Paul Nettesheim

Rat tracheal epithelial cell differentiation in vitro

SummaryIn vitro culture conditions enabling rat tracheal epithelial (RTE) cells to differentiate to mucociliary, mucous, or squamous phenotypes are described. Medium composition for rapid cell growth to confluence in membrane insert cultures was determined, and the effects of major modifiers of differentiation were tested. Retinoic acid (RA), collagen gel substratum, and an air-liquid interface at the level of the cell layer were required for expression of a mucociliary phenotype which most closely approximated the morphology of the tracheal epithelium in vivo. Large quantities of high molecular weight, hyaluronidase-resistant glycoconjugates, most likely mucin glycoproteins, were produced in the presence of RA when the cells were grown with or without a collagen gel and in submerged as well as in interface cultures. However, extensive ciliagenesis was dependent on the simultaneous presence of RA, collagen gel, and an air-liquid interface. When RA was omitted from the media, the cells became stratified squamous and developed a cornified apical layer in air-liquid interface cultures. This phenotype was accompanied by loss of transglutaminase (TGase) type II and keratin 18 and expression of the squamous markers TGase type I and keratin 13. The ability to modulate RTE cell phenotypes in culture will facilitate future studies investigating molecular regulation of tracheal cell proliferation, differentiation, and function.In vitro culture conditions enabling rat tracheal epithelial (RTE) cells to differentiate to mucociliary, mucous, or squamous phenotypes are described. Medium composition for rapid cell growth to confluence in membrane insert cultures was determined, and the effects of major modifiers of differentiation were tested. Retinoic acid (RA), collagen gel substratum, and an air-liquid interface at the level of the cell layer were required for expression of a mucociliary phenotype which most closely approximated the morphology of the tracheal epithelium in vivo. Large quantities of high molecular weight, hyaluronidase-resistant glycoconjugates, most likely mucin glycoproteins, were produced in the presence of RA when the cells were grown with or without a collagen gel and in submerged as well as in interface cultures. However, extensive ciliagenesis was dependent on the simultaneous presence of RA, collagen gel, and an air-liquid interface. When RA was omitted from the media, the cells became stratified squamous and developed a cornified apical layer in air-liquid interface cultures. This phenotype was accompanied by loss of transglutaminase (TGase) type II and keratin 18 and expression of the squamous markers TGase type I and keratin 13. The ability to modulate RTE cell phenotypes in culture will facilitate future studies investigating molecular regulation of tracheal cell proliferation, differentiation, and function.

Growth and differentiation of hamster tracheal epithelial cells in culture.

The purpose of these studies was to define culture conditions that support growth and differentiation of normal epithelial cells obtained from hamster tracheas. Epithelial cells from tracheas of adult hamsters were collected using enzymatic procedures and cultured under various conditions. The medium used consisted of a 1:1 mixture of medium 199 and Dulbeccos modified Eagles medium with 2% fetal bovine serum, which was conditioned by mouse 3T3 cells before use. Insulin, transferrin, hydrocortisone, epidermal growth factor, and an extract from bovine hypothalamus were used as supplements. When seeded on uncoated or collagen-coated tissue culture dishes, the hamster cells grew only poorly. When the cells were seeded on collagen gels, however, rapid and prolonged growth ensued. The cultures had a population doubling time of 20 hr and a colony-forming efficiency of 7-10%, and they could be grown for up to three passages. Growth was dependent on the presence of transferrin, insulin, epidermal growth factor, and 3T3 conditioning factors in the medium. The latter could be omitted if the concentration of serum was increased. Less important for growth was the presence of hydrocortisone and bovine hypothalamus extract. In contrast to results with tracheal epithelial cells from adult rabbits, rats, and mice, differentiation into ciliated cells regularly occurred in cultures of cells derived from hamster tracheas. The appearance of ciliated cells in the cultures was dependent on the presence of collagen gel as a substratum and of 3T3 conditioning factors in the medium. In addition, there were numerous cells that contained electron-dense cytoplasmic granules. The granules were not stained by dialyzed iron, which stains acidic glycoproteins, but were stained positively by periodic acid-Schiff reagents and the periodic acid-thiocarbohydrazide-silver proteinate method, suggesting the presence of secretory granules containing neutral glycoproteins. A similar staining pattern was observed for the secretory granules of intact hamster tracheas. The culture system described supports growth and cellular differentiation of normal tracheal epithelial cells of hamsters. We believe therefore that it will be a useful model for studying the regulation of tracheal cell function on the cellular and biochemical level.

