Human Tracheal Gland Research Articles

Retinoic acid and calcium regulation of p53, transforming growth factor-beta 1, and transforming growth factor-alpha gene expression and growth in adenovirus 12-SV40-transformed human tracheal gland epithelial cells.

Vitamin A and calcium are important regulators of growth and differentiation of epithelial cells and are intimately involved in preneoplastic and neoplastic transformation. It has been proposed that their effects are mediated by autocrine/paracrine positive and negative regulators of growth. The objectives of this investigation were to examine the effects of all-trans retinoic acid (RA) and Ca2+ on cell proliferation, anchorage-independent growth (AIG), and on the expression of transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta 1 (TGF-beta 1), and p53 tumor suppressor genes in human tracheal gland epithelial (HTGE) cells immortalized by adenovirus 12-simian virus 40 (Ad12-SV40) hybrid. Cells exhibiting the transformed phenotype, AIG, were maintained in serum-free culture conditions. Calcium effects were examined at 0.15, 0.50, 1.0, and 2.0 mM concentrations. The effects of RA were determined with 10(-9), 10(-7), and 10(-6) M concentrations. Gene expression was examined by Northern and Western analyses. Ca2+ had no significant effect on cell proliferation, but it enhanced the expression of TGF-beta 1 gene and slightly inhibited p53 expression. Ca2+ had no effect on TGF-alpha. RA inhibited both cell proliferation and AIG growth, which was accompanied by enhanced expression of p53. RA had no significant effect on the expression of TGF-alpha and TGF-beta 1 genes. These results demonstrate that RA regulates growth of HTGE cells mainly by upregulating the p53 gene; Ca2+, which enhances TGF-beta 1 expression, had no effect on growth.

Localization of the cystic fibrosis transmembrane conductance regulator in airway secretory glands

Cystic fibrosis (CF) is caused by mutations in the gene coding for the CF transmembrane conductance regulator (CFTR). From human normal tracheal submucosal gland cells in culture, we identified endogenous CFTR as a 170 kDa protein, consistent with that of fully glycosylated, mature CFTR molecule. This observation led to the hypothesis that airway secretory glands could be an important site for the CFTR expression. Using anti-human CFTR polyclonal and monoclonal antibodies, we examined the cellular and subcellular localization of the CFTR protein in airway submucosal glands from human and bovine tracheal tissues as well as in tracheal gland cell cultures. In human tracheal tissue, CFTR immunolabelling was present along both the apical and basolateral plasma membranes of glandular mucous cells. In contrast, CFTR was associated with the secretory granules of glandular serous cells. Using immunogold electron microscopy, we demonstrated that CFTR protein was more specifically associated with the membrane of serous cell secretory granules. In bovine tracheal tissue CFTR labelling was also identified in the secretory granules of glandular serous cells. In contrast, when bovine and human tracheal gland cells were cultured, no mature secretory granules were present, but a predominantly intracytoplasmic distribution of CFTR was observed. Our data thus suggest that in airway tissues, CFTR could be involved in intracellular processes of the mucus exocytosis in submucosal secretory glands.

Secretory proteins and glycoconjugates synthesized by human tracheal gland cells in culture.

To study the proteins and glycoconjugates synthesized by serous cells from human tracheal glands (HTG), isolated HTG cells were cultured in the presence of radiolabeled precursors 14C-proline, Na2(35)SO4, and 3H-fucose. The secretory 14C/35S/3H-radiolabeled proteins and glycoproteins, de novo synthesized by HTG cells, were analyzed by gel filtration chromatography. We observed the incorporation of 14C-proline into antileukoprotease and an unknown 30 kD protein, and the incorporation of 35SO4-- and 3H-fucose into high molecular weight glycoconjugates and sulfoconjugates (M(r) > 1,000,000) and into components with apparent M(r) of approximately 250 and 100 kD. After specific chemical and enzymatic treatment, the 35S- and 3H-glycoconjugates were shown to be -O-linked mucin-like glycoproteins and proteoglycans. These results show that cultured HTG cells synthesize some of the macromolecules identified in bronchial secretions.

