Nasal Epithelial Cells Can Act as a Physiological Surrogate for Paediatric Asthma Studies

Introduction

Differentiated paediatric epithelial cells can be used to study the role of epithelial cells in asthma. Nasal epithelial cells are easier to obtain and may act as a surrogate for bronchial epithelium in asthma studies. We assessed the suitability of nasal epithelium from asthmatic children to be a surrogate for bronchial epithelium using air-liquid interface cultures.

Methods

Paired nasal and bronchial epithelial cells from asthmatic children (n = 9) were differentiated for 28 days under unstimulated and IL-13-stimulated conditions. Morphological and physiological markers were analysed using immunocytochemistry, transepithelial-electrical-resistance, Quantitative Real-time-PCR, ELISA and multiplex cytokine/chemokine analysis.

Results

Physiologically, nasal epithelial cells from asthmatic children exhibit similar cytokine responses to stimulation with IL-13 compared with paired bronchial epithelial cells. Morphologically however, nasal epithelial cells differed significantly from bronchial epithelial cells from asthmatic patients under unstimulated and IL-13-stimulated conditions. Nasal epithelial cells exhibited lower proliferation/differentiation rates and lower percentages of goblet and ciliated cells when unstimulated, while exhibiting a diminished and varied response to IL-13.

Conclusions

We conclude that morphologically, nasal epithelial cells would not be a suitable surrogate due to a significantly lower rate of proliferation and differentiation of goblet and ciliated cells. Physiologically, nasal epithelial cells respond similarly to exogenous stimulation with IL-13 in cytokine production and could be used as a physiological surrogate in the event that bronchial epithelial cells are not available.     

Establishment of a mouse thymic epithelial cell line, IT-76MHC and a brief review on cultured thymic epithelial cells.

Interactions between thymocytes and thymic stromal cells are essential for thymocyte differentiation, but little evidence has been presented to directly show in vivo functions or interactions of the stromal cells. Among the stromal cells, the thymic epithelial cell has been considered to have profound effect on thymocyte differentiation and maturation. The calcium-depleted medium, originally developed for the culture of mouse epidermal cells, was applied for the culture of the mouse thymic epithelial cells, and successfully, an epithelial cell line, IT-76MHC was obtained from the mouse thymus. IT-76MHC cells were identified as distinct mouse thymic epithelial cells by 1/ mosaic-like arrangement, 2/ presence of well-developed desmosome and 3/ tonofilaments, 4/ positivity for cytokeratin, and 5/ induced expression of MHC class I and II by IFN-gamma treatment. IGF-1, IGF-2, oxytocin and vasopressin were also detected immunohistochemically in IT-76MHC cells. Furthermore, the IT-76MHC thymic epithelial cells, when injected intrathymically in the allogeneic mouse, prolonged the survival of skin graft from the same donor strain that IT-76MHC cells were derived. These results demonstrate that the thymic epithelial cell line IT-76MHC produces modest thymocyte survival factors as well as a growth suppressor, and that IT-76MHC cells have the ability to induce transplantation tolerance probably through their expression of MHC class I and II molecules. Taken altogether, the IT-76MHC thymic epithelial cells have been proved to be useful tools to better understand the in vivo functions of thymic epithelial cells, and to gain a deep insight into their involvement in the critical selection process of thymocytes which still remains obscure. Finally and additionally, literatures so far reported on thymic epithelial cells in culture, especially lines and clones, are reviewed and their identity as well as their functions are discussed.     

Monoclonal antibody identification of a type II alveolar epithelial cell antigen and expression of the antigen during lung development

A monoclonal antibody identifying an antigen expressed by rat type II alveolar epithelial cells, but not by type I epithelial cells or other mature lung cells, was produced by immunization of mice with cells of the rat L2 cell line. The antigen recognized by the antibody was present on the microvillous luminal surface of type II epithelial cells. In adult rat lung, only type II epithelial cells bound the antibody. During fetal development the antigen was expressed by cuboidal epithelial cells lining the respiratory ducts of the first divisions of the tracheal bud, but not by epithelial cells lining the esophagus or trachea. The antigen continued to be expressed by cuboidal epithelial cells lining the larger respiratory ducts until approximately 19 days gestational age. Thereafter, expression was increasingly limited to selected single cells or clusters of two to four cuboidal cells in the smallest ducts. By the 21st postnatal day, the antigen was expressed only by type II alveolar epithelial cells. Type II alveolar epithelial cells isolated from adult lung and the L2 cell line in culture expressed the antigen on the cell surface. A protein of approximately 146,000 Mr was isolated by immunoadsorption of the antigen from non-ionic detergent extracts of type II cells and L2 cells. Preliminary studies of the binding of the antibody to other rat tissues indicate that the antibody binds to renal proximal tubular epithelial cells of the kidney and the luminal surface of the small bowel epithelial cells.     