Regulation of mucous differentiation and mucin gene expression in the tracheobronchial epithelium

The goal of our studies is to elucidate mechanisms that control and modulate mucous differentiation and mucin gene expression in the conducting airways. We used cultures of normal human tracheobronchial epithelial (NHTBE) cells that were shown to secrete two major airway mucins, namely MUC5AC and MUC5B as well as several other secretory products. Mucous differentiation and expression of MUC2, MUC5AC, MUC5B and MUC7, but not MUCi, MUC4, and MUC8 mucin genes, were shown to be retinoic acid- (RA) or retinol-dependent. We found that RA control of mucin genes was mediated by the retinoid acid receptors RAR alpha and, to a lesser extent, by RAR gamma. Our studies also showed that other important bioregulators such as thyroid hormone (T3) and epidermal growth factor (EGF) modulate basal expression of mucin genes, interacting with RA in a concentration-dependent manner. T3, which binds to thyroid receptors (TRs) belonging to the same superfamily of steroid hormone nuclear receptors as the RARs, inhibits mucin gene expression, particularly MUC5AC. One possible mechanism of this T3 effect is downregulation of RAR proteins, which are critical for mucin gene expression. However, we also found that T3 inhibits MUC5AC transcription.EGF, which had previously been shown to stimulate mucin expression and mucin secretion in cultured rat tracheal epithelial (RTE) cells, inhibited mucin secretion in human bronchial epithelial cell cultures. This effect was EGF concentration- and time-dependent and was progressively abolished by increasing the RA concentration. Subsequent studies suggested that the inhibitory effects of high concentrations of EGF may result from selective reduction of MUC5AC expression. These studies thus point to potentially important species differences in the mechanisms regulating mucous production, and they also confirm previous findings indicating differential regulation of MUC5AC and MUC5B gene expression.

Distribution of Nonciliated Bronchiolar Epithelial (Clara) Cells in Intra- and Extrapulmonary Airways of the Rabbit

Three types of nonciliated secretory epithelial cells are thought to contribute to the mucous lining of intrapulmonary airways: mucous cells, serous cells, and Clara cells. The Clara cell is distinguished from the other two by low cuboidal shape, presence of membrane-bound electron-dense ovoid secretory granuLes, abundant apical agranular endoplasmic reticulum (AER), and its location as the lining cell of distal conducting airways (bronchioles). The present study was designed to define the distribution of the Clara cell within the trachea and intrapulmonary airway tree. We correlated airway size, generation of branching, and airway wall components with Clara cell morphology and abundance. Lungs of 6 male rabbits (8-15 weeks old) free of respiratory disease were fixed by airway infusion of glutaraldehyde/paraformaldehyde at 30 cm pressure. The trachea, primary bronchi, and airways of the right cranial lobe of each lung were exposed by microdissection. The branching pattern was recorded and numbered. One half of the dissected lung was processed for scanning electron microscopy and the other half embedded as large epoxy blocks for simultaneous transmission electron and light microscopy. The percentages of ciliated, mucous, nonciliated, and basal epithelial cells were quantitated by light microscopy. Basal cells and peribronchial cartilage were absent distal to the fourth intrapulmonary generation in both the axial airway and its first major branch. Mucous (goblet) cells were present (less than 2%) only in trachea and primary and lobar bronchi. Nonciliated cells were more abundant distally, constituting 21%-45% of the epithelium in cartilaginous airways and 35%-65% in noncartilaginous ones. In the first six airway generations including trachea, nonciliated cells were without large apical protrusions but were covered by long microvilli. Cells of more distal airways had prominent apical protrusions and lacked microvilli. Nonciliated cells in all airways had abundant apical AER, granular endoplasmic reticulum (GER), and electron-dense ovoid granules. Cells in proximal airways were columnar rather than low cuboidal and had more GER. Only nonciliated cell shape, luminal surface, and granule abundance varied with airway size and generation. We concluded that the nonciliated secretory cell lining all intrapulmonary airways in the rabbit lung is one cell type, the Clara cell.

