Terminal Bronchioles Harbor a Unique Airway Stem Cell Population That Localizes to the Bronchoalveolar Duct Junction
2002; Elsevier BV; Volume: 161; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64169-7
ISSN1525-2191
AutoresAdam Giangreco, Susan D. Reynolds, Barry R. Stripp,
Tópico(s)Neuroscience of respiration and sleep
ResumoCellular 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. 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. Dramatic differences in the cellular composition of conducting airway epithelia exist in a continuum between the trachea and terminal bronchioles; a feature that contributes to functional heterogeneity essential for normal airway homeostasis.1Barth PJ Wolf M Ramaswamy A Distribution and number of Clara cells in the normal and disturbed development of the human fetal lung.Pediatr Pathol. 1994; 4: 637-651Crossref Scopus (19) Google Scholar, 2Plopper CG Mariassy AT Hill LH Ultrastructure of the nonciliated bronchiolar epithelial (Clara) cell of mammalian lung: I. A comparison of rabbit, guinea pig, rat, hamster, and mouse.Exp Lung Res. 1980; 1: 139-154Crossref PubMed Scopus (110) Google Scholar, 3Plopper CG Mariassy AT Hill LH Ultrastructure of the nonciliated bronchiolar epithelial (Clara) cell of mammalian lung: II. 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Injury models that target terminally differentiated cell populations identify the importance of the transit-amplifying (TA) subset of progenitor cells in the regeneration process. Proliferation and differentiation of TA cells results in rapid restoration of a normal epithelium. In contrast, selective depletion of an abundant TA cell pool results in activation of rare stem cell populations.9Enver T Greaves M Loops, lineage, and leukemia.Cell. 1998; 94: 9-12Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 10Lehrer MS Sun TT Lavker RM Strategies of epithelial repair: modulation of stem cell and transit amplifying cell proliferation.J Cell Sci. 1998; 111: 2867-2875Crossref PubMed Google Scholar, 11Petkov PM Kim K Sandhu J Shafritz DA Dabeva MD Identification of differentially expressed genes in epithelial stem/progenitor cells of fetal rat liver.Genomics. 2000; 68: 197-209Crossref PubMed Scopus (11) Google Scholar, 12Saam JR Gordon JI Inducible gene knockouts in the small intestinal and colonic epithelium.J Biol Chem. 1999; 274: 38071-38082Crossref PubMed Scopus (166) Google Scholar, 13Watt FM Hogan BL Out of Eden: stem cells and their niches.Science. 2000; 287: 1427-1430Crossref PubMed Scopus (1453) Google Scholar, 14Watt FM Stem cell fate and patterning in mammalian epidermis.Curr Opin Gene Dev. 2001; 11: 410-417Crossref PubMed Scopus (206) Google Scholar, 15Weissman IL Anderson DJ Gage F Stem and progenitor cells: origins, phenotypes, lineage commitments, and transdifferentiations.Annu Rev Cell Dev Biol. 2001; 17: 387-403Crossref PubMed Scopus (754) Google Scholar To date, stem cells have been identified that are restricted to the trachea, submucosal gland ducts, and neuroepithelial bodies (NEBs) of the bronchiolar epithelium.16Borthwick DW Shahbazian M Krantz QT Dorin JR Randell SH Evidence for stem-cell niches in the tracheal epithelium.Am J Respir Cell Mol Biol. 2001; 24: 662-670Crossref PubMed Scopus (329) Google Scholar, 17Engelhardt JF Schlossberg H Yankaskas JR Dudus L Progenitor cells of the adult human airway involved in submucosal gland development.Development. 1995; 121: 2031-2046PubMed Google Scholar, 18Engelhardt JF Stem cell niches in the mouse airway.Am J Respir Cell Mol Biol. 2001; 24: 649-652Crossref PubMed Scopus (85) Google Scholar, 19Hong KU Reynolds SD Giangreco A Hurley CM Stripp BR Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion.Am J Respir Cell Mol Biol. 2001; 24: 671-681Crossref PubMed Scopus (367) Google Scholar, 20Reynolds SD Giangreco A Power JH Stripp BR Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration.Am J Pathol. 