Motility of endodermal epithelial cells plays a major role in reorganizing the two epithelial layers in Hydra

Cell-cell interactions and cell rearrangements play important roles during development. Aggregates of Hydra cells reorganize into the two epithelial layers and subsequently form a normal animal. Examination of the formation of the two layers under various situations, indicates that the motility of endodermal epithelial cells, but not the differential adhesive forces of the two types of epithelial cells, plays the critical role in setting up the two epithelial layers. (1) When aggregates of ectodermal cells and of endodermal cells were placed in direct contact, the endodermal cells migrated into the interior of the ectodermal aggregate. This process was completely inhibited by cytochalasin B although initial firm attachment between the two aggregates was not blocked. (2) A single endodermal epithelial cell placed in contact with an ectodermal aggregate, actively extended pseudopod-like structures and migrated toward the center of the ectodermal aggregate. In contrast, an ectodermal epithelial cell remained in contact with an endodermal aggregate and never exhibited migratory behavior. Cytochalasin treatment of only endodermal epithelial cells abolished the migration. (3) One to 4 endodermal epithelial cells and/or ectodermal epithelial cells were placed in contact with one another forming up to 4-cell aggregates. Endodermal epithelial cells exhibited high motility that can be attributed to the migratory movement described above. Finally, formation of actin bundles, as visualized with rhodamine-phalloidin, was always correlated with pseudopod formation in endodermal epithelial cells during early and mid stages of aggregate formation.     

Transient transfection of mammary epithelial cells with a PEI/DNA/adenovirus system

Studies on epithelial cells often require the transient expression of exogenous proteins in polarized epithelial cells. However, the major limitation of this approach has been the difficulty of obtaining transient gene expression in polarized epithelial cell cultures. We report here on the application of a polyethylenimine (PEI)/DNA/adenovirus system for efficient transient gene expression in mammary epithelial cells. Based on luciferase assay and FACScan analysis the PEI/DNA/adenovirus system is shown to be an effective and simple method for transfecting epithelial cells.     

Human amniotic epithelial cells induce apoptosis of cancer cells: a new anti-tumor therapeutic strategy

Background aimsAmniotic membrane (AM), the innermost layer of human placenta, is composed of a single layer of epithelial cells, a basement membrane and an avascular stroma. The AM has many functions and properties, among which angiogenic modulatory and immunoregulatory effects are applicable in cancer therapy. Because these functions belong to amniotic epithelial cells, in this study we compared the anti-cancer effect of amniotic epithelial cells and the whole AM.MethodsThe effect of the AM and the amniotic epithelial cells on cancer cell apoptosis was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunocytochemistry. The effect of the AM on angiogenesis in conditions both with and without epithelial cells was also evaluated using rat aortic ring assay.ResultsThere was a decrease in cancer cell viability after adding either AM or amniotic epithelial cell supernatant to cancer cells. A significant increase in caspase-3 and caspase-8 expression in cancer cells treated with amniotic epithelial cell supernatant was observed. The recorded media also demonstrated the possible induction of apoptosis in cancer cells treated with the amniotic epithelial cell supernatant. In the aorta ring assay, the AM showed an anti-angiogenic effect in the presence of its epithelial cells; however, this effect was altered to initiate angiogenesis when amniotic epithelial cells were removed from the AM.ConclusionsThese results suggest that amniotic epithelial cells, with their anti-angiogenic effect and induction of apoptosis, are candidates for cancer therapeutic agents in the near future.     

Heparin-binding keratinocyte growth factor is a candidate stromal-to-epithelial-cell andromedin.

The growth of isolated epithelial and stromal cells from both androgen-dependent normal rat prostate and an androgen-responsive model rat prostate tumor is androgen-independent. When added to co-cultures of epithelial and stromal cells separated by a semipermeable membrane, androgen stimulated epithelial cell growth without an effect on stromal cell growth. Northern blot and nuclease protection analysis of mRNA revealed that stromal cells specifically expressed an androgen-sensitive secreted member of the heparin-binding fibroblast growth factor family [keratinocyte growth factor (KGF)/fibroblast growth factor-7]. KGF was mitogenic for epithelial cells, but not for stromal cells. Epithelial cells expressed specifically a splice variant of the bek receptor gene that specifically binds KGF. Expression of the bek receptor gene in stromal cells was undetectable by Northern blot and nuclease protection analyses. The results suggest that stromal cell-derived KGF has the properties of an andromedin, which mediates the indirect control of epithelial cell proliferation by androgen through a directional stromal-to-epithelial cell paracrine mechanism.     