Immortalization of human tracheal gland epithelial cells by adenovirus12-SV40 hybrid virus

Immortalization of human tracheal gland epithelial cells by adenovirus12-SV40 hybrid virus

Characterization of epithelial cell cultures derived from human tracheal glands

Cultures of normal human tracheal gland epithelial cells that exhibit functional differentiation have been propagated in serum-free medium supplemented with insulin (5 micrograms/ml), epidermal growth factor (10 ng/ml), hydrocortisone (0.5 micrograms/ml), and bovine pituitary extract (25 micrograms/ml). The cells retain many characteristics of epithelial cells including microvilli on cell surfaces, desmosomes between cells, and tonofilaments in the cytoplasm. In addition, they exhibit keratin-positive titers and react positively with Peanut agglutinin, which is specific for the disaccharide beta-D-galactose-(1----3)N-acetyl D-galactosamine, a major component of mucin glycoprotein. The cells also exhibit normal Cl- channel activity which was enhanced by the cAMP agonist Forskolin. The major component of the cellular secretion was hyaluronic acid; approximately 10% of the void volume material was resistant to hyaluronidase and may contain material similar to mucin glycoprotein. Some of the cell cultures have been maintained in serum-free conditions for 6 to 7 passages. This model will be important to study regulation of ion-channel activities and mucous glycoprotein secretion and to compare such regulations with the tracheal mucosal epithelial cells already established.

ヒト気管腺の加齢による変化

The histochemical and ultrastructural differences between young and aged human tracheal glands were studied. The materials were obtained at necropsy or during surgical procedures of the larynx and trachea. In human tracheal gland, remarkable differences between young (10 to 21-year-old) and aged (61 to 81-year-old) ones were especially seen in serous cells. Mucosubstances of young tracheal serous cells were classified as serum glycoprotein types, but those of the aged cells were mainly mucin types. Ultrastructurally, young tracheal serous cells contained homogeneously electron dense or biphasic secretory granules. On the other hand, the aged cells contained not only homogeneous electron dense granules but also multiform ones, hiphasic or electron lucent, resembling mucous cell granules. In aged human tracheal serous cells, alterations of histochemical and ultrastructural properties of secretory materials reflected depression of host defence mechanism of the trachea. By these results, the reason why various airway deseases increase with aging could be explained. In aged human, in addition, oncocytes were found within the mucous tubule and they occasion ally contained secretory granules. Although appearance of oncocytes in the tracheal gland was closely related with aging, this phenomenon seemed not to directly participate in depres sion of the defence mechanism.

シンポジウム ２ 高齢者の喉頭，下気道の生理と病態 Ａ． 加齢に伴う喉頭・気管の変化

シンポジウム ２ 高齢者の喉頭，下気道の生理と病態 Ａ． 加齢に伴う喉頭・気管の変化

Production of lipocortin-like proteins by cultured human tracheal submucosal gland cells

Evidence is obtained for the presence of lipocortin-like proteins in human tracheal gland cells in culture. Using polyclonal antibodies to lipocortin I. indirect immunofluorescence studies demonstrate that lipocortin I is mainly confined to the tracheal gland cell surface. From cell membranes, four Ca 2+-dependent proteins (35, 40, 45 and 67 kDa) were identified as lipocortin related proteins by using immunoblotting and fluorography following [ 35S]methionine metabolic labeling experiments. A strong immunoreactivity for the 35 kDa protein was observed. In addition, lipocortinlike proteins with apparent M r33, 35, 37 and 67 kDa, respectively, were released in the apical culture medium by trachéal gland cells cultured on microporous membrane of a double chamber culture system.

