Rat tracheal epithelial cell differentiation in vitro

Summary 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.     

Identification of an apical Cl-/HCO3- exchanger in gastric surface mucous and duodenal villus cells

The molecular identity of the apical HCO3(-)-secreting transporter in gastric mucous cells remains unknown despite its essential role in preventing injury and ulcer by gastric acid. Here we report the identification of a Cl-/HCO3- exchanger that is located on apical membranes of gastric surface epithelial cells. RT-PCR studies of mouse gastrointestinal tract mRNAs demonstrated that this transporter, known as anion exchanger isoform 4 (AE4), is expressed in both stomach and duodenum. Northern blot analysis of RNA from purified stomach epithelial cells indicated that AE4 is expressed at higher levels in mucous cells than in parietal cells. Immunoblotting experiments identified AE4 as a approximately 110- to 120-kDa protein in membranes from stomach epithelium and apical membranes from duodenum. Immunocytochemical staining demonstrated that AE4 is expressed in apical membranes of surface cells in both mouse and rabbit stomach and duodenum. Functional studies in oocytes indicated that AE4 functions as a Cl-/HCO3- exchanger. These data show that AE4 is an apical Cl-/HCO3- exchanger in gastric mucous cells and duodenal villus cells. On the basis of its function and location, we propose that AE4 may play an important role in mucosal protection.     

Murine nasal septa for respiratory epithelial air-liquid interface cultures

Air-liquid interface models using murine tracheal respiratory epithelium have revolutionized the in vitro study of pulmonary diseases. This model is often impractical because of the small number of respiratory epithelial cells that can be isolated from the mouse trachea. We describe a simple technique to harvest the murine nasal septum and grow the epithelial cells in an air-liquid interface. The degree of ciliation of mouse trachea, nasal septum, and their respective cultured epithelium at an air-liquid interface were compared by scanning electron microscopy (SEM). Immunocytochemistry for type IV beta-tubulin and zona occludens-1 (Zo-1) are performed to determine differentiation and confluence, respectively. To rule out contamination with olfactory epithelium (OE), immunocytochemistry for olfactory marker protein (OMP) was performed. Transepithelial resistance and potential measurements were determined using a modified vertical Ussing chamber SEM reveals approximately 90% ciliated respiratory epithelium in the nasal septum as compared with 35% in the mouse trachea. The septal air-liquid interface culture demonstrates comparable ciliated respiratory epithelium to the nasal septum. Immunocytochemistry demonstrates an intact monolayer and diffuse differentiated ciliated epithelium. These cultures exhibit a transepithelial resistance and potential confirming a confluent monolayer with electrically active airway epitheliumn containing both a sodium-absorptive pathway and a chloride-secretory pathway. To increase the yield of respiratory epithelial cells harvested from mice, we have found the nasal septum is a superior source when compared with the trachea. The nasal septum increases the yield of respiratory epithelial cells up to 8-fold.     

Ammonia-induced apoptosis is accelerated at higher pH in gastric surface mucous cells

Gastric luminal ammonia produced by Helicobacter pylori has been shown to cause gastric mucosal injury. This study was conducted to examine the mechanisms by which gastric luminal ammonia causes apoptosis of gastric epithelial cells. Monolayers of GSM06 cells, developed from murine gastric surface mucous cells, were cultured in the absence or presence of 10-30 mM NH(4)Cl at ambient pH of 5.0, 6.0, and 7.0. In the presence of luminal NH(4)Cl, GSM06 cells showed 1) cell shrinkage and nuclear chromatin condensation, 2) DNA fragmentation into oligonucleosomes, 3) leakage of cytochrome c into cytosolic fraction without affecting bax expression, and 4) increases in activity of caspases-3 and -9. These changes were accentuated when the cells were cultured at pH 7.0. In the absence of NH(4)Cl, none of these changes was detected at any pH examined. These results suggest that gastric luminal ammonia, at concentrations detected in H. pylori-infected subjects, induces apoptosis of gastric epithelial cells by release of cytochrome c from mitochondria, followed by activation of caspases-9 and -3, especially at higher ambient pH.     

