoid tissue in rat intrapulmonary airways.

The distribution of mucous cells in rat intrapulmonary airways was investigated by conventional light microscopic technique. A striking association was found between differentiation of epithelial mucous cells and accumulation of lymphoid tissue in the lamina propria of bronchioli. Nodular accumulations of lymphoid tissue, covered by a lymphoepithelium, as well as differentiation of mucous cells, increased with age. This normal development was accelerated in rats exposed to aerosolized ovalbumin.

Sensitizing ability of derivatives of picryl chloride after exposure of mice on the skin and in the lung.

In the present study the immunizing efficacy of antigen or hapten administered by the respiratory route and the resulting inflammatory reactions in the lungs after challenge were studied. We compared the induction of delayed hypersensitivity (DH) and the IgG, IgM, IgA, and IgE antibody formation after immunization with three different trinitrobenzene derivatives by the epicutaneous, the intradermal, and the intraperitoneal routes with the intranasal route. Mice sensitized with picryl chloride (PiCl) epicutaneously or intradermally, or with picryl sulfonic acid (PSA) epicutaneously showed strong DH responses. Moderate DH responses were obtained after sensitization with PSA intradermally and with trinitrophenylated human serum albumin (TNP-HSA) intraperitoneally. Intranasal administration of PSA and TNP-HSA also resulted in moderate DH responses. Strong IgG responses were seen in mice sensitized with PiCl and PSA epicutaneously, intradermally, and also intranasally. Administration of TNP-HSA epicutaneously, intradermally, and intraperitoneally also gave high IgG responses, while intranasal administration only induced moderate ones. Considerable IgM responses were seen only in mice sensitized epicutaneously with PSA. Antibody responses of the IgA isotype were only seen after epicutaneous sensitization with PiCl or PSA and intradermally with PiCl. Histological examination of lung tissue from mice sensitized with PiCl epicutaneously or with PSA intranasally and challenged with PSA intranasally revealed some inflammatory cells around bronchioli, capillaries and in the interstitial tissue. Differentiation of mucous cells in the epithelium covering inflammatory cells was also seen, and a slight increase in mast cell numbers around vessels. However, after sensitization with PiCl epicutaneously, TNP-HSA intranasally, and challenge with 2% TNP-HSA intranasally, a weaker inflammation was seen. In conclusion, intranasal administration of PSA or epicutaneous administration of PSA and PiCl gave strong DH and IgG responses. These sensitization regimes also resulted in pronounced inflammatory reactions in the lungs.

Histogenesis of the oropharyngeal and oesophageal mucosa as related to early feeding in rainbow trout, Salmo gairdneri Richardson.

The histogenesis of the oropharyngeal and oesophageal mucosa of rainbow trout, Salmo gairdneri, eleutheroembryos was studied from hatching to day 40 in relation to early feeding. Numbers of epithelial layers of the mucosa increased appreciably until day 20 but little thereafter. Differentiation of mucous cells was evident by day 10 and increased rapidly in number until about day 20. The first structurally distinct taste buds were observed in the oropharyngeal mucosa at day 8 and appeared by day 20 to be fully differentiated. An epithelial cell plug that occluded the upper alimentary canal and would have prevented passage of food into the stomach disappeared at about day 17. Teeth, although developing under the mucosa by day 3, had not yet erupted at day 22, the time of first feeding.It is suggested that the onset of first feeding in rainbow trout is in synchrony with the histogenesis of the oropharyngeal mucosa, notably the mucous cells and taste buds. The oropharyngeal mucosa of the fish that failed to feed by day 37 appeared to have undergone extensive hyperplasia while mucous cells were atypical, and taste buds seemingly deformed.

Ultrastructural and carbohydrate histochemical studies on the differentiation and renewal of mucous cells in the rat gastric fundus.