Culture and Characterization of Human Tracheal Gland Cells

In order to study the composition and regulation of human tracheal gland (HTG) cell secretion, we cultured HTG cells isolated by enzymatic digestion from tracheal mucosa obtained 30 to 60 min after death. On microscopic observation, isolated cells were mainly composed of secretory glandular cells. Maximal HTG cell growth was observed when cells were cultured on type I collagen in the presence of 2% Ultroser G. Under these conditions, 3 to 6 HTG cell passages, corresponding to 20 to 30 population doublings, could be achieved. Lysozyme and bronchial inhibitor (Brl), two secretory protein markers specific to the serous HTG cells, were released in the culture medium, maximal secretion being observed 7 days after the cells had reached confluency. At that time, Brl could be detected, with an immunoperoxidase technique, in about 90% of the cells in culture, suggesting that most cells in culture were serous cells. Using transmission electron microscopy, after in situ fixation, HTG cells exhibited an epithelioid appearance at confluency. Using the biotin-streptavidin gold technique, we identified Brl in cytoplasmic vesicles and in small, immature electron-dense secretory granules. In high cell density cultures, we observed dome formation, suggesting active ion transport mechanisms in HTG cell culture. At confluency, a dose-dependent increase of Brl secretion was induced by phenylephrine, isoproterenol, and carbochol. These results suggest that HTG cell culture provides a useful tool to study the biochemistry and regulation of human tracheobronchial gland cell secretion.

Immunocytochemical demonstration of quantitative differences in the distribution of lysozyme in human airway secretory granule phenotypes

The distribution of lysozyme in the different secretory granules (SG) of human tracheal and bronchial submucosal gland serous cells was studied by light and electron microscopy, using a post-embedding immunogold technique. SG were differentiated into 5 phenotypes according to their structure and staining electron density. All the SG-phenotypes were reactive to lysozyme. In the heterogeneous SG-phenotypes, quantitative immunocytochemistry showed that the density of lysozyme labeling was significantly higher in the electron-dense central core compared to the electron-lucent peripheral rim. At the tracheal level, the density of lysozyme did not vary significantly within the different SG-phenotypes, whereas at the bronchial level, the differences were significant. Moreover, the lysozyme labeling density was much higher in the bronchial than in the tracheal SG.

Human Tracheobronchial Submucosal Gland Cells in Culture

Cellular mechanisms regulating airway secretion, secretory products of individual airway cell types, and control of airway cell growth and differentiation are poorly understood. In order to aid studies of these questions, we have established a system for culturing human tracheobronchial submucosal gland cells. Gland acini were isolated by enzymatic disaggregation from submucosal tissue obtained postmortem from patients without pulmonary diseases and from patients with cystic fibrosis. In culture, acini attached to a collagen substratum, and gland cells proliferated and formed confluent monolayers which were homogeneous by phase microscopy. In contrast to cells of freshly disaggregated acini which expressed either serous or mucous gland cell secretory antigens, in culture virtually all cells (greater than or equal to 95%) concurrently expressed both antigens as assessed by immunocytochemical staining with serous and mucous cell-specific antibodies. Similarly, electron microscopy revealed cells with serous- or mucous-type secretory granules, and cells containing both types of granules. Cultures incorporated 35S into high (greater than 10(6) D) and lower (greater than 700 kD; 150 kD) molecular weight molecules. Cholinergic and adrenergic agonists increased release of radio-labeled secretions. These findings demonstrate that human tracheal gland cells in culture retain immunocytochemical, ultrastructural, and functional features of both differentiated serous and mucous gland cells. This culture system will be useful for studying the biology and pathology of human tracheobronchial submucosal gland cells.

Light-microscopic examination of ABH and Lewis antigens in human tracheal and epiglottic glands using the avidin-biotin-peroxidase complex technique.

The localization of ABH and Lewis antigens was examined in formalin-fixed, paraffin-embedded human tracheal and epiglottic glands using monoclonal anti A, B, H, Lea and Leb antibodies. The mucous cells of the glands showed reactivity with antibodies corresponding to the respective ABO blood groups of the tissue donors. The mucous cells from one blood group A, Le(a-b-) individual showed no reactivity with any antibodies and those from another blood group A, Le(a-b-) individual showed reactivity only with anti A antibody. In individuals from blood group Le(a + b-) of all ABO groups, the mucous cells reacted exclusively with anti Lea. In blood group O, Le(a-b+) individuals, the mucous cells showed intense reaction with anti H and Leb antibodies and weak to moderate reactivity with anti Lea. In Le(a-b+) individuals of A1, B and A1B blood groups, the mucous cells showed strong reactivity with anti A and/or B antibodies, moderate with anti Leb, weak or no activity with anti Lea and absent with anti H. In blood group A2 Le(a-b+) individuals, the mucous cells stained with anti A were weakly stained or completely unstained with anti H antibody, but cells negative with anti A gave strong positive reactions with anti H antibody.