Regulation of 15-lipoxygenase expression and mucus secretion by IL-4 in human bronchial epithelial cells

Our laboratory has recently shown that mucus differentiation of cultured normal human tracheobronchial epithelial (NHTBE) cells is accompanied by the increased expression of 15-lipoxygenase (15-LO). We used differentiated NHTBE cells to investigate the regulation of 15-LO expression and mucus secretion by inflammatory cytokines. Interleukin (IL)-4 and IL-13 dramatically enhanced the expression of 15-LO, whereas tumor necrosis factor-α, IL-1β, and interferon (IFN)-γ had no effect. These cytokines did not increase the expression of cyclooxygenase-2, with the exception of a modest induction by IL-1β. The IL-4-induced 15-LO expression was concentration dependent, and mRNA and protein expression increased within 3 and 6 h, respectively, after IL-4 treatment. In metabolism studies with intact cells, 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid (13-HODE) were the major metabolites formed from exogenous arachidonic acid and linoleic acid. No prostaglandins were detected. IL-4 treatment dramatically increased the formation of 13-HODE and 15-HETE compared with that in untreated NHTBE cells, and several additional 15-LO metabolites were observed. Pretreatment of NHTBE cells with IFN-γ or dexamethasone did not inhibit the IL-4-induced expression of 15-LO except at high concentrations (100 ng/ml of IFN-γ and 10 μM dexamethasone). IL-4 treatment inhibited mucus secretion and attenuated the expression of the mucin genes MUC5AC and MUC5B at 12-24 h after treatment. Addition of 15-HETE precursor and 13-HODE precursor to the cultures did not alter mucin secretion or mucin gene expression. On the basis of the data presented, we conclude that the increase in 15-LO expression by IL-4 and attenuation of mucus secretion may be independent biological events.Our laboratory has recently shown that mucus differentiation of cultured normal human tracheobronchial epithelial (NHTBE) cells is accompanied by the increased expression of 15-lipoxygenase (15-LO). We used differentiated NHTBE cells to investigate the regulation of 15-LO expression and mucus secretion by inflammatory cytokines. Interleukin (IL)-4 and IL-13 dramatically enhanced the expression of 15-LO, whereas tumor necrosis factor-alpha, IL-1beta, and interferon (IFN)-gamma had no effect. These cytokines did not increase the expression of cyclooxygenase-2, with the exception of a modest induction by IL-1beta. The IL-4-induced 15-LO expression was concentration dependent, and mRNA and protein expression increased within 3 and 6 h, respectively, after IL-4 treatment. In metabolism studies with intact cells, 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid (13-HODE) were the major metabolites formed from exogenous arachidonic acid and linoleic acid. No prostaglandins were detected. IL-4 treatment dramatically increased the formation of 13-HODE and 15-HETE compared with that in untreated NHTBE cells, and several additional 15-LO metabolites were observed. Pretreatment of NHTBE cells with IFN-gamma or dexamethasone did not inhibit the IL-4-induced expression of 15-LO except at high concentrations (100 ng/ml of IFN-gamma and 10 microM dexamethasone). IL-4 treatment inhibited mucus secretion and attenuated the expression of the mucin genes MUC5AC and MUC5B at 12-24 h after treatment. Addition of 15-HETE precursor and 13-HODE precursor to the cultures did not alter mucin secretion or mucin gene expression. On the basis of the data presented, we conclude that the increase in 15-LO expression by IL-4 and attenuation of mucus secretion may be independent biological events.

