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CC16 is almost exclusively expressed in non-ciliated epithelial Clara cells, and widely used as a Clara cell marker. Diesel exhaust particles (DEPs), the fine particulate matters produced by diesel engines, cause or exacerbate airway-related diseases. Our previous study documented that DEP inhibits the CC16 expression in the immortalized mouse Clara cell line through methylation of C/EBPα promoter. However, the molecular mechanism by which DEP regulates CC16 secretion is unclear. Here, we isolated CC16 containing Clara cells (CC16 + ) from human distal lung, and found that DEP inhibited CC16 secretion from CC16 + cells via methylation of C/EBPα and inhibition of Munc18b transcription. CC16 + cell conditioned media containing different concentrations of CC16 was prepared and used for culture of airway epithelial cells BEAS-2B with no expression of CC16. A positive correlation was observed between CC16 level and DEP-induced autophagy activity, and a negative correlation between CC16 level and DEP-induced pro-inflammatory cytokine TNF-α, IL-6, and IL-8 level, suggesting that CC16 might mitigate DEP-induced inflammation via promoting autophagy in BEAS-2B cells. This result was further confirmed by adding recombinant CC16 to BEAS-2B cells exposed to DEP. Moreover, CC16 level was significantly increased when CC16 + cells were cultured in BEAS-2B cell conditioned medium containing TNF-α or the normal medium supplemented with recombinant TNF-α, suggesting that TNF-α induced CC16 production and secretion from CC16 + cells. Collectively, these data point that CC16 and TNF-α form a negative feedback loop, and this negative feedback loop between Clara cells and normal airway epithelial cells protects against DEP exposure-induced inflammation.

Club cells are critical in maintaining airway integrity via, in part, secretion of immunomodulatory Club cell 10 kd protein (CC10) and xenobiotic detoxification. Aryl hydrocarbon receptor (AhR) is important in xenobiotic metabolism, but its role in Club cell function is unclear. To this end, an AhR ligand, 6-formylindolo[3,2-b]carbazole (FICZ, 10 nM) was found to induce, in a ligand and AhR-dependent manner, endoplasmic reticulum stress, phospholipid remodeling, free fatty acid and triglyceride synthesis, leading to perilipin 2-dependent lipid droplet (LD) biogenesis in a Club cell-like cell line, NL20. The increase in LDs was due, in part, to the blockade of adipose triglyceride lipase to LDs, while perilipin 5 facilitated LDs-mitochondria connection, leading to the breakdown of LDs via mitochondrial β-oxidation and acetyl-coA generation. In FICZ-treated cells, increased CC10 secretion and its intracellular association with LDs were noted. Administration of low (0.28 ng), medium (1.42 ng), and high (7.10 ng) doses of FICZ in C57BL/6 mice significantly enhanced lipopolysaccharide (LPS, 0.1 μg)-induced airway inflammation, mucin secretion, pro-inflammatory cytokines and CC10 in the bronchoalveolar lavage fluids, as compared to those seen in mice receiving LPS alone, suggesting the importance of AhR signaling in controlling the metabolic homeostasis and functions of Club cells.

Thioredoxin reductase-1 (TXNRD1) inhibition activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2) responses and prevents acute lung injury (ALI). Heme oxygenase-1 (HO-1) induction following TXNRD1 inhibition is Nrf2-dependent in airway epithelial (club) cells in vitro . The influence of club cell HO-1 on lung development and lung injury responses is poorly understood. The present studies characterized the effects of hyperoxia on club cell-specific HO-1 knockout (KO) mice. These mice were generated by crossing Hmox1 flox mice with transgenic mice expressing cre recombinase under control of the club cell-specific Scgb1a1 promoter. Baseline analyses of lung architecture and function performed in age-matched adult wild-type and KO mice indicated an increased alveolar size and airway resistance in HO-1 KO mice. In subsequent experiments, adult wild-type and HO-1 KO mice were either continuously exposed to >95% hyperoxia or room air for 72 h or exposed to >95 hyperoxia for 48 h followed by recovery in room air for 48 h. Injury was quantitatively assessed by calculating right lung/body weight ratios (g/kg). Analyses indicated an independent effect of hyperoxia but not genotype on right lung/body weight ratios in both wild-type and HO-1 KO mice. The magnitude of increases in right lung/body weight ratios was similar in mice of both genotypes. In the recovery model, an independent effect of hyperoxia but not genotype was also detected. In contrast to the continuous exposure model, right lung/body weight ratio mice were significantly elevated in HO-1 KO but not wild-type mice. Though club cell HO-1 does not alter hyperoxic sensitivity in adult mice, it significantly influences lung development and resolution of lung injury following acute hyperoxic exposure., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Katelyn Dunigan-Russell et al.)