2000; 156: 269-278Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar Even though cells that contribute to the renewal of terminal bronchioles have also been shown to exist using a model of lung progenitor cell depletion, the precise location and characteristics of putative stem cells have not been described. The terminal bronchiole is a particularly resilient region of the airway that exhibits rapid epithelial renewal in response to depletion of either terminally differentiated (ciliated) or TA cell populations. Clara cells function as the sole TA cell that participates in maintenance of both secretory and ciliated cell types after oxidant-mediated damage.21Evans MJ Cabral-Anderson LJ Freeman G Role of the Clara cell in renewal of the bronchiolar epithelium.Lab Invest. 1978; 38: 648-653PubMed Google Scholar Importantly, depletion of this TA cell population through administration of the Clara cell-specific cytotoxicant naphthalene fails to diminish the regenerative potential of terminal bronchioles.20Reynolds SD Giangreco A Power JH Stripp BR Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration.Am J Pathol. 2000; 156: 269-278Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 22Van Winkle LS Buckpitt AR Nishio SJ Isaac JM Plopper CG Cellular response in naphthalene-induced Clara cell injury and bronchiolar epithelial repair in mice.Am J Physiol. 1995; 269: L800-L818PubMed Google Scholar, 23Van Winkle LS Isaac JM Plopper CG Distribution of epidermal growth factor receptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse.Am J Pathol. 1997; 151: 443-459PubMed Google Scholar After Clara cell depletion, residual cells of terminal bronchioles are fully capable of epithelial renewal and restoration of differentiated cell phenotypes appropriate for this region.22Van Winkle LS Buckpitt AR Nishio SJ Isaac JM Plopper CG Cellular response in naphthalene-induced Clara cell injury and bronchiolar epithelial repair in mice.Am J Physiol. 1995; 269: L800-L818PubMed Google Scholar, 23Van Winkle LS Isaac JM Plopper CG Distribution of epidermal growth factor receptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse.Am J Pathol. 1997; 151: 443-459PubMed Google Scholar These data support the notion that a population of pollutant-resistant stem cells participate in regeneration of the surrounding terminal bronchiolar epithelium after TA cell depletion. Even though the molecular and phenotypic characteristics of stem cell populations vary according to the tissue in which they reside, a number of unifying properties are used to distinguish this category of regenerative cells from their TA counterparts.13Watt FM Hogan BL Out of Eden: stem cells and their niches.Science. 2000; 287: 1427-1430Crossref PubMed Scopus (1453) Google Scholar Characteristics of stem cells include a relatively undifferentiated phenotype, pluripotent differentiation potential, a low rate of steady-state proliferation, an extended life span, and localization to a specific microenvironment commonly referred to as a stem cell niche.10Lehrer MS Sun TT Lavker RM Strategies of epithelial repair: modulation of stem cell and transit amplifying cell proliferation.J Cell Sci. 1998; 111: 2867-2875Crossref PubMed Google Scholar, 13Watt FM Hogan BL Out of Eden: stem cells and their niches.Science. 2000; 287: 1427-1430Crossref PubMed Scopus (1453) Google Scholar, 24Spradling A Drummond-Barbosa D Kai T Stem cells find their niche.Nature. 2001; 414: 98-104Crossref PubMed Scopus (1201) Google Scholar, 25Quesenberry PJ Becker PS Stem cell homing: rolling, crawling, and nesting.Proc Natl Acad Sci USA. 1998; 95: 15155-15157Crossref PubMed Scopus (123) Google Scholar Using these criteria, we have previously demonstrated the existence of a Clara cell secretory protein (CCSP)-expressing (CE) cell population within NEB microenvironments of the bronchiolar epithelium that functions as an airway stem cell.20Reynolds SD Giangreco A Power JH Stripp BR Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration.Am J Pathol. 