Cellular mechanisms contributing to renewal of terminal bronchioles remain poorly defined. Our previous studies identified pollutant-resistant Clara cell secretory protein (CCSP)-expressing stem cells that localize to the neuroepithelial body (NEB) and contribute to renewal of the proximal bronchiolar epithelium. However, activation of NEB-associated stem cells is unlikely to contribute to renewal of terminal bronchiolar epithelium because of the paucity of NEBs at this location. Goals of this study were to determine the location and properties of cells contributing to renewal of terminal bronchioles after Clara cell depletion. Pollutant-resistant CCSP-expressing cells were identified that localized to the bronchoalveolar duct junction (BADJ) and contribute to restoration of a phenotypically diverse epithelium. CCSP-expressing cells comprise the predominant proliferative population in initial terminal bronchiolar repair and include a population of label-retaining cells suggesting that they maintain characteristics of a stem cell population. Furthermore, immunohistochemical co-localization studies involving CCSP and the NEB-specific marker calcitonin gene-related peptide indicate that BADJ-associated CCSP-expressing stem cells function independently of NEB microenvironments. These studies identify a BADJ-associated, NEB-independent, CCSP-expressing stem cell population in terminal bronchioles and support the notion that regiospecific stem cell niches function to maintain epithelial diversity after injury.

Terminal bronchioles harbor a unique airway stem cell population that localizes to the bronchoalveolar duct junction.

Remodeling of the conducting airway epithelium is a common finding in the chronically injured lung and has been associated with increased risk for developing lung cancer. Pulmonary neuroendocrine cells and clusters of these cells termed neuroepithelial bodies (NEBs) play a central role in each of these processes. We previously developed an adult mouse model of airway injury and repair in which epithelial regeneration after naphthalene-induced Clara cell ablation occurred preferentially at airway branch points and gave rise to nascent Clara cells. Continued repair was accompanied by NEB hyperplasia. We now provide the following evidence that the NEB microenvironment serves as a source of airway progenitor cells that contribute to focal regeneration of the airway epithelium: 1) nascent Clara cells and NEBs localize to the same spatial domain; 2) within NEB, both Clara cell secretory protein- and calcitonin gene-related peptide-immunopositive cells are proliferative; 3) the NEB microenvironment of both the steady-state and repairing lung includes cells that are dually immunopositive for Clara cell secretory protein and calcitonin gene-related peptide, which were previously identified only within the embryonic lung; and 4) NEBs harbor variant Clara cells deficient in cytochrome P450 2F2-immunoreactive protein. These data suggest that the NEB microenvironment is a reservoir of pollutant-resistant progenitor cells responsive to depletion of an abundant airway progenitor such as the Clara cell.

/pdf/neuroepithelial-bodies-of-pulmonary-airways-serve-as-a-1tvecsb6na.pdf

Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration.

Murine lung epithelial (MLE) cell lines representing the distal bronchiolar and alveolar epithelium were produced from lung tumors generated in transgenic mice harboring the viral oncogene simian virus 40 (SV40) large tumor antigen under transcriptional control of a promoter region from the human surfactant protein C (SP-C) gene. The cell lines exhibited rapid growth, lack of contact inhibition, and an epithelial cell morphology for 30-40 passages in culture. Microvilli, cytoplasmic multivesicular bodies, and multilamellar inclusion bodies (morphologic characteristics of alveolar type II cells) were detected in some of the MLE cell lines by electron microscopic analysis. The MLE cells also maintained functional characteristics of distal respiratory epithelial cells including the expression of surfactant proteins and mRNAs and the ability to secrete phospholipids. Expression of the exogenous SV40 large tumor antigen gene was detected in all of the generated cell lines. The SP-C/SV40 large tumor antigen transgenic mice and the MLE cell lines will be useful for the study of pulmonary surfactant production and regulation as well as lung development and tumorigenesis.

https://www.pnas.org/content/pnas/90/23/11029.full.pdf

Production of immortalized distal respiratory epithelial cell lines from surfactant protein C/simian virus 40 large tumor antigen transgenic mice

Most reviews of adult stem cells focus on the relatively undifferentiated cells dedicated to the renewal of rapidly proliferating tissues, such as the skin, gut and blood. By contrast, there is mounting evidence that organs and tissues such as the liver and pancreatic islets, which turn over more slowly, use alternative strategies, including the self-renewal of differentiated cells. The response of these organs to injury may also reveal the potential of differentiated cells to act as stem cells. The lung shows both slow turnover and rapid repair. New experimental approaches, including those based on studies of embryonic development, are needed to identify putative lung stem cells and strategies of lung homeostasis and repair.