Gastric surface mucous cells (SMC), mucous neck cells (MNC) and their undifferentiated and immature precursors were studied by light and electron microscopic histochemistry. The secretory granules of SMC were smaller, more electron dense and more reactive to PAS and its analogues than those of MNC. Alcian blue demonstrated that the mucus of SMC was acidic and that of MNC was neutral. The periodic acid-thiocarbohydrazide-silver proteinate method revealed the pressence of carbohydrates in the golgi apparatus, condensing vacuoles, secretory granules, apical vesicles and tubules and cell coat. Maturation of SMC during their migration towards the free surface was reflected by an increase in size and number of secretory granules, an increase of RER and microfilaments, and a decrease of microvilli and apical vesicles and tubules. The secretory granules of older SMC were less acidic and possessed a proteinaceous core. Most MNC were fully differentiated, but some immature MNC containing only a few granules were found. Furthermore, undifferentiated cells and intermediates between SMC and MNC were also observed. The presence of both transitional and intermediate forms indicates that both SMC, and MNC arise from the same population of undifferentiated cells. Incorporation of 3H-thymidine revealed that undifferentiated cells, use isthmic SMC, MNC and intermediate cells are proliferative. No proliferative activity was found in foveolar SMC, parietal, chief, fibrillovesicular or endocrine cells.

Ciliogenesis in the mucous cells of the quail oviduct. II. Hormonal control

The hormonal control of ciliogenesis and transformation of mucous cells was studied in the oviduct (magnum) of ovariectomized quails. Estradiol benzoate induces ciliogenesis with doses varying from 10 mug/day to 100 mug/day after 6 days of treatment. With 100 mug/day, differentiation of some mucous cells is also induced as well as the formation of transitory "mixed cells" which are in the process of ciliogenesis and contain mucous granules. Associated with progesterone (1 mg/day), estradiol benzoate (10 mug/day) induces the differentiation of mucous cells and ciliated cells. The luminal epithelium of quails injected with this mixture is similar to the luminal epithelium observed in the oviduct of laying quails. With the same dose of progesterone (1 mg/day) and 20 mug/day of estradiol benzoate for 6 days, ciliogenesis is completely inhibited. All epithelial cells are secretory cells. Transformation of 50% of the mucous cells into ciliated cells is obtained by following the previous estradiol-progesterone treatment with the injection of estradiol benzoate (20 mug/day) for 3 days. Divisions of mucous cells were also observed. It is also possible to induce ciliogenesis in some mucous cells by withdrawing both hormones for 3 days. In this case, no cell divisions were observed.

Differentiation of immature mucous cells into parietal, argyrophil, and chief cells in stomach grafts.

Microscopic and submicroscopic studies on regenerating gastric mucosa neonatally grafted in the subcutaneous tissue of littermate mice have revealed that immature mucous cells are totipotent; ultimately they transform into mature mucous, parietal, argyrophil, and chief cells in the gastric glands.

The Effects of Vitamin A and Citral on Epithelial Differentiation in vitro 1. The Chick Tracheal Epithelium

ABSTRACT Differentiation of the tracheal epithelium of embryonic chicks was studied in organ cultures in a normal medium and in media containing added vitamin A and citral. In normal medium, the tracheal epithelium differentiated well, but usually fewer mucous cells developed than in the intact chick. High concentrations of vitamin A alcohol inhibited the differentiation of mucous cells and favoured that of ciliated cells. Citral, in fairly low concentrations, had opposite effects: it stimulated mucus-secretion and inhibited the differentiation of ciliated cells. In higher concentrations it stimulated division of the basal cells, and the epithelium became stratified and sometimes keratinized. Vitamin A and citral added together to the culture medium produced a citral effect, which varied in intensity according to the proportions of the two compounds, but was always milder than that resulting from treatment with the same concentration of citral alone. These results suggest that citral inhibits both the natural vitamin A in the plasma and the added synthetic vitamin A, thus producing changes in the tracheal epithelium characteristic of vitamin A deficiency. The results indicate that secretion of mucus by the tracheal epithelium is maximal at a lower concentration of vitamin A than that to which it is normally exposed in the body. It is suggested that vitamin A may influence epithelial differentiation partly by affecting the mitotic rate.