OVEREXPRESSION OF MUCIN GENES INDUCED BY INTERLEUKIN1-1β, TUMOR NECROSIS FACTOR-α, LIPOPOLYSACCHARIDE, AND NEUTROPHIL ELASTASE IS INHIBITED BY A RETINOIC ACID RECEPTOR α ANTAGONIST

Proinflammatory cytokines, lipopolysaccharide (LPS), and neutrophil elastase (NE) have been implicated in the induction of hypersecretion of respiratory mucus. In this study, we demonstrated that interleukin-1 β (IL-1 β) increased MUC2 and MUC5AC mRNA levels 2- to 3-fold in a time- and dose-dependent manner in NCI-H292 cells. In contrast, MUC5B mRNA was not significantly changed. A transcription inhibitor blocked the stimulation of MUC2 and MUC5AC gene expression by IL-1 β. A translation inhibitor did not interfere with the induction of MUC2 mRNA expression, whereas stimulation of MUC5AC mRNA was blocked, suggesting de novo protein synthesis is required for the stimulation of MUC5AC mRNA. We previously reported that induction of MUC2, MUC5AC, and MUC5B gene expressions by retinoic acid is mediated by the retinoic acid receptor (RAR α) , and inhibited by the specific RAR α antagonist Ro 41-5253. Here, we demonstrate that the RAR α antagonist can effectively inhibit IL-1 β -induced MUC2 and MUC5AC gene expression and reduce intracellular MUC5AC protein. Further investigation showed that the RAR α antagonist also inhibited the stimulation of MUC2 and MUC5AC mRNA expression by tumor necrosis factor- α, LPS, and NE.

Biosynthesis of prostaglandins by isolated and cultured airway epithelial cells

The metabolism of arachidonic acid to prostaglandins and thromboxane by freshly isolated and cultured respiratory tract epithelial cells was examined by HPLC methods. Homogenates prepared from freshly isolated rat and rabbit tracheal epithelial cells did not convert arachidonic acid to prostaglandins. Rat tracheal epithelial cells however, did convert arachidonic acid to uncharacterized metabolites possibly hydroxyfatty acids. In contrast, rat tracheal epithelial cells grown in culture for 9 days acquired the capacity to convert arachidonic acid to PGE2 and related products while cultured rabbit tracheal epithelial cells converted arachidonic acid to TXB2. The conversion of arachidonic acid to PGE2 by rat tracheal cells was studied at various times in culture. The formation of PGE2 appeared to parallel the growth of the cultures. In contrast to freshly isolated rat tracheal cells, enriched rat Clara cell fractions were able to convert 14C-arachidonic acid to prostacyclin (PGI2) ans measured by HPLC analysis of its stable end product 6-keto PGF1 alpha. PGI2 was also the major metabolite of arachidonic acid produced by enriched rat alveolar type II cell fractions. PGF2 alpha, and hydroxyfatty acids were also formed. These results suggest that arachidonic acid metabolism differs in various types of respiratory tract cells and that maintenance of such cells in culture alters the pattern of arachidonic acid metabolism.

Inhibition of Ciliated Cell Differentiation by Fluid Submersion

Rat tracheal epithelial (RTE) cells, plated at low density on collagen gel-coated membranes, differentiate into a mucociliary epithelium when cultured at an air-liquid interface (ALI). However, when RTE cells are cultured submerged in media, ciliated cell differentiation is drastically reduced. This study examined possible mechanisms for the inhibition of ciliated cell differentiation by submersion. Ciliated cell differentiation was measured using a monoclonal antibody specific for rat ciliated cells. Removing growth stimulatory compounds from both the basal and apical media increased ciliated cell differentiation in submerged cultures, indicating that submersion inhibits, but does not prevent, ciliogenesis. However, the effect of submersion was independent of the composition of the apical media. The depth of apical fluid was important, with depths > or = 1 mm causing almost complete inhibition of ciliated cell differentiation, while a depth of 0.5 mm allowed significant ciliogenesis. Submersion appeared to block ciliated cell differentiation at an early step, because ciliated cell development required several days following creation of an ALI. Once ciliogenesis was initiated in ALI cultures, submersion did not reverse or inhibit the development off ciliated cells. These studies have provided new information on the inhibition of ciliated cell differentiation by fluid submersion.