Reconstituted human gingival epithelium: Nonsubmerged in vitro model

Summary Many studies have shown that human gingival keratinocytes grown in submerged culture fail to attain optimal differentiation. This study reports an in vitro culture system for oral gingival epithelial cells, in which they are grown at the air-liquid interface, on polycarbonate inserts, in the presence of an NIH-3T3 feeder layer. This model was compared with two submerged culture methods for gingival keratinocytes, on type I collagen gel and on an NIH-3T3 feeder layer. Transmission electron microscopy showed an advanced level of stratification (over six layers of cells) for cultures grown at the air-liquid interface. Immunofluorescence and electrophoretic patterns showed the presence of cytokeratins 10 and 11 in cytoskeletal protein extracts of these cultured keratinocytes. In this air-liquid interface culture model, in the presence of NIH-3T3 feeder cells, keratinocytes can achieve an advanced level of stratification and differentiation and a resemblance to in vivo gingiva. The obtention of a highly differentiated epithelium will permit in vitro pharmacological studies and studies on the biocompatability of certain alloys with the superficial periodontium; it will also provide grafts for patients undergoing periodontal surgery.     

Mucociliary Clearance Defects in a Murine In Vitro Model of Pneumococcal Airway Infection

Mucociliary airway clearance is an innate defense mechanism that protects the lung from harmful effects of inhaled pathogens. In order to escape mechanical clearance, airway pathogens including Streptococcus pneumoniae (pneumococcus) are thought to inactivate mucociliary clearance by mechanisms such as slowing of ciliary beating and lytic damage of epithelial cells. Pore-forming toxins like pneumolysin, may be instrumental in these processes. In a murine in vitro airway infection model using tracheal epithelial cells grown in air-liquid interface cultures, we investigated the functional consequences on the ciliated respiratory epithelium when the first contact with pneumococci is established. High-speed video microscopy and live-cell imaging showed that the apical infection with both wildtype and pneumolysin-deficient pneumococci caused insufficient fluid flow along the epithelial surface and loss of efficient clearance, whereas ciliary beat frequency remained within the normal range. Three-dimensional confocal microscopy demonstrated that pneumococci caused specific morphologic aberrations of two key elements in the F-actin cytoskeleton: the junctional F-actin at the apical cortex of the lateral cell borders and the apical F-actin, localized within the planes of the apical cell sides at the ciliary bases. The lesions affected the columnar shape of the polarized respiratory epithelial cells. In addition, the planar architecture of the entire ciliated respiratory epithelium was irregularly distorted. Our observations indicate that the mechanical supports essential for both effective cilia strokes and stability of the epithelial barrier were weakened. We provide a new model, where - in pneumococcal infection - persistent ciliary beating generates turbulent fluid flow at non-planar distorted epithelial surface areas, which enables pneumococci to resist mechanical cilia-mediated clearance.     

Activation of Toll-like receptor 2 on human tracheobronchial epithelial cells induces the antimicrobial peptide human beta defensin-2

As pattern recognition receptors capable of eliciting responses to a diverse array of microbial products, Toll-like receptors (TLRs) participate in the activation of host defense mechanisms that protect against infectious pathogens. Given that epithelial cells lie at the interface between the host and its environment, we designed experiments to determine whether human airway epithelial cells express TLRs and respond to TLR agonists. Immunohistochemical labeling of TLR2 in normal human airways revealed TLR2 expression throughout the epithelium, with an apparently higher level of expression on noncolumnar basal epithelial cells. Two-color immunofluorescent labeling of TLR2 and cytokeratins 8 and 15 revealed that TLR2 is coexpressed with the epithelial cell markers. In addition, airway epithelial cells grown at air-liquid interface responded to bacterial lipopeptide in a TLR2-dependent manner with induction of mRNA and protein of the antimicrobial peptide human beta defensin-2. Stimulation of epithelial cell cultures with lipopeptide resulted in a small and variable reduction of bacteria on the apical surface. Together, these data suggest that TLRs monitor epithelial surfaces to enhance host defense by inducing the production of an antimicrobial peptide.     