2000; 156: 269-278Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar CE stem cells and TA cell populations within the airway epithelium have been distinguished primarily by differences in pollutant susceptibility, proliferative and differentiation potential, and spatial localization. Depletion of both of these populations via transgene-mediated ablation of all CCSP-positive cells results in an overall failure of bronchiolar epithelial renewal.19Hong KU Reynolds SD Giangreco A Hurley CM Stripp BR Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion.Am J Respir Cell Mol Biol. 2001; 24: 671-681Crossref PubMed Scopus (367) Google Scholar, 26Reynolds SD Hong KU Giangreco A Mango GW Guron C Morimoto Y Stripp BR Conditional Clara cell ablation reveals a self-renewing progenitor function of pulmonary neuroendocrine cells.Am J Physiol. 2000; 278: L1256-L1263PubMed Google Scholar These results indicate the importance of CE cells in bronchiolar regeneration and led us to hypothesize that phenotypically similar populations of stem cells may regulate epithelial renewal in both the proximal and terminal bronchioles.19Hong KU Reynolds SD Giangreco A Hurley CM Stripp BR Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion.Am J Respir Cell Mol Biol. 2001; 24: 671-681Crossref PubMed Scopus (367) Google Scholar, 20Reynolds SD Giangreco A Power JH Stripp BR Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration.Am J Pathol. 2000; 156: 269-278Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar Goals of the present study were to investigate this hypothesis through defining the molecular phenotype of cells contributing to repair of terminal bronchioles, their localization, and their rate of proliferation in the steady-state and after injury. Results indicate that regeneration of the terminal bronchiole after injury is mediated by CE stem cells with characteristics similar to those located within NEB microenvironment, but that this stem cell functions within a unique NEB-independent niche located at the bronchoalveolar duct junction (BADJ). Adult male FVB/n wild-type mice (2 to 4 months of age) were housed under specific pathogen-free conditions and tested quarterly using a 16-agent serological sentinel-screening program. Animals were maintained under a 12-hour light/dark cycle, and allowed access to both food and water ad libitum. Naphthalene (Sigma, St. Louis, MO) was dissolved in Mazola corn oil and administered to mice via intraperitoneal injection before 10:00 a.m. Animals were briefly anesthetized with aerosolized halothane before exposure to facilitate weighing and injections. Mice received 275 mg of naphthalene/kg body weight; control animals received a comparable volume (10 ml/kg body weight) of corn oil alone. [3H]-Thymidine (2.5 mCi/kg) was injected intraperitoneally to mice 1 hour before sacrifice for pulse labeling. For continuous labeling studies, [3H]-thymidine (1.0 mCi/ml) was administered throughout a 7-day period via a subcutaneous miniosmotic pump (Alzet model 2002, 0.5 μl/hour). Alzet pumps were implanted 6 hours after naphthalene administration and removed after the labeling period. Paraffin-embedded lung tissue was sectioned at 5 μm to reveal the major axial pathway and adhered to glass slides. Slides were blocked with 0.5% bovine serum albumin (Sigma)/5% normal goat serum (Vector Laboratories, Burlingame, CA) in phosphate-buffered saline (PBS) (blocking solution) for 30 minutes. Adjacent serial sections were incubated with polyclonal rabbit anti-CCSP (1:30,000)26Reynolds SD Hong KU Giangreco A Mango GW Guron C Morimoto Y Stripp BR Conditional Clara cell ablation reveals a self-renewing progenitor function of pulmonary neuroendocrine cells.Am J Physiol. 2000; 278: L1256-L1263PubMed Google Scholar or rabbit anti-calcitonin gene-related peptide (CGRP) (l:16,000, Sigma)26Reynolds SD Hong KU Giangreco A Mango GW Guron C Morimoto Y Stripp BR Conditional Clara cell ablation reveals a self-renewing progenitor function of pulmonary neuroendocrine cells.Am J Physiol. 