The Non-Ciliated Bronchiolar Cell (NCBC) is responsible for the defense and maintenance of the bronchiolar epithelium. Several cellular defense mechanisms have been associated with an increase in the secretion of CC16 and changes in the phenotype of the cell; these mechanisms could be linked to tolerance to the damage due to exposure to inhaled Particulate Matter (PM) of the epithelium. These defense mechanisms have not been sufficiently explored. In this article, we studied the response of the NCBC to inhaled vanadium, an element which adheres to PM. This response was measured by the changes in the phenotype of the NCBC and the secretion of CC16 in a mouse model. Mice were exposed in two phases to different vanadium concentrations; 1.27 mg/m³ in the first phase and 2.56 mg/m³ in the second phase. Mice were sacrificed on the 2nd, 4th, 5th, 6th and 8th weeks. In the second phase, we observed the following: sloughing of the NCBC, hyperplasia and small inflammatory foci remained without changes and that the expression of CC16 was higher in this phase than in phase I. We also observed a change in the phenotype with a slow decrease in both phases. The increase in the secretion of CC16 and the phenotype reversion could be due to the anti-inflammatory activity of CC16. The changes observed in the second phase could be attributed to the tolerance to inhaled vanadium.

Bisphenol A (BPA) is an endocrine disrupting compound that is a pervasive environmental contaminant. Although it has been reported to affect the development of a variety of fetal reproductive tissues, data on the effect of fetal BPA exposure on oviducts were extremely limited and were only available in mice. To determine if there are adverse effects of gestational BPA exposure on fetal oviduct, we exposed pregnant rhesus macaques with female fetuses to oral or nonoral BPA during the last trimester of gestation (day 100 to term). After the treatment, fetal oviducts were collected for morphology evaluation. BPA exposure altered the percentages of different cell types (ciliated, nonciliated, and secretory) in the fetal oviduct and resulted in a significant high ciliated cell population in the BPA-exposed fetal oviduct. The distribution of ciliated cells on the epithelium in the BPA-exposed fetal oviduct was also altered. Gestational BPA exposure reduced the expression of mucosubstance and uteroglobin in secretory cells in the fetal oviduct. A comparison of the outcome of the fetal oviduct studies with similar outcomes previously reported in the lung from the same fetuses demonstrates that BPA exhibits opposite effects in these two organs. In conclusion, the BPA-associated alterations in the fetal oviduct could potentially affect the oviduct morphology and function later in life with a negative impact on fertility. The mechanisms of action of the differential response in the oviduct and the lung to BPA exposure require further investigation.

Epithelial-mesenchymal transition (EMT) occurring in alveolar epithelial cells plays an important role in the development and progression of pulmonary fibrosis. Previous studies showed that antiflammin-1 (the active fragment of uteroglobin) effectively inhibited bleomycin-induced pulmonary fibrosis. However, its mechanism is still far from being clarified. In this study, we investigated the effects of antiflammin-1 on EMT in A549 cells induced by transforming growth factor-β1 (TGF-β1) and the underlying mechanism by using morphological observation and Western blot. The results showed that the expression of α-smooth muscle actin (α-SMA) increased significantly while the expression of E-cadherin decreased significantly in A549 cells following treatment with TGF-β1 concomitant with morphological change of A549 cells from pebble-like shape epithelial cells to spindle-like mesenchymal shape. This process of EMT in A549 cells induced by TGF-β1 was significantly inhibited when A549 cells were co-incubated with TGF-β1 and antiflammin-1. Furthermore, the anti-lipocalin interacting membrane receptor (LIMR) antibody and PD98059 (an ERK signaling pathway blocker) attenuated the inhibitory effect of antiflammin-1 on TGF-β1-induced EMT, respectively. Our findings indicate that antiflammin-1 can inhibit EMT in A549 cells induced by TGF-β1, which is related to LIMR and its downstream ERK signaling pathway.