Monoclonal antibodies recognize a cell surface marker of epithelial differentiation in the rabbit reproductive tract

Monoclonal antibodies against the cell surface were produced by immunizing mice with endometrial scrapings prepared from 6-day pregnant rabbits. Spleen cells from an immune mouse were fused with myeloma cells and cultured by standard hybridoma technology methods. Hybridoma supernatants were screened for reaction with the apical epithelial surface by immunohistochemistry on frozen sections of uterus from 6-day pregnant rabbits, and positive colonies were cloned by limiting dilution. Ascites fluid was produced in mice from hybridoma clones that gave a consistent pattern of apical epithelial surface staining through 6 sub-clonings. Antibodies in the ascites fluid were tested by immunohistochemistry on frozen sections of uterus, oviduct, lung, liver and kidney from nonpregnant or 6-day pregnant rabbits. At a dilution of 1:5000, the antibodies recognized an antigen that was specific to the apical surface of luminal but not glandular epithelium of the 6-day pregnant uterus and could not be detected in the nonpregnant uterine epithelium. At higher concentrations of antibody (1:100 to 1:1000), crossreaction was seen with antigens in stromal and myometrial cells of pregnant and nonpregnant uterus. At a dilution of 1:5000, the antibody also crossreacted with some components of lung, liver and kidney but without discriminating between the two reproductive states. In the oviduct, staining of the surface epithelium was specific to the pregnant state. We conclude that this monoclonal antibody has a high affinity for a luminal epithelial cell surface antigen in the reproductive tract of the pregnant rabbit and shows multiple organ reactivity with other tissues that is not affected by pregnancy. This antigen will provide a useful cell surface marker of epithelial differentiation in the progestational reproductive tract.     

Organotypic culture of human amnion cells in air-liquid interface as a potential substitute for skin regeneration

Background aimsThe aim of the present study was to evaluate the effects of air-liquid interface on the differentiation potential of human amnion epithelial cells (HAECs) to skin-like substitute in organotypic culture.MethodsHAECs at passage 1–2 were seeded onto a fibrin layer populated with human amnion mesenchymal cells to form the organotypic cultures. The organotypic HAECs were then cultured for 7, 14 and 21 d in two types of culture system: the submerged culture and the air-liquid interface culture. Cell morphogenesis was examined under the light and electron microscopes (transmission and scanning) and analyzed by immunohistochemistry.ResultsOrganotypic HAECs formed a single layer epithelium after 3 wk in submerged as well as air-liquid interface cultures. Ultrastructurally, desmosomes were observed in organotypic HAECs cultured in the air-liquid interface but not in the submerged culture. The presence of desmosomes marked the onset of early epidermal differentiation. Organotypic HAECs were positive against anti-CK18 and anti-CK14 in both the submerged and the air-liquid interface cultures. The co-expression of CK14 and CK18 suggested that differentiation of HAECs into skin may follow the process of embryonic skin development. However, weak expression of CK14 was observed after 2 and 3 wk of culture in air-liquid interface. CK10, involucrin, type IV collagen and laminin-5 expression was absent in organotypic HAECs. This observation reflects the initial process of embryonic epidermal differentiation and stratification.ConclusionsResults from the present study suggest that the air-liquid interface could stimulate early differentiation of organotypic HAECs to epidermal cells, with a potential use for skin regeneration.     

Helicobacter pylori infection increases cell kinetics in human gastric epithelial cells without adhering to proliferating cells

We investigated the effect of H. pylori infection on cell proliferation of gastric mucosa using immunostaining for H. pylori or Ki67. H. pylori cells attached to surface mucous cells covering luminal surface and the upper part of gastric foveolae, and up-regulated the proliferative activity of gastric epithelial cells without adhering to the proliferating epithelial cells.     