2000; 278: L1256-L1263PubMed Google Scholar overnight at 4°C in blocking solution. Slides were washed three times in PBS, and incubated with biotinylated goat anti-rabbit Ig (1:4000 in blocking solution, Sigma). Slides were washed and streptavidin-horseradish peroxidase (Sigma) was added at a 1:4000 dilution in PBS. Antigen-antibody complexes were detected using a diaminobenzidine substrate detection kit (Vector Laboratories). For detection of incorporated [3H]-thymidine, immunostained slides were washed overnight in PBS, dehydrated, and dried. Slides were coated with NTB-2 emulsion (Kodak, Rochester, NY), dried, stored at 4°C for 2 weeks, and developed according to the manufacturer's instructions. All slides are counterstained with Mayer's hematoxylin to identify nuclei. Standard immunofluorescence methods were used to simultaneously detect CGRP and CCSP on 5-μm tissue sections.19Hong KU Reynolds SD Giangreco A Hurley CM Stripp BR Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion.Am J Respir Cell Mol Biol. 2001; 24: 671-681Crossref PubMed Scopus (367) Google Scholar Rabbit anti-CGRP (1:4000) and goat-anti-CCSP (1:10,000) were diluted in 3% bovine serum albumin/PBS. After a 24-hour incubation at 4°C, slides were incubated simultaneously with Alexa 488-conjugated donkey anti-goat Ig and Alexa 594-conjugated donkey anti-rabbit Ig (1:125 dilution; Molecular Probes, Eugene, OR) overnight at 4°C in the dark. All slides were mounted with 2 μg/ml of 4,6-diamidino-2-phenylindole (Sigma) in Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL) and visualized with an AX70 microscope equipped with a 4,6-diamidino-2-phenylindole/Texas Red dual optical excitation filter cube and a fluorescein isothiocyanate optical excitation filter cube (Olympus, Melville, NY). CCSP mRNA was detected on 5-μm paraffin sections of lung tissue using [35S] anti-sense riboprobes. Conditions for hybridization were essentially as described.27Wert SE Glasser SW Korfhagen TR Whitsett JA Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice.Dev Biol. 1993; 156: 426-443Crossref PubMed Scopus (268) Google Scholar Slides were washed under high stringency conditions, coated with Kodak NTB2 emulsion, exposed for 16 hours, and developed according to the manufacturer's instructions. Terminal bronchioles were defined by the presence of an intact BADJ, at least 200 μm of conducting airway epithelium, and a visible alveolar duct. All cells within 200 μm of the BADJ exhibiting a nuclear profile and clear attachment to the basement membrane were included in cell density analyses. Previous studies have used a similar methodology for analysis of neuroepithelial body (NEB) microenvironments.20Reynolds SD Giangreco A Power JH Stripp BR Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration.Am J Pathol. 2000; 156: 269-278Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar For proliferation analysis, cells were defined as “labeled” if they contained at least five grains localized over a visible nucleus. “Label retaining cells” were defined as those containing >15 grains/nucleus. Mean values for cellular density, percent Clara cells, and proliferative index were compared to control values using the Student's t-test. Statistical significance for all comparisons was accepted at P < 0.05. The terminal bronchiolar epithelium of mice is a particularly sensitive site for naphthalene toxicity, exposure to which results in rapid and extensive depletion of Clara cells.22Van Winkle LS Buckpitt AR Nishio SJ Isaac JM Plopper CG Cellular response in naphthalene-induced Clara cell injury and bronchiolar epithelial repair in mice.Am J Physiol. 1995; 269: L800-L818PubMed Google Scholar, 28Stripp BR Maxson K Mera R Singh G Plasticity of airway cell proliferation and gene expression after acute naphthalene injury.Am J Physiol. 1995; 269: L791-L799PubMed Google Scholar Although Clara cells are the principal progenitor for this region of the conducting airway,21Evans MJ Cabral-Anderson LJ Freeman G Role of the Clara cell in renewal of the bronchiolar epithelium.Lab Invest. 1978; 38: 648-653PubMed Google Scholar repair of terminal bronchioles is rapidly induced and virtually complete 2 weeks after administration of 200 mg/kg of naphthalene.22Van Winkle LS Buckpitt AR Nishio SJ Isaac JM Plopper CG Cellular response in naphthalene-induced Clara cell injury and bronchiolar epithelial repair in mice.Am J Physiol. 