Background: Chronic persistent airway inflammation has been associated with the comorbidity of asthma and bipolar disorder (BD). However, the direct relevance between airway inflammation and BD-like psychiatric comorbidity is almost unknown. Integrin β4 (ITGB4) is downregulated on the airway epithelial of asthma patients, which might play a critical role in the parthenogenesis of airway inflammation. So this study aimed to examine the role of ITGB4 deficiency in mediating airway inflammation and further leading to the BD-like behaviors., Methods: ITGB4 -/- mice were generated by mating ITGB4 fl/fl mice with CCSP-rtTA tg/- /TetO-Cretg/tg mice. Mania-like behavior tests were performed, including hyperlocomotion, D-amphetamine-induced hyperactivity, open-field test, and elevated plus-maze test. Depressive-like behavior tests were carried out, including sucrose preference, forced swimming, and learned helplessness. Inflammatory cells (Th17, Th1, Th2) in the lung were examined by flow cytometry. Futhermore, inflammatory cytokines (IL-4, IL-13) in bronchoalveolar lavage fluid and sera were detected by ELISA. Protein expression of the IL-4Rα on choroid plexus, microglial marker (IBA1), and synapse-associated proteins (synaptophysin, SYP) in the hippocampus and prefrontal cortex were examined by western blotting. Additionally, proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in the hippocampus and prefrontal cortex were detected by immunohistochemistry. Inflammatory disorder in the lung, hippocampus, and prefrontal cortex was tested by hematoxylin and eosin (H&E) staining. And cell apoptosis in the hippocampus and prefrontal cortex was measured by TUNEL test., Results: ITGB4 -/- mice exhibited mania-like behavior, including hyperlocomotion, D-amphetamine-induced hyperactivity, and reduced anxiety-like behavior. While under stressful conditions, ITGB4 -/- mice manifested depressive-like behavior, including anhedonia, behavioral despair, and enhanced learned helplessness. At the same time, ITGB4 -/- mice mainly exerted Th2-type inflammation in periphery, like the number and major cytokines IL-4 and IL-13 of Th2-type inflammation. ITGB4 -/- mice also showed a significant increase of microglia and pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α in the hippocampus and prefrontal cortex. Additionally, neuron damage, increased neuron apoptosis, and the decrease of SYP were found in ITGB4 -/- mice., Conclusions: These findings confirmed that airway inflammatory induced by ITGB4 deficiency is the important incentive for the BD-like behavior during asthma pathogenesis. The ITGB4-deficient mice provide a validated animal model for us to study the possible mechanism of BD-like psychiatric comorbidity of asthma patients.

Background: Club cell protein-16 (CC16) expression has been associated with smoking-related lung function decline. The study hypothesis was that CC16 expression in both serum and bronchial epithelium is associated with lung function decline in smokers, and exposure to cigarette smoke will lead to reduction in CC16 expression in bronchial epithelial cells., Methods: In a cohort of community-based male Chinese subjects recruited for lung function test in 2000, we reassessed their lung function ten years later and measured serum levels of CC16. CC16 expression was further assayed in bronchial epithelium from endobronchial biopsies taken from an independent cohort of subjects undergoing autofluorescence bronchoscopy, and tested for correlation between CC16 immunostaining intensity and lung function. In an in-vitro model, bronchial epithelial cells were exposed to cigarette smoke extract (CSE), and the expression levels of CC16 were measured in bronchial epithelial cells before and after exposure to CSE., Results: There was a significant association between FEV 1 decline and serum CC16 levels in smokers. Expression of CC16 in bronchial epithelium showed significant correlation with FEV 1 /FVC. Bronchial epithelial cells showed significant decrease in CC16 expression after exposure to CSE, followed by a subsequent rise in CC16 expression upon removal of CSE., Conclusions: Results of these clinical and laboratory investigations suggested that low serum CC16 was associated with smoking-related decline in lung function, demonstrated the first time in a Chinese cohort. The data also lend support to the putative role of CC16 in protection against smoking-related bronchial epithelial damage. (Abstract word count: 243) US CLINICAL TRIAL REGISTRY: NCT01185652 , first posted 20 August, 2010.

We report here the establishment and characterization of an in vitro human small airway model (SmallAir™). The epithelial cells were isolated from the distal lungs by enzymatic digestion. After amplification, the cells were seeded on the microporous membrane of Transwell inserts. Once confluent, the cultures were switched to air-liquid interface. After 3weeks of culture, the epithelium became fully differentiated, with morphology of columnar epithelium, and a thickness of 10-15μm. Most significantly, CC-10, a specific marker of Club cells, was highly expressed in SmallAir™. CC-10 was detected by both immune-cytochemistry and Western Blot. As expected, SmallAir™ contained few Muc5-Ac positive cells (goblet cells). In contrast, CC-10 was not detected in MucilAir™, an in vitro model of the human nasal and bronchial epithelial model. Instead, Muc5-Ac was highly expressed in MucilAir™. However, both MucilAir™ and SmallAir™ contain basal cells and ciliated cells, showing cilia beating and mucociliary clearance. Clearly, MucilAir™ and SmallAir™ are two distinct airway epithelial models., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Klebsiella pneumoniae is a common cause of pneumonia. Previous studies have documented an important role for Toll-like receptors (TLRs) expressed by myeloid cells in the recognition of K. pneumoniae and the initiation of a protective immune response. Lung epithelial cells also express TLRs and can participate in innate immune defense. The aim of this study was to examine the role of the common TLR adaptor protein myeloid-differentiation factor (MyD) 88 in lung epithelium during host defense against K. pneumoniae-induced pneumonia. To this end, we first crossed mice expressing cre recombinase under the control of the surfactant protein C (SftpCcre) or the club cell 10 kD (CC10cre) promoter with reporter mice to show that SftpCcre mice mainly express cre in type II alveolar cells, whereas CC10cre mice express cre almost exclusively in bronchiolar epithelial cells. We then generated mice with cell-targeted deletion of MyD88 in type II alveolar (SftpCcre-MyD88-lox) and bronchiolar epithelial (CC10cre-MyD88-lox) cells, and infected them with K. pneumoniae via the airways. Bacterial growth and dissemination were not affected by the loss of MyD88 in SftpCcre-MyD88-lox or CC10cre-MyD88-lox mice compared with control littermates. Furthermore, inflammatory responses induced by K. pneumoniae in the lung were not dependent on MyD88 expression in type II alveolar or bronchiolar epithelial cells. These results indicate that MyD88 expression in two distinct lung epithelial cell types does not contribute to host defense during pneumonia caused by a common human gram-negative pathogen.