A collagen IV matrix is required for guinea pig gastric epithelial cell monolayers to provide an optimal model of the stomach surface for biopharmaceutical screening

The surface epithelial cells of the stomach represent a major component of the gastric barrier. A cell culture model of the gastric epithelial cell surface would prove useful for biopharmaceutical screening of new chemical entities and dosage forms. Primary cultures of guinea pig gastric mucous epithelial cells were grown on filter inserts (Transwells) for 3 days. Tight-junction formation, assessed by transepithelial electrical resistance (TEER) and permeability of mannitol and fluorescein, was enhanced when collagen IV rather than collagen I was used to coat the polycarbonate filter. TEER for cells grown on collagen IV was close to that obtained with intact guinea pig gastric epithelium in vitro. Differentiation was assessed by incorporation of [3H]glucosamine into glycoprotein and by activity of NADPH oxidase, which produces superoxide. Both of these measures were greater for cells grown on filters coated with collagen I than for cells grown on plastic culture plates, but no major difference was found between cells grown on collagens I and IV. The proportion of cells, which stained positively for mucin with periodic acid Schiff reagent, was greater than 95% for all culture conditions. Monolayers grown on membranes coated with collagen IV exhibited apically polarized secretion of mucin and superoxide, and were resistant to acidification of the apical medium to pH 3.0 for 30 min. A screen of nonsteroidal anti-inflammatory drugs revealed a novel effect of diclofenac and niflumic acid in reversibly reducing permeability by the paracellular route. In conclusion, the mucous cell preparation grown on collagen IV represents a good model of the gastric surface epithelium suitable for screening procedures.     

Reconstruction of a rabbit corneal epithelium on a lyophilized amniotic membrane using tilting air-liquid interface culture followed by tilting submerged culture

Herein, we reconstructed a rabbit corneal epithelium on a lyophilized amniotic membrane (LAM) using a modified version of two Teflon rings (the Ahn’s supporter). We compared the corneal epithelial cells we had differentiated in vitro using air-liquid interface (6 days, 12 days) and submerged (6 days, 12 days) cultures and followed a six-day tilting dynamic air-liquid interface culture with a six-day tilting submerged culture. We characterized the reconstructed corneal epithelium using digital photography, histological imaging, and transmission electron microscopy. The reconstructed corneal epithelium created under air-liquid interface culture exhibited a healthier basal corneal epithelial layer than that created under submerged culture. The reconstructed corneal epithelium on the LAM that was produced using the tilting dymanic culture exhibited a healthy basal layer. We therefore proposed that tilting submerged culture not only supplied nutrients from the medium to the corneal epithelial cells on the LAM, but it also removed the horny layer in the upper part of the reconstructed corneal epithelium, presumably by mimicking the effects of blinking. This study demonstrated that corneal epithelium reconstruction on a LAM using a tilting submerged culture after a tilting air-liquid interface culture may be useful not only for allogeneic or autologous transplantation, but also for in vitro toxicological test kits.     

Physiological and morphological effects of alendronate on rabbit esophageal epithelium