1995; 269: L800-L818PubMed Google Scholar, 28Stripp BR Maxson K Mera R Singh G Plasticity of airway cell proliferation and gene expression after acute naphthalene injury.Am J Physiol. 1995; 269: L791-L799PubMed Google Scholar, 29Born SL Fix AS Caudill D Lehman-McKeeman LD Selective Clara cell injury in mouse lung following acute administration of coumarin.Toxicol Appl Pharmacol. 1998; 151: 45-56Crossref PubMed Scopus (39) Google Scholar, 30Van Winkle LS Isaac JM Plopper CG Repair of naphthalene-injured microdissected airways in vitro.Am J Respir Cell Mol Biol. 1996; 15: 1-8Crossref PubMed Scopus (27) Google Scholar, 31Van Winkle LS Johnson ZA Nishio SJ Brown CD Plopper CG Early events in naphthalene-induced acute Clara cell toxicity: comparison of membrane permeability and ultrastructure.Am J Respir Cell Mol Biol. 1999; 21: 44-53Crossref PubMed Scopus (82) Google Scholar Even though NEB-associated CCSP-expressing (CE) cells have been identified as a naphthalene-resistant stem cell pool that contributes to renewal of proximal bronchiolar epithelium, there is still debate over the identity and properties of cells contributing to renewal of terminal bronchiolar (TB) epithelium. To identify regions of the TB epithelium that maintained naphthalene-resistant cells, mice were exposed to 275 mg/kg of naphthalene, recovered 24 hours, and the phenotype of pollutant-resistant cells assessed by dual-color fluorescence immunostaining for the Clara cell-specific marker (CCSP, green immunofluorescence) or the pulmonary neuroendocrine cell (PNEC)-specific marker calcitonin gene-related peptide (CGRP, red immunofluorescence). In the distal airway of untreated animals, CE cells accounted for 60 to 90% of epithelial cells whereas PNECs comprise only a small percentage of this population (Figure 1A). Naphthalene exposure resulted in extensive exfoliation of CE cells within 24 hours (Figure 1B). Clusters of pollutant-resistant CE cells that have been previously shown to serve as regenerative foci are maintained adjacent to NEBs of bronchi (Figure 1B, asterisk).19Hong KU Reynolds SD Giangreco A Hurley CM Stripp BR Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion.Am J Respir Cell Mol Biol. 2001; 24: 671-681Crossref PubMed Scopus (367) Google Scholar, 20Reynolds SD Giangreco A Power JH Stripp BR Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration.Am J Pathol. 2000; 156: 269-278Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar Interestingly, a second population of pollutant-resistant CE cells that did not appear to co-localize with CGRP-immunoreactive PNECs was located within TBs adjacent to the BADJ. (Figure 1B, arrow). This observation suggested that the BADJ may represent a second distinct regenerative microenvironment within bronchioles in addition to the previously characterized NEB microenvironment. To determine whether NEB-independent pollutant-resistant cells of terminal bronchioles are capable of appropriate epithelial renewal, airways of mice were compared 45 days after administration of 275 mg/kg of naphthalene or vehicle (corn oil). Efficacy of epithelial renewal within terminal bronchioles was determined by comparison of epithelial cell density and the representation of CCSP-immunoreactive cells in untreated and treated animals. No qualitative differences in the abundance of CCSP within immunoreactive cells of corn oil and naphthalene-exposed groups were observed (Figure 2, A and B, respectively). A small yet significant reduction in terminal bronchiolar cell density was observed in treated animals (Figure 2C). This decrease in cell density was not associated with a shift in the representation of CE cells at this location (Figure 2D). These data demonstrate that terminal bronchioles contain a regenerative cell population capable of restoring an airway of appropriate cellular diversity after depletion of the TA (Clara) cell population. To investigate the kinetics of Clara cell ablation within terminal bronchioles and to identify the location and cell type