Clara cell protein (CC16) is an anti-inflammatory protein, which is expressed in the airway epithelium. It is involved in the development of airway inflammatory diseases, including chronic obstructive pulmonary disease and asthma. However, the exact molecular mechanism underlying its anti‑inflammatory action remains to be fully elucidated. The aim of the present study was to define the protein profiles of the anti‑inflammatory effect of CC16 in lipopolysaccharide (LPS)‑treated rat tracheal epithelial (RTE) cells using shotgun proteomics. Protein extracts were obtained from control RTE cells, RTE cells treated with LPS and RTE cells treated with LPS and recombinant CC16 (rCC16). Subsequent label‑free quantification and bioinformatics analyses identified 12 proteins that were differentially expressed in the three treatment groups as a cluster of five distinct groups according to their molecular functions. Five of the twelve proteins were revealed to be associated with the cytoskeleton: Matrix metalloproteinase‑9, myosin heavy chain 10, actin‑related protein‑3 homolog, elongation factor 1‑α‑1 (EF‑1‑α‑1), and acidic ribosomal phosphoprotein P0. Five of the twelve proteins were associated with cellular proliferation: DNA‑dependent protein kinase catalytic subunit, EF‑1‑α‑1, tyrosine 3‑monooxygenase, caspase recruitment domain (CARD) protein 12 and adenosylhomocysteinase (SAHH) 3. Three proteins were associated with gene regulation: EF‑1‑α‑1, SAHH 3 and acidic ribosomal phosphoprotein P0. Three proteins were associated with inflammation: Tyrosine 3‑monooxygenase, CARD protein 12 and statin‑related protein. ATPase (H+‑transporting, V1 subunit A, isoform 1) was revealed to be associated with energy metabolism, and uridine diphosphate glycosyltransferase 1 family polypeptide A8 with drug metabolism and detoxification. The identified proteins were further validated using reverse transcription‑quantitative polymerase chain reaction. These protein profiles, and their interacting protein network, may facilitate the elucidation of the molecular mechanisms underlying the anti‑inflammatory effects of CC16.

Protein adduction is considered to be critical to the loss of cellular homeostasis associated with environmental chemicals undergoing metabolic activation. Despite considerable effort, our understanding of the key proteins mediating the pathologic consequences from protein modification by electrophiles is incomplete. This work focused on naphthalene (NA) induced acute injury of respiratory epithelial cells and tolerance which arises after multiple toxicant doses to define the initial cellular proteomic response and later protective actions related to tolerance. Airways and nasal olfactory epithelium from mice exposed to 15 ppm NA either for 4 h (acute) or for 4 h/day × 7 days (tolerant) were used for label-free protein quantitation by LC/MS/MS. Cytochrome P450 2F2 and secretoglobin 1A1 are decreased dramatically in airways of mice exposed for 4 h, a finding consistent with the fact that CYPs are localized primarily in Clara cells. A number of heat shock proteins and protein disulfide isomerases, which had previously been identified as adduct targets for reactive metabolites from several lung toxicants, were upregulated in airways but not olfactory epithelium of tolerant mice. Protein targets that are upregulated in tolerance may be key players in the pathophysiology associated with reactive metabolite protein adduction. All MS data have been deposited in the ProteomeXchange with identifier PXD000846 (http://proteomecentral.proteomexchange.org/dataset/PXD000846)., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The contribution of bone marrow cells (BMC) in lung repair is controversial. We previously reported a subpopulation of BMC that express Clara cell secretory protein (CCSP). To determine the contribution of endogenous CCSP(+) BMC to airway regeneration, we performed bone marrow transplantation studies using the CCtk mouse, which expresses a thymidine kinase suicide gene under regulation of the CCSP promoter. Mice were transplanted with wild-type or CCtk BMC and treated with ganciclovir to eliminate CCSP(+) cells. After airway injury using naphthalene, mice depleted of CCSP(+) BMC had more inflammatory cells in lung and decreased levels of oxygen in arterial blood. They also had reduced expression of airway epithelial genes and less Clara cells compared to control mice that had intact CCSP(+) BMC and bone marrow derived CCSP(+) cells in the airways. After naphthalene injury, administration of CCSP reproduced the beneficial effect of CCSP(+) BMC by improving recovery of airway epithelium, reducing lung inflammation and increasing oxygen in arterial blood from mice depleted of CCSP(+) BMC. Our data demonstrate that ablation of CCSP(+) BMC delays airway recovery and suggests the beneficial effect of CCSP(+) BMC in lung recovery is in part due to production of CCSP itself.