Culture of human tracheal gland cells : Ultrastructural characterization and secretory products

Culture of human tracheal gland cells : Ultrastructural characterization and secretory products

Electron microscope studies of human tracheal glands, with special reference to oncocytes and oncocytic cells

Twenty-nine cases of human tracheal glands were studied in electron microscopy. The materials used in this study were taken from patients whose ages ranged from 1 to 90 years old of both sexes.The tracheal glands were composed of occasionally branched tubulo-alveolar glands consisting of the duct and the terminal portion of both mucous and serous type, and furthermore, smoothmuscle type myoepithelial cells surrounding the outside of the terminal portions.Both oncocytes and oncocytic cells appeared everywhere in the gland, both in the duct and also in the terminal portion epithelium. They were generally seen in a cluster, but sometimes in solitary form within the glandular epithelium.Oncocytes were tightly packed with an unusually large number of atypical hypertrophied mitochondria. On the other hand, the oncocytic cells contained variable numbers of such atypical large mitochondria in addition to usual cell organelles. These findings in the oncocytic cells are strongly suggestive that the oncocytic cell is a transitional type cell between the normal glandular cell and the typical oncocyte within the gland. Atypical mitochondria in such cells were almost devoid of intramitochondrial granules, and had conspicuously larger sizes than those of usual ones.These atypical hypertrophied mitochondria were often provided with tightly packed bundles of longitudinally or arcuately arranged cristae, as well as a dilated matrix with either dense globules or crystalloid inclusions. The cells containing such peculiar mitochondria had to some extent similar characteristics to those of the usual glandular cells. However, in this study no myoepithelial cells exhibited any morphological signs of transforming into oncocytes or oncocytic cells.Increased numbers of oncocytes and oncocytic cells tended to be seen more frequently in aged individuals, and thus this oncocytic degeneration of the gland may be considered as one of the changes due to aging within the glandular tissues of man.In some cases, a few annulate lamellae connected to the limiting membrane of the rough endoplasmic reticulum were observed, however, their functional significance remains obscure.

Presence of salivary protein C and salivary peptide P-C-like immunoreactivity in the laryngo-tracheo-bronchial glands.

An indirect immunofluorescence technique using antisera against salivary peptide P-C and against salivary Protein C was carried out on the laryngeal, tracheal and bronchial glands to examine whether salivary peptide P-C-like immunoreactivity, recently demonstrated in the serous cells of the human salivary glands, was also present in those of laryngeal, tracheal and bronchial glands and to ascertain whether salivary peptide P-C is a fragment of salivary Protein C or not. Salivary peptide P-C-like immunoreactivity was present in the serous cells of the human laryngeal, tracheal and bronchial glands. Observation of serial sections immunostained with two kinds of antisera revealed that cells reacting with antisera against salivary peptide P-C were identical to those reacting with antisera against salivary Protein C pre-incubated with salivary peptide P-C. The finding implied that salivary peptide P-C and salivary Protein C, originally isolated from human saliva, were also present in the serous cells of tissues other than the salivary glands. Furthermore, analysis of the primary structure of salivary peptide P-C and salivary Protein C together with the present morphological finding suggests that salivary peptide P-C is a COOH-terminal fragment of salivary Protein C. Thus, salivary Protein C and salivary peptide P-C may play some role in the function of the serous cells of the salivary and laryngo-tracheo-bronchial glands.

Light microscopic histochemical detection of sugar residues in secretory glycoproteins of rodent and human tracheal glands with lectin-horseradish peroxidase conjugates and the galactose oxidase-Schiff sequence.