/pdf/epithelial-stem-cells-of-the-lung-privileged-few-or-48kh7wka2l.pdf

Epithelial stem cells of the lung: privileged few or opportunities for many?

Epithelial organs, including the lung, are known to possess regenerative abilities through activation of endogenous stem cell populations, but the molecular pathways regulating stem cell expansion and regeneration are not well understood. Here we show that Gata6 regulates the temporal appearance and number of bronchioalveolar stem cells (BASCs) in the lung, its absence in Gata6-null lung epithelium leading to the precocious appearance of BASCs and concurrent loss in epithelial differentiation. This expansion of BASCs was the result of a pronounced increase in canonical Wnt signaling in lung epithelium upon loss of Gata6. Expression of the noncanonical Wnt receptor Fzd2 was downregulated in Gata6 mutants and increased Fzd2 or decreased beta-catenin expression rescued, in part, the lung epithelial defects in Gata6 mutants. During lung epithelial regeneration, canonical Wnt signaling was activated in the niche containing BASCs and forced activation of Wnt signaling led to a large increase in BASC numbers. Moreover, Gata6 was required for proper lung epithelial regeneration, and postnatal loss of Gata6 led to increased BASC expansion and decreased differentiation. Together, these data demonstrate that Gata6-regulated Wnt signaling controls the balance between progenitor expansion and epithelial differentiation required for both lung development and regeneration.

/pdf/a-gata6-wnt-pathway-required-for-epithelial-stem-cell-3yqusjpb0f.pdf

A Gata6-Wnt pathway required for epithelial stem cell development and airway regeneration

The purpose of this study was to define the sites of cytotoxicity within the respiratory tract (nasal cavity and tracheobronchial airway tree) resulting from administration of naphthalene, an organic chemical whose cytotoxic properties require metabolic activation via the cytochrome P-450 monooxygenase system. Three species were compared: mouse, hamster and rat. Naphthalene was administered in corn oil at these doses: mouse (0-400 mg/kg), hamster (0-800 mg/kg) and rat (0-1600 mg/kg), and the animals were sacrificed 24 hr later. In mice, naphthalene produced Clara cell cytotoxicity at 50 mg/kg. The primary alteration was swelling and vacuolation of Clara cells in terminal bronchioles. At 100 mg/kg, the number of terminal bronchioles with vacuolated Clara cells and the number of Clara cells within terminal bronchioles which showed vacuolation increased. At 200 and 300 mg/kg, almost all of the nonciliated cells lining terminal bronchioles in mice were exfoliated and necrotic. In contrast, there was no apparent effect on Clara cells or ciliated cells of terminal bronchioles in rats treated with up to 1600 mg/kg. At 800 mg/kg, minor alterations in Clara cells in some terminal bronchioles were observed in hamsters. At 300 mg/kg, lobar bronchus and trachea showed swelling 'and vacuolation of nonciliated cells in mice, but no detectable change at 200 mg/kg or below. The trachea and lobar bronchus were unaffected in rats, but showed cytotoxic changes in hamsters. In the nasal cavity of mice, cytotoxicity was observed only in the olfactory epithelium and only in animals treated with 400 mg/kg. There were minimal alterations in the respiratory epithelium. The only epithelial population showing cytotoxicity in the rat was the olfactory epithelium. Complete necrosis was observed at 200 mg/kg and higher. In the hamster there was no discernible alteration in the olfactory epithelium at 100 and 200 mg/kg. At 400 mg/kg, the olfactory epithelium was necrotic. Naphthalene injury to the tracheobronchial epithelium of the mouse is: 1) Clara cell specific; 2) dose-related in the terminal bronchiole; and 3) involves more proximal airways in a dose-dependent fashion. The tracheobronchial epithelium of the rat is refractory to Clara cell injury even at the LD50, but proximal airways are more susceptible than distal airways in the hamster. The nasal cavity shows specific injury in one zone (olfactory epithelium) in a dose and species specific manner. The susceptibility to naphthalene-induced injury in the olfactory epithelium does not correlate with the susceptibility of Clara cells in more distal portions of the respiratory tract.(ABSTRACT TRUNCATED AT 400 WORDS)

Relationship of cytochrome P-450 activity to Clara cell cytotoxicity. I. Histopathologic comparison of the respiratory tract of mice, rats and hamsters after parenteral administration of naphthalene.

C. Suverkropp

Papers

Relationship of cytochrome P-450 activity to Clara cell cytotoxicity. I. Histopathologic comparison of the respiratory tract of mice, rats and hamsters after parenteral administration of naphthalene.