Alendronate, an aminobisphosphonate, produces as a side effect a topical (pill induced) esophagitis. To gain insight into this phenomenon, we assessed the effects of luminal alendronate on both esophageal epithelial structure and function. Sections of rabbit esophageal epithelium were exposed to luminal alendronate at neutral or acidic pH while mounted in Ussing chambers to monitor transmural electrical potential difference (PD), short-circuit current (I(sc)), and resistance (R). Morphological changes were sought by light microscopy in hematoxylin and eosin-stained sections. Impedance analysis was used for localization of alendronate-induced effects on ion transport. Luminal, but not serosal, alendronate (pH 6.9-7.2), increased PD and I(sc) in a dose- and time-dependent manner, with little change in R and mild edema of surface cell layers. The changes in I(sc) (and PD) were reversible with drug washout and could be prevented either by inhibition of Na,K-ATPase activity with serosal ouabain or by inhibition of apical Na channels with luminal acidification to pH 2.0 with HCl. An effect on apical Na channel activity was also supported by impedance analysis. Luminal alendronate at acidic pH was more damaging than either alendronate at neutral pH or acidic pH alone. These data suggest that alendronate stimulates net ion (Na) transport in esophageal epithelium by increasing apical membrane sodium channel activity and that this occurs with limited morphological change and no alteration in barrier function. Also alendronate is far more damaging at acidic than at neutral pH, suggesting its association with esophagitis requires gastric acid for expression. This expression may occur either by potentiation between the damaging effects of (refluxed) gastric acid and drug or by acid-induced conversion of the drug to a more toxic form.     

A novel in vitro infection model of Helicobacter pylori using mucin-producing murine gastric surface mucous cells

BACKGROUND: Helicobacter pylori is found within the gastric surface mucous gel layer and in the epithelial surface. Gastric cancer cells have been used in experimental H. pylori infection in vitro, although cancer cells have some abnormalities in cellular properties. The aim of this study was to develop an in vitro H. pylori infection model using normal gastric surface cells that produce gastric mucin. MATERIALS AND METHODS: Normal murine gastric surface mucous cells (GSM06) were cultured by the liquid interface method using a serum-free medium and a collagen gel containing a fibroblast cell line (L929) and infected with H. pylori. Infection by H. pylori was assessed by enumerating the colony-forming units (CFU) of H. pylori adhered to GSM06 cells and by transmission electron microscopy. The production of mucin was determined by a lectin binding assay, sugar analysis, and MUC5AC gene expression. RESULTS: GSM06 cells cultured under these conditions produced mucin containing N-acetylgalactosamine and MUC5AC as the core protein. Significantly higher numbers of H. pylori adhered to GSM06 cells under mucin-producing conditions than under nonproducing conditions. Microscopic observation showed a filamentous structure resembling a type IV secretion system apparatus formed between the surface of GSM06 cells and H. pylori. CONCLUSIONS: This study demonstrates a novel in vitro H. pylori infection model using mucin-producing murine GSM06 cells for early stages of infection.     

A study of the anatomy, histology and ultrastructure of the digestive tract of Orthrias angorae Steindachner, 1897

The anatomy, histology and ultrastructure of the digestive tract of Orthrias angorae (Steindachner, 1897) were investigated using light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The histological structure consists of four layers: mucosa, submucosa, muscularis and serosa. The esophageal mucosa consists of undifferentiated basal epithelial cells, mucous cells and surface epithelial cells. It was observed that the J-shaped stomach had a meshwork of folds in the cardiac region, and longitudinal folds in the fundic and pyloric regions. A single layer of columnar cells, PAS positive only in their apical portions, forms the epithelium. The convoluted tube-shape intestine is lined by simple columnar epithelial cells, which have microvilli at the apical surface. The wall of the esophagus and stomach are thicker than that of the intestine because of the thick muscle layer. There were numerous goblet cells in the intestine. There were numerous gastric glands in the submucosa layer ofthe cardiac stomach, but none were present in the pyloric region of the stomach. There were no pyloric caeca between the stomach and intestine. The enterocytes with microvilli contained rough endoplasmic reticulum, ribosomes and rounded bodies, and the gastric cells contained a well-developed Golgi apparatus.     

Integrated duodenal protective response to acid.