The differentiated human airway epithelium consists of different cell types forming a polarized and pseudostratified epithelium. This is dramatically altered in chronic obstructive pulmonary disease (COPD), characterized by basal and goblet cell hyperplasia, and squamous cell metaplasia. The effect of cigarette smoke on human bronchial epithelial cell (HBEC) differentiation remains to be elucidated. We analysed whether cigarette smoke extract (CSE) affected primary (p)HBEC differentiation and function. pHBEC were differentiated at the air-liquid interface (ALI) and differentiation was quantified after 7, 14, 21, or 28 days by assessing acetylated tubulin, CC10, or MUC5AC for ciliated, Clara, or goblet cells, respectively. Exposure of differentiating pHBEC to CSE impaired epithelial barrier formation, as assessed by resistance measurements (TEER). Importantly, CSE exposure significantly reduced the number of ciliated cells, while it increased the number of Clara and goblet cells. CSE-dependent cell number changes were reflected by a reduction of acetylated tubulin levels, an increased expression of the basal cell marker KRT14, and increased secretion of CC10, but not by changes in transcript levels of CC10, MUC5AC, or FOXJ1. Our data demonstrate that cigarette smoke specifically alters the cellular composition of the airway epithelium by affecting basal cell differentiation in a post-transcriptional manner.

Objectives: Little is known about the role of Wnt/β-catenin in postnatal airway homeostasis and basal cell function. This study aimed to investigate the role of Wnt signaling in the self-renewal of basal cells and the involvement of β-catenin in tracheal repair after naphthalene-induced injury., Methods: Mice were treated with naphthalene and injected with 4-hydroxytamoxifen. Injury and repair of the tracheal epithelium after naphthalene-mediated secretory cell depletion was assessed by a immunohistochemical study. The involvement of Wnt and β-catenin signaling in basal cell proliferation was investigated during in vitro expansion., Results: Immunohistochemical analysis of tracheal epithelium in wild-type mice showed a reduction in the number of Clara cell secretory protein (CCSP+) and forkhead box transcription factor (Fox-J1+) cells on days 2 to 5 after naphthalene-induced injury; this cell population was regenerated by day 10. After flush labeling, bromodeoxyuridine-positive (BrdU+) cells and Ki67+ cells were observed in tracheal epithelium on days 2 to 5 but not on days 10 and 21. Confocal microscopy visualizing K5+ and BrdU+ cells showed that Wnt3a promotes proliferation of K5+ cells. Immunohistochemical analysis of K5+ and CCSP+ in tracheal epithelial cells from wild-type littermate and K5-Cre-mediated β-catenin knock-out mice showed that on day 3, the number of CCSP+ cells was decreased in all mice. On day 10, CCSP+ cells were present in wild-type littermate mice but absent in conditional knock-out mice., Conclusions: Basal cells serve as stem cells in the tracheal epithelium, regenerating and maintaining tracheal epithelial cells in a mouse model of tracheal injury. β-Catenin is required for proliferation and self-renewal of tracheal epithelial cells., (Copyright © 2014 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Transformation-related protein 63-expressing (p63(+)) basal cells are confined to the trachea in the mouse lung. However, after influenza virus infection or bleomycin treatment, patches of p63(+) cells were observed in the damaged lung parenchyma. To address whether the newly induced p63(+) cells are derived from the p63(+) basal cells, we performed lineage tracing. In a keratin 5 promoter-driven CreER system, although preexisting p63(+) basal cells were labeled by enhanced green fluorescent protein (EGFP) after tamoxifen treatment, none or only a small fraction (∼ 15%) of the p63(+) patches was labeled by EGFP after bleomycin treatment or influenza virus infection, respectively. In contrast, > 60% of p63(+) patches contained EGFP(+) cells in Scgb1a1-CreER transgenic system where club cells are labeled. Many p63(+) cells were found in bronchiole-like lumen structures with columnar cells at the lumen side. The columnar cells were positive for club cell marker Cyp2f2 and could be traced to the newly induced p63(+) cells. These results suggest that most of the newly induced p63(+) cells in the damaged parenchyma are likely derived from club cells rather than from p63(+) basal cells and that newly induced p63(+) cells may be involved in the regeneration of bronchioles.

