Myofibroblasts have been thought to participate in subepithelial fibrosis in asthma, but the mechanism of myofibroblast induction has not been fully understood. In this study we investigated injury-related myofibroblast induction in a coculture system of guinea-pig epithelial cells and fibroblasts cocultured in a human amnion chamber. After pseudostratified epithelial cells were mechanically scraped, migrated flat epithelial cells differentiated into cuboidal appearances on Day 4 and then returned to their original shapes on Day 8. During the course of the epithelial redifferentiation, it was found by Northern blot analysis, immunohistochemistry for alpha-smooth muscle actin, and electron microscopic observation that the myofibroblasts were transiently induced on Day 4. The myofibroblast induction was inhibited by the blocking of transforming growth factor (TGF)-beta1 and thrombospondin (TSP)-1, indicating that the activation of TGF-beta1 by TSP-1 would induce myofibroblasts. This finding was also supported by a transient upregulation of TSP immunoreactivity and TSP-1 messenger RNA (mRNA) in fibroblasts. Interestingly, epithelial injury reduced TGF-beta1 immunoreactivity in the amnion membrane but did not affect TGF-beta1 mRNA in epithelial cells and fibroblasts, indicating that TGF-beta1 supplied from the extracellular matrix can participate in myofibroblast induction. Concurrently with myofibroblast induction, procollagen type I and III mRNAs were upregulated in fibroblasts, and obvious collagen deposition was observed ultrastructurally around the myofibroblasts compared with the fibroblasts. These results indicate that induced myofibroblasts can be functionally more active in producing collagen than are resting fibroblasts. The present study suggests that epithelial injury stimulates TGF-beta1 release from the extracellular matrix and its activation via TSP-1 production, causing collagen synthesis through myofibroblast induction.

The airway epithelium plays a critical role in asthma. E-cadherin, located on the basolateral side of the epithelial cells, forms adherent junctions. To investigate the role of E-cadherin on the regulation of permeability of molecules and fluid in asthmatic responses, we observed the dynamics of E-cadherin after an immunochallenge against guinea pigs. Immunohistochemical studies revealed that E-cadherin was expressed on the lateral sides of epithelial cells before the immunochallenge and after immediate airway responses (IAR). However, E-cadherin immunoreactivities decreased from the basolateral region in late airway responses (LAR) 6 h after the challenge. Simultaneously, soluble E-cadherin immunoreactivities were detected in lavage fluid only in LAR, suggesting that E-cadherin is partly cleaved and released into the lumen in LAR. Airway permeability, which was examined by penetration of horseradish peroxidase from the airway side into the epithelium, increased in both IAR and LAR. These results suggest that E-cadherin detachment from the lateral side of the epithelial cells increased airway permeability in LAR but not IAR. We conclude that an antigen challenge causes an opening of adherent junctions as well as increases airway permeability in LAR. This mechanism would participate in airflow limitation during attacks and the increase of airway permeability and hyperresponsiveness in asthmatics.

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We have developed a unique in vitro reconstitution system for tracheal epithelia of guinea pigs. In the system, a human amnion membrane was used as a basement membrane and the tracheal epithelial cells were cultured on the epithelial side of the membrane. Three weeks later, the tracheal fibroblasts were co-cultured on the serosal side of the amnion membrane and the culturing was continued for an additional 10 d. The morphology of the cultured epithelial cells consisted of a pseudostratified columnar ciliated epithelium from cuboidal ciliated epithelium during the last 10 d of the culture period. Epithelial cells included both goblet-like and basal cells. In addition, the frequency of each type of differentiated cells was almost identical to that of in vivo tracheas. Interestingly, the same results were obtained when the conditioned medium of the tracheal fibroblasts was used instead of the fibroblasts themselves. These results suggest that epithelial-mesenchymal interaction is likely involved in growth and differentiation of epithelial cells in vivo in a soluble factor(s)-mediated manner. As well as the epithelial cells, the fibroblasts also formed a multilayer during the last 10 d of co-culturing. This indicates that in vitro reconstitution of tracheal epithelia is achieved without addition of any exogenous growth or differentiation factors. The reconstitution system is shown to be useful for investigating the cellular and molecular interaction of epithelial and mesenchymal cells. Possible applications of the culture system and possible factors involved in growth and differentiation of epithelial cells are discussed.

The present study suggests that NO, probably produced by iNOS, is one of the factors involved in the delay of gastric emptying in the LPS-treated rats and probably in those with sepsis.