CXCL8 modulates human intestinal epithelial cells through a CXCR1 dependent pathway

BACKGROUND: CXCL8 (previously known as Interleukin-8), a member of the alpha-chemokine family of chemotactic cytokines, stimulates intestinal neutrophil activation and chemotaxis. As intestinal epithelial cells have been recently shown to produce CXCL8, the aim of this study was to identify functional activities of CXCL8 on intestinal epithelial cells. METHODS: The expression of CXCL8 receptors CXCR1 and CXCR2 was assessed by RT-PCR and FACS analysis in human Caco-2 and HT-29 cells. The effects of CXCL8 on intestinal epithelial proliferation were assessed with colorimetric MTT assays and the effects on epithelial restitution with an in vitro migration model using Caco-2 and HT-29 cells. RESULTS: While the expression of both CXCR1 mRNA and protein could be demonstrated by RT-PCR and FACS analysis in human Caco-2 and HT-29 cells, no expression of CXCR2 was observed in these cell lines. Colorimetric MTT assays revealed that CXCL8 does not modulate cell proliferation in HT-29 and Caco-2 cells. In contrast, CXCL8 significantly enhanced intestinal epithelial migration in an in vitro migration model of HT-29 and Caco-2 cells. Enhancement of intestinal epithelial cell migration by CXCL8 was partially CXCR1-dependent and TGFbeta-independent. CONCLUSION: CXCL8 exerts functional effects on intestinal epithelial cells that may be relevant for intestinal inflammation and mucosal healing.     

Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population

Highly regulated programs for airway epithelial cell proliferation and differentiation during development and repair are often disrupted in disease. These processes have been studied in mouse models; however, it is difficult to isolate and identify epithelial cell-specific responses in vivo. To investigate these processes in vitro, we characterized a model for primary culture of mouse tracheal epithelial cells. Small numbers of cells seeded at low density (7.5 x 10(4) cells/cm2) rapidly proliferated and became polarized. Subsequently, supplemented media and air-liquid interface conditions resulted in development of highly differentiated epithelia composed of ciliated and nonciliated cells with gene expression characteristic of native airways. Genetically altered or injured mouse tracheal epithelial cells also reflected in vivo patterns of airway epithelial cell gene expression. Passage of cells resulted in continued proliferation but limited differentiation after the first passage, suggesting that transit-amplifying cell populations were present but with independent programs for proliferation and differentiation. This approach provides a high-fidelity in vitro model for evaluation of gene regulation and expression in mouse airway epithelial cells.     

Formation of septa in the bulbus cordis of a turtle and a lizard

The epithelium lining the intrahepatic bile ducts of normal adult mice consists of a single layer of cuboidal or low columnar cells and has ultrastructure comparable to that described previously (Rouiller and Jézéquel, '65). Some of the epithelial cells, however, exhibit such particular features as dilatation of granular endoplasmic reticulum cisternae, polysome formation of ribosomes and the presence of active forms of the Golgi apparatus, numerous lysosome-like bodies and apical projections and blebs. Postnatal cholecystectomy does not induce any qualitative changes in the epithelial fine structure, but results in a significant increase in number of the particular structures mentioned. Therefore, the cholecystectomy is thought to stimulate the secretory activity of the epithelial cells, and such stimulation appears due to the absence of a possible activity of epithelial secretion in the gallbladder.     

Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells

Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.     

Epidermal growth factor promotes a neural phenotype in thymic epithelial cells and enhances neuropoietic cytokine expression

Neural crest-derived cells populate the thymus, and their coexistence with epithelial cells is required for proper organ development and T cell education function. We show here that epidermal growth factor (EGF), a major epithelial cell growth-enhancing agent, has a morphogenetic action to promote the expression of a neuronal phenotype (e.g., neurofilament expression) in cultured thymic epithelial cells that are characterized by a cytokeratin-positive epithelial cell background. The proliferation of such neurodifferentiated cells is also enhanced by EGF. Furthermore, the growth factor enhances cells that express the genes encoding the preprotachykinin A-generated neuropeptides and bipotential neuropoietic and lymphopoietic cytokines ciliary neurotrophic factor and interleukin-6. These cytokines also enhance the neuronal phenotype of thymic epithelial cells. Therefore, EGF appears to be a composite autocrine/paracrine neuromodulator in thymic stroma. This suggests that EGF may regulate thymus-dependent immune functions by promoting neuronal gene expression in neural crest- derived cells.     