We have previously reported a subpopulation of bone marrow cells (BMC) that express Clara cell secretory protein (CCSP), generally felt to be specific to lung Clara cells. Ablation of lung Clara cells has been reported using a transgenic mouse that expresses thymidine kinase under control of the CCSP promoter. Treatment with ganciclovir results in permanent elimination of CCSP(+) cells, failure of airway regeneration, and death. To determine if transtracheal delivery of wild-type bone marrow CCSP(+) cells is beneficial after ablation of lung CCSP(+) cells, transgenic mice were treated with ganciclovir followed by transtracheal administration of CCSP(+) or CCSP(-) BMC. Compared with mice administered CCSP(-) cells, mice treated with CCSP(+) cells had more donor cells lining the airway epithelium, where they expressed epithelial markers including CCSP. Although donor CCSP(+) cells did not substantially repopulate the airway, their administration resulted in increased host ciliated cells, better preservation of airway epithelium, reduction of inflammatory cells, and an increase in animal survival time. Administration of CCSP(+) BMC is beneficial after permanent ablation of lung Clara cells by increasing bronchial epithelial repair. Therefore, CCSP(+) BMC could be important for treatment of lung diseases where airways re-epithelialization is compromised.

Background: Chlorine is a widely used toxic compound that is considered a chemical threat agent. Chlorine inhalation injures airway epithelial cells, leading to pulmonary abnormalities. Efficient repair of injured epithelium is necessary to restore normal lung structure and function. The objective of the current study was to characterize repair of the tracheal epithelium after acute chlorine injury., Methods: C57BL/6 mice were exposed to chlorine and injected with 5-ethynyl-2'-deoxyuridine (EdU) to label proliferating cells prior to sacrifice and collection of tracheas on days 2, 4, 7, and 10 after exposure. Airway repair and restoration of a differentiated epithelium were examined by co-localization of EdU labeling with markers for the three major tracheal epithelial cell types [keratin 5 (K5) and keratin 14 (K14) for basal cells, Clara cell secretory protein (CCSP) for Clara cells, and acetylated tubulin (AcTub) for ciliated cells]. Morphometric analysis was used to measure proliferation and restoration of a pseudostratified epithelium., Results: Epithelial repair was fastest and most extensive in proximal trachea compared with middle and distal trachea. In unexposed mice, cell proliferation was minimal, all basal cells expressed K5, and K14-expressing basal cells were absent from most sections. Chlorine exposure resulted in the sloughing of Clara and ciliated cells from the tracheal epithelium. Two to four days after chlorine exposure, cell proliferation occurred in K5- and K14-expressing basal cells, and the number of K14 cells was dramatically increased. In the period of peak cell proliferation, few if any ciliated or Clara cells were detected in repairing trachea. Expression of ciliated and Clara cell markers was detected at later times (days 7-10), but cell proliferation was not detected in areas in which these differentiated markers were re-expressed. Fibrotic lesions were observed at days 7-10 primarily in distal trachea., Conclusion: The data are consistent with a model where surviving basal cells function as progenitor cells to repopulate the tracheal epithelium after chlorine injury. In areas with few remaining basal cells, repair is inefficient, leading to airway fibrosis. These studies establish a model for understanding regenerative processes in the respiratory epithelium useful for testing therapies for airway injury.