Cell type-specific lectin staining of the tracheobronchial epithelium of the rat: Quantitative studies with Griffonia simplicifolia I isolectin B4

We compared lectin staining patterns to cell population densities, as determined by morphological criteria in rat airways. Eight lectins were studied: Griffonia simplicifolia I isolectin B4 (GSI-B4), Arachis hypogaea (PNA), Wisteria floribunda (WFA), Glycine maximus (SBA), Dolichos biflorus (DBA), Helix pomatia (HPA), Ulex europaeus (UEA-1), and Maclura pomifera (MPA). Two of the lectins strongly stained morphologically distinct cell subpopulations. GSI-B4 stained basal cells, and MPA stained non-ciliated bronchiolar (Clara) cells. The specificity and sensitivity of GSI-B4 as a marker for basal cells was examined. In the trachea, 35% of all cells were GSI-B4 positive; 84% of these were basal cells, 7% were unidentified cells, 5% were serous/mucous cells, and 4% were ciliated, brush, or inflammatory cells. Comparison to cell population density data strongly suggested that all basal cells were GSI-B4 positive. The segmental bronchus was a transitional area; GSI-B4 positive basal cells were present in the region closest to the lobar bronchus but were absent in the distal region; instead, MPA-positive Clara cells appeared. When dissociated tracheal cells were obtained by pronase digestion, 43% were GSI-B4 positive. These results show that GSI-B4 is a sensitive and relatively specific marker for basal cells in the rat trachea which can be used to study dissociated epithelial cells.

Repopulation of Denuded Tracheas by Clara Cells Isolated from the Lungs of Rabbits

An experimental approach was examined to study the growth and differentiation potential of different epithelial cell types isolated from the airways of adult rabbits. Clara cells isolated from the lungs of rabbits and a mixed cell population obtained from rabbit tracheas were injected (separately) into denuded rat tracheas which were then grafted into nude mice. Epithelial cells were obtained from rabbit tracheas by digestion of tracheal epithelium with proteases. The resulting cell suspension contained mucous cells, Claralike cells, ciliated cells, basal cells and a small number of inflammatory and unidentified cells. This mixed cell suspension was inoculated into rat tracheas denuded of their own epithelium which were then grafted subcutaneously onto the backs of nude mice. The tracheas were recovered two weeks later and were prepared for light and electron microscopy. These grafts were found to be lined by a tall columnar pseudostratified mucociliary epithelium, indicating that the rat tracheal grafts transplanted into nude mice supported the growth and differentiation of tracheal epithelial cells. Similarly Clara cells, isolated and purified to 85.9 +/- 2.8% (n = 9) purity from rabbit lungs by a combination of centrifugation and elutriation procedures, were inoculated into rat tracheas which were transplanted into nude mice. Two weeks later the grafts were recovered and prepared for histological and ultrastructural examination. These tracheas were lined with a low cuboidal epithelium reminiscent of bronchiolar epithelium. The epithelium was composed of ciliated cells and Claralike cells rich in smooth endoplasmic reticulum but containing only a few secretory granules. These results indicate that the tracheal graft model is suited to examine the differentiative potential of specific cell types isolated from the airways. Furthermore, they show that Clara cell isolates are able to establish an epithelium resembling bronchiolar epithelium in denuded tracheas while tracheal cells containing at least four different epithelial cell types are able to establish a mucociliary epithelium resembling the pseudostratified tracheal lining.