responsible for initial epithelial renewal, mice were exposed to either corn oil or 275 mg/kg of naphthalene, recovered for 6, 12, 24, or 36 hours, and injected with 2.5 mCi/kg [3H]-thymidine (TdR) 1 hour before sacrifice. Clara cells were identified by immunohistochemistry for CCSP and proliferating cells were identified by autoradiographic detection of incorporated [3H]-TdR (Figure 3). Six hours after naphthalene exposure Clara cells exhibited a loss of apical projections without significant alterations in overall cell density as determined by CCSP immunoreactivity (Figure 3, compare A and B). Exfoliating CCSP-immunoreactive cells were evident beginning at the 12-hour time point (Figure 3C). This change was associated with a reduction in both the overall cell density and percentage of Clara cells within the epithelial population. Within 24 hours, maximal CE cell ablation was achieved as determined by the absence of exfoliating cell bodies (Figure 3D). The previously identified population of pollutant-resistant CE cells (Figure 1B) was identifiable directly adjacent to the BADJ at this time point (Figure 3D). Although no epithelial proliferation was observed in any terminal bronchiolar cell populations at this early recovery time point, proliferating fibroblasts that serve as a control for appropriate [3H]-TdR incorporation and detection were identified at this and all other time points analyzed (Figure 3, B, E arrowheads). Proliferative epithelial cells were first observed 36 hours after naphthalene administration and exhibited a rounded morphology (Figure 3E). At this time point 0.6 ± 0.1 proliferative cells were detected per 100 μm (approximately one labeled cell per terminal bronchiole), of which CE pollutant-resistant cells represent 90 ± 15%. These data suggest a critical role for these cells in renewal of the distal conducting airway. A caveat to identification of BADJ-associated CCSP-positive cells as CE cells is the relatively long half-life of CCSP that may result in immunoreactivity in cells that have undergone a de- or trans-differentiative process.32Miele L Cordella-Miele E Mantile G Peri A Mukherjee AB Uteroglobin and uteroglobin-like proteins: the uteroglobin family of proteins.J Endocrinol Invest. 1994; 17: 679-692PubMed Google Scholar, 33Warburton D Wuenschell C Flores-Delgado G Anderson K Commitment and differentiation of lung cell lineages.Biochem Cell Biol. 1998; 76: 971-995Crossref PubMed Scopus (64) Google Scholar To ascertain whether pollutant-resistant, BADJ-associated, CCSP-immunopositive cells actively transcribe CCSP, as well as to more fully characterize their molecular phenotype, the distribution of CCSP mRNA in TBs of control and naphthalene-exposed mice was determined by in situ hybridization (Figure 4, A and B). CE cells present 24 hours after exposure were analyzed as these cells represent a residual, resistant population rather than nascent cells that may be the product of epithelial regeneration. Nonspecific hybridization signal was minimal as determined by anti-sense [35S]-labeled CCSP probe hybridization to tissue from CCSP−/− mice (Figure 4C). CCSP mRNA was detected throughout the terminal bronchiole of untreated wild-type mice (Figure 4A). In contrast, terminal bronchioles from naphthalene-treated mice maintained a subset of CCSP mRNA-positive cells directly adjacent to the BADJ (Figure 4B). The levels of CCSP mRNA in this region were reduced compared with those of control, yet they are significantly greater than background levels. Although the cellular specificity of this transcript cannot be determined using [35S]-labeled probes, the proximity of CCSP mRNA-positive cells to the BADJ correlates with immunohistochemical data (Figure 1, Figure 3) . These data support the notion that pollutant-resistant cells of the BADJ are indeed a CE population. The phenotype, frequency, and spatial distribution of the initial cohort of proliferating cells within the TBs were determined 36 hours after naphthalene exposure using the BADJ as a reference po
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