Lectins conjugated to horseradish peroxidase were used to stain paraffin sections of mouse, rat, and human respiratory tract tissues. An additional method was applied utilizing galactose oxidase to oxidize the C-6 hydroxyl of galactose and N-acetylgalactosamine residues and the resulting aldehyde was visualized with 2% Schiff's reagent. Sections were stained prior to and after removal of sialic acid residues. Oligosaccharides with terminal beta-galactose and alpha-N-acetylgalactosamine residues were found in all serous cells in the mouse trachea but were never seen in human tracheal serous cells. About 5-10% of serous cells in the rat trachea contained terminal beta-galactose, whereas all human tracheal serous cells and the remainder of those in the rat contained oligosaccharides with terminal sialic acid and penultimate beta-galactose residues. Fucose was not detected in tracheal serous cells of any species. Mucous cells in the mouse and rat trachea produced heterogeneous oligosaccharides containing terminal alpha-N-acetylgalactosamine and/or terminal sialic acid residues in various proportions. The structure of oligosaccharides in human tracheal mucous cells varied between individuals and was related to ABO blood group reactivity. The majority of oligosaccharides in type A individuals contained terminal alpha-N-acetylgalactosamine, whereas type AB individuals had approximately equal amounts of terminal alpha-galactose and alpha-N-acetylgalactosamine residues. Mucous cells in the two type O specimens examined contained a large amount of terminal beta-galactose and surprisingly, terminal fucose was not detected in these individuals. These results support biochemical studies showing structural diversity in oligosaccharide chains of respiratory tract secretions and reveal differences in glycoprotein secretion of different cell types.

Electron microscopic studies on human tracheal glands (author's transl)

An electron microscopic study on fine structure of the human tracheal glands, on which few reports have been published, was carried out.The materials were obtained duing surgical procedures of the larynx and trachea.The human tracheal gland mainly locates in submucosa of the trachea.The shape of gland is simple or compound tubulo-alveolar, and the gland has a duct and a secretory portion(acinus).The secretory portion of the gland comprise of mucous cells and serous cells consistently in a regular arrangement.The mucous cells adjoin directly distal to the branched duct and followed by serous cells.Serous cells form frequently a deanilune.The secretory portion of the gland is surrounded by myoepithelial cell sresembling smooth musclefiber, and they are encased further by metnbrana propria comprised of a basement membrane and connective tissue.The duct of the tracheal gland has double layers of epithelial cells, viz.tall columnar cells facing the lumen and basal cells outside.The tall columnar cell frequently contains round granules suggestive of anactive secretory function.In our observation ultrastructural characteristics of the unicous cells and of the serous cells are investigated separately, and the mechanism of secretion is morphologically elucidated through the electron microscopic investigation of their morphological changes corresponding to the secretory cycle.No appreciable morphological difference exists between the serous cells constituting serous tubules and demilune.Mitochondria-rich cells(oncocytes)are occasionally found within the epithelium of the secretory portion and in a rare instance they contained secretory granules.Several findings on myoepithelial cells resembling smooth muscle fiber which are found in a considerable number at the base of the glandular cells of the secretory portion are described.

The effect of pH on Alcian Blue staining of epithelial acid glycoproteins. I. Sialomucins and sulphomucins (singly or in simple combinations)

This study is concerned with the staining of epithelial acid glycoproteins by Alcian Blue at various pH levels. A detailed analysis of the effect of pH on Alcian Blue staining of epithelial tissues at selected sites was made. Alcian Blue was combined with the periodic acid-Schiff technique, the Alcian Blue being used at pH levels between 2.6 and 0.5.Animal salivary glands, human foetal tracheal gland and epithelial goblet cells of the adult bronchial mucosa were chosen for study because the nature of their acid glycoprotein is known and is relatively simple.

HISTOLOGICAL AND CYTOLOGICAL STUDIES ON HUMAN TRACHEAL GLANDS

HISTOLOGICAL AND CYTOLOGICAL STUDIES ON HUMAN TRACHEAL GLANDS