In-vivo expression of a C-type RNA virus in rat ventral prostate epithelial cells.

During evaluation of a procedure for separating rat ventral prostate epithelial from connective tissue cells, isolated fractions were examined by transmission electron microscopy. Characteristic C-type RNA viruses were seen budding from or in close proximity to the plasma membranes of isolated epithelial cells. Dissociation of rat prostate cells and concentration of the epithelial fraction facilitated detection of this virus.     

EEDA: a protein associated with an early stage of stratified epithelial differentiation

Using suppressive subtractive hybridization, we have identified a novel gene, which we named early epithelial differentiation associated (EEDA), which is uniquely associated with an early stage of stratified epithelial differentiation. In epidermis, esophageal epithelium, and tongue epithelium, EEDA mRNA, and antigen was abundant in suprabasal cells, but was barely detectable in more differentiated cells. Consistent with the limbal location of corneal epithelial stem cells, EEDA was expressed in basal corneal epithelial cells that are out of the stem cell compartment, as well as the suprabasal corneal epithelial cells. The strongest EEDA expression occurred in suprabasal precortical cells of mouse, bovine, and human anagen follicles. Developmental studies showed that the appearance of EEDA in embryonic mouse epidermis (E 15.5) coincided with morphological keratinization. Interestingly, EEDA expression is turned off when epithelia were perturbed by wounding and by cultivation under both low and high Ca2+ conditions. Our results indicate that EEDA is involved in the early stages of normal epithelial differentiation, and that EEDA is important for the "normal" differentiation pathway in a wide range of stratified epithelia.     

Characterization of cultured epithelial cells using a novel technique not requiring enzymatic digestion for subculturing

Our laboratory had developed a methodology to expand epithelial cells in culture by growing keratinocyte monolayers, under large volumes of medium that produces large numbers of keratinocytes that leave the monolayer and move into suspension. The cells have been defined as epithelial Pop Up Keratinocytes or ePUKs cells and appear to be highly suitable for clinical applications. In this publication we extend the characterization of the cells with a detailed analysis of the capabilities of the monolayer of a single culture flask to produce, over time, ePUK cells. The cells were characterized using standard epithelial markers for proliferation and differentiation. Analysis of morphology of the monolayer formed and total number of cells produced is presented for a variety of human epithelial cell strains. These keratinocytes provide an additional controlled human cell system for investigation of the mechanisms regulating epithelia cell growth and differentiation and since they are produced in large numbers, they are highly suitable for use in epithelial cell banking.     

Endogenous N-cadherin in a subpopulation of MDCK cells: distribution and catenin complex composition

Epithelial (E)-cadherin plays a critical role in developing a normal epithelial phenotype but neural (N)-cadherin can disrupt epithelial shape, at least in carcinoma-derived cells. Here the normal epithelial cell line MDCK was used to select for a trypsin-sensitive (TS-MDCK) subpopulation that expresses low levels of endogenous N-cadherin. Similar amounts of E-cadherin and all catenins are found in both TS-MDCK and trypsin-resistant cells (TR-MDCK), but TS-MDCK are less phenotypically epithelioid and more motile, and junctional proteins are more detergent soluble. In TS-MDCK, N-cadherin is largely nonjunctional; a similar N-cadherin distribution and mesenchymal phenotype are found in TR-MDCK transfected to express low levels of exogenous N-cadherin. Little N-cadherin was attracted to junctions between TS-MDCK and hTERT-RPE1 cells, a retinal pigment epithelium-derived line that expresses dominantly N-cadherin. No differences were seen in E-cadherin-catenin complexes in TS- and TR-MDCK, but N-cadherin-catenin complexes in TS-MDCK have more abundant p120 catenin. Overall, the results indicate that E- and N-cadherin assemble stoichiometrically different complexes with p120 in the same cells. Further, N-cadherin does not participate with E-cadherin in a zonular epithelial junction in normal MDCK epithelial cells. Rather, even low levels of endogenous N-cadherin contribute to a disrupted epithelial phenotype, resembling the effect of N-cadherin on carcinoma-derived epithelial cells.     

Problems of being a cell in a soft body

Many soft bodied coelenterates are highly deformable or contractile. In the absence of hard skeletal elements, the epithelia are subjected to mechanical forces which cause a wide range of structural changes in the component epithelial cells. What kinds of structural change occur and how are the cells adapted to them? These questions are addressed with reference to cell surface area, cell membranes, cell junctions and epithelial cilia.