The proximal duodenum is unique in that it is the only leaky epithelium regularly exposed to concentrated gastric acid. To prevent injury from occurring, numerous duodenal defense mechanisms have evolved. The most studied is bicarbonate secretion, which is presumed to neutralize luminal acid. Less well studied in their protective roles are the mucus gel layer and blood flow. Measuring duodenal epithelial intracellular pH [pHi], blood flow and mucus gel thickness (MGT), we studied duodenal defense mechanisms in vivo so as to more fully understand the mucosal response to luminal acid. Exposure of the mucosa to physiologic acid solutions promptly lowered pHi, followed by recovery after acid was removed, indicating that acid at physiologic concentrations readily diffuses into, but does not damage duodenal epithelial cells. Cellular acid then exits the cell via an amiloride-inhibitable process, presumably sodium-proton exchange (NHE). MGT and blood flow increase promptly during acid perfusion; both decrease after acid challenge and are inhibited by vanilloid receptor antagonists or by sensory afferent denervation. Bicarbonate secretion is not affected by acid superfusion but increases after challenge. Inhibition of cellular base loading lowers pHi, whereas inhibition of apical base extrusion alkalinizes pHi. These observations support the following hypothesis: luminal acid diffuses into the epithelial cells, lowering pHi. Acidic pHi increases the activity of a basolateral NHE, acidifying the submucosal space and increasing cellular base loading. The acidic submucosal space activates capsaicin receptors on afferent nerves, increasing MGT and blood flow. With concontinued acid exposure, a new steady state with thickened mucus gel, increased blood flow, and a higher cellular buffering power protects against acid injury. After acid challenge, mucus secretion decreases, blood flow slows, and pHi returns to normal, the latter occurring via apical bicarbonate extrusion, increasing bicarbonate secretion. Through these integrated mechanisms, the epithelial cells are protected from damage due to repeated pulses of concentrated gastric acid.     

Expression of cellCAM-105 in the apical surface of rat uterine epithelium is controlled by ovarian steroid hormones

Affinity-purified antibodies to cellCAM-105, an adhesive cell surface glycoprotein, were used in immunohistochemical investigations of rat uteri at various functional stages: (i) the oestrous, pro-oestrous, metoestrous, and dioestrous stages of the oestrous cycle, (ii) Days 1-8 of normal pregnancy, (iii) delayed implantation, (iv) 18 h after oestrogen reactivation from delay of implantation, and (v) juvenile rats, and normal ovariectomized adults, respectively, before and after experimental injection of progesterone and/or oestrogen. CellCAM-105 was present in the apical zones of the luminal and glandular epithelium cells in a stage-specific and hormone-dependent manner. The results indicate that: (1) steroid hormones are essential for the expression of cellCAM-105 in the uterine epithelial cells; (2) progesterone induces cellCAM-105 expression in the glandular epithelium, and oestrogen induces cellCAM-105 expression in the luminal epithelium; (3) progesterone induces down-regulation of cellCAM-105 from the surface of the uterine luminal epithelium of juvenile rats; (4) cellCAM-105 is absent in the luminal epithelial cells but present in the glandular epithelial cells of the rat uterus at the time of blastocyst implantation.     

Amniotic membrane inhibits squamous metaplasia of human conjunctival epithelium

Squamous metaplasia is a common pathological process that occurs in the ocular surface epithelium. At present, there is no effective treatment for this abnormality. In the current study, we established an ex vivo conjunctival squamous metaplasia model by culturing human conjunctival tissues at an air-liquid interface for durations of up to 12 days. We then investigated the effects of amniotic membrane (AM) on squamous metaplasia through coculture of conjunctival tissues with AM or AM extract. We found that metaplasia features such as hyperproliferation and abnormal epidermal differentiation of conjunctival epithelium could be inhibited by AM or its extract. In addition, existing squamous metaplasia of conjunctival epithelium could be reversed to a nearly normal phenotype by AM. The mechanism by which AM prevents squamous metaplasia may involve downregulation of p38 mitogen-activated protein kinase and Wnt signaling pathways, which were activated in conjunctival explants cultured with an airlift technique. In conclusion, AM can inhibit and reverse squamous metaplasia of conjunctival epithelium. This finding may shed new light on prevention and treatment of diseases that involve epithelial squamous metaplasia.