Bronchiolitis obliterans syndrome (BOS) is a condition of progressive airflow obstruction that affects a majority of lung transplant recipients and limits long-term posttransplant survival. Although epithelial injury appears central to the development of BOS, little is known regarding the specific epithelial cell types that are affected in this condition. We hypothesized that BOS would involve preferential injury to the secretory Clara cells that function in innate defense and epithelial repair. To test this hypothesis, we assessed tissue transcript, tissue protein and lung fluid protein expression of Clara cell secretory protein (CCSP), a marker for Clara cells, in lung transplant recipients with BOS, BOS-free patients and in donor controls. Our results demonstrate that CCSP tissue transcript and protein expression are significantly reduced in lung transplant recipients with BOS compared to BOS-free or donor controls. In addition, we demonstrate that CCSP protein levels are significantly reduced in the lung fluid of patients with BOS compared to BOS-free controls, in cross-sectional and longitudinal analysis. Collectively, these complementary results illustrate that BOS involves a selective alteration in the distribution and function of bronchiolar Clara cells., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Clara cell 10-kD protein (CC10) is well known to be an immuno-suppressive protein secreted from airway epithelial cells after inflammatory stimulation and is involved in the development of allergic disorders. Although histamine H1 receptor antagonists are used for the treatment of allergic disorders, the influence of the agents on CC10 production is not well understood. In the present study, we examined the influence of a histamine H1 receptor antagonist, fexofenadine hydrochloride (FEX) on CC10 production in vitro and in vivo., Methods: Nasal epithelial cells (5 x 10(6) cells/ml) were stimulated with 20 ng/ml TNF-alpha in the presence of various concentrations of FEX for 24 hours. CC10 levels in culture supernatants were examined by ELISA. Patients with Japanese cedar pollinosis were treated orally with FEX twice a day at a single dose of 60 mg for two weeks during Japanese cedar pollen season (February 2011 to April 2011). CC10 levels in nasal secretions were also examined by ELISA., Results: The addition of FEX into cell cultures caused increase in CC10 production induced by TNF-alpha stimulation, and the minimum concentration that caused significant increase was 200 ng/ml. Oral administration of FEX also increased CC10 levels in nasal secretions from pollinosis patients along with attenuation of clinical symptoms., Conclusion: The ability of FEX to enhance CC10 production may account, at least in part, for the clinical efficacy of the agent in allergic disorders, including allergic rhinitis.

Background: Clara cell 10-kDa protein (CC10) is a multifunctional protein with anti-inflammatory and immunomodulatory effects. Induction of CC10 expression by gene transfection may possess potential therapeutic effect. Nuclear factor κB (NF-κB) plays a key role in the inflammatory processes of airway diseases., Method/results: To investigate potential therapeutic effect of CC10 gene transfection in controlling airway inflammation and the underlying intracellular mechanisms, in this study, we constructed CC10 plasmid and transfected it into bronchial epithelial cell line BEAS-2B cells and CC10 knockout mice. In BEAS-2B cells, CC10's effect on interleukin (IL)-1β induced IL-8 expression was explored by means of RT-PCR and ELISA and its effect on NF-κB classical signaling pathway was studied by luciferase reporter, western blot, and immunoprecipitation assay. The effect of endogenous CC10 on IL-1β evoked IL-8 expression was studied by means of nasal explant culture. In mice, CC10's effect on IL-1β induced IL-8 and nuclear p65 expression was examined by immunohistochemistry. First, we found that the CC10 gene transfer could inhibit IL-1β induced IL-8 expression in BEAS-2B cells. Furthermore, we found that CC10 repressed IL-1β induced NF-κB activation by inhibiting the phosphorylation of IκB-α but not IκB kinase-α/β in BEAS-2B cells. Nevertheless, we did not observe a direct interaction between CC10 and p65 subunit in BEAS-2B cells. In nasal explant culture, we found that IL-1β induced IL-8 expression was inversely correlated with CC10 levels in human sinonasal mucosa. In vivo study revealed that CC10 gene transfer could attenuate the increase of IL-8 and nuclear p65 staining in nasal epithelial cells in CC10 knockout mice evoked by IL-1β administration., Conclusion: These results indicate that CC10 gene transfer may inhibit airway inflammation through suppressing the activation of NF-κB, which may provide us a new consideration in the therapy of airway inflammation.

The guinea pig (Cavea porcellus) is a mammalian non-rodent species in the Caviidae family. The sensitivity of the respiratory system and the susceptibility to infectious diseases allows the guinea pig to be a useful model for both infectious and non-infectious lung diseases such as asthma and tuberculosis. In this report, we demonstrated for the first time, the major cell types and composition in the guinea pig airway epithelium, using cell type-specific markers by immunohistochemical staining using the commercial available immunological reagents that cross-react with guinea pig. Our results revealed the availability of antibodies cross-reacting with airway epithelial cell types of basal, non-ciliated columnar, ciliated, Clara, goblet and alveolar type II cells, as well as those cells expressing Mucin 5AC, Mucin 2, Aquaporin 4 and Calcitonin Gene Related Peptide. The distribution of these various cell types were quantified in the guinea pig airway by immunohistochemical staining and were comparable with morphometric studies using an electron microscopy assay. Moreover, this study also demonstrated that goblet cells are the main secretory cell type in the guinea pig's airway, distinguishing this species from rats and mice. These results provide useful information for the understanding of airway epithelial cell biology and mechanisms of epithelial-immune integration in guinea pig models., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Laminins and their integrin receptors are implicated in epithelial cell differentiation and progenitor cell maintenance. We report here that a previously unrecognized subpopulation of mouse alveolar epithelial cells (AECs) expressing the laminin receptor α6β4, but little or no pro-surfactant C (pro-SPC), is endowed with regenerative potential. Ex vivo, this subpopulation expanded clonally as progenitors but also differentiated toward mature cell types. Integrin β4 itself was not required for AEC proliferation or differentiation. An in vivo embryonic lung organoid assay, which we believe to be novel, was used to show that purified β4+ adult AECs admixed with E14.5 lung single-cell suspensions and implanted under kidney capsules self-organized into distinct Clara cell 10-kDa secretory protein (CC10+) airway-like and SPC+ saccular structures within 6 days. Using a bleomycin model of lung injury and an SPC-driven inducible cre to fate-map AECs, we found the majority of type II AECs in fibrotic areas were not derived from preexisting type II AECs, demonstrating that SPC- progenitor cells replenished type II AECs during repair. Our findings support the idea that there is a stable AEC progenitor population in the adult lung, provide in vivo evidence of AEC progenitor cell differentiation after parenchymal injury, and identify a strong candidate progenitor cell for maintenance of type II AECs during lung repair.

Reactive oxidants such as nitrogen dioxide (NO(2)) injure the pulmonary epithelium, causing airway damage and inflammation. We previously demonstrated that nuclear factor-κ B (NF-κB) activation within airway epithelial cells occurs in response to NO(2) inhalation, and is critical for lipopolysaccharide-induced or antigen-induced inflammatory responses. Here, we investigated whether manipulation of NF-κB activity in lung epithelium affected severe lung injuries induced by NO(2) inhalation. Wild-type C57BL/6J, CC10-IκBα(SR) transgenic mice with repressed airway epithelial NF-κB function, or transgenic mice expressing a doxycycline-inducible, constitutively active I κ B kinase β (CC10-rTet-(CA)IKKβ) with augmented NF-κB function in airway epithelium, were exposed to toxic levels of 25 ppm or 50 ppm NO(2) for 6 hours a day for 1 or 3 days. In wild-type mice, NO(2) caused the activation of NF-κB in airway epithelium after 6 hours, and after 3 days resulted in severe acute lung injury, characterized by neutrophilia, peribronchiolar lesions, and increased protein, lactate dehydrogenase, and inflammatory cytokines. Compared with wild-type mice, neutrophilic inflammation and elastase activity, lung injury, and several proinflammatory cytokines were significantly suppressed in CC10-IκBα(SR) mice exposed to 25 or 50 ppm NO(2). Paradoxically, CC10-rTet-(CA)IKKβ mice that received doxycycline showed no further increase in NO(2)-induced lung injury compared with wild-type mice exposed to NO(2), instead displaying significant reductions in histologic parameters of lung injury, despite elevations in several proinflammatory cytokines. These intriguing findings demonstrate distinct functions of airway epithelial NF-κB activities in oxidant-induced severe acute lung injury, and suggest that although airway epithelial NF-κB activities modulate NO(2)-induced pulmonary inflammation, additional NF-κB-regulated functions confer partial protection from lung injury.

Objective: To observe the expression of Clara cell secretory protein(CCSP) in the Kunming mouse model of n-hexane long-term inhalation, and to discuss the functions of Clara cell in injury lung induced by n-hexane., Methods: 24 healthy mice were randomly divided into 4 groups: one control group and three n-hexane groups (4 w, 8 w and 12 w), 6 each group. Primary concentration of n-hexane was 17.6 g/m3, 8 hours per day, 6 d per week. After inhalation, n-hexane concentration of blood from celiac artery was detected. The lungs were embedded with paraffin and HE staining in the routine. The ratio of Clara cells with CCSP reaction in bronchiole and the number of macrophage cells with lysozyme reaction were determined by immuno-histochemistry., Results: In the poisoning groups, the average n-hexane concentration of blood was significantly higher than that of the control group (P < 0.01). There were apparent pathologic damages in lungs of the poisoning mice. In poisoning 4 w, 8 w and 12 w groups, the ratio of Clara cells was significantly decreased [(73.33 +/- 4.21)%, (60.98 +/- 4.94)%, (34.04 +/- 2.33)% in terminal bronchiole, and (75.44 +/- 7.91)%, (58.54 +/- 4.86)%, (33.35 +/- 2.67)% in respiratory bronchiole] as compared with the control mice [(80.26 +/- 6.43)% and (81.74 +/- 7.75)%, P < 0.05 or P < 0.01], meanwhile the numbers of macrophage cells were gradually increased [(21.39 +/- 7.41), (28.54 +/- 10.73), (48.97 +/- 19.55) per microscopic field at 200x] in poisoning mice than those in control mice [(7.84 +/- 3.12) per microscopic field at 200x, P < 0.05 or P < 0.01]., Conclusion: In injury lungs after n-hexane inhalation, Clara cells are the target cells of n-hexane toxicity effect. Clara cells play an extensive protective role in lung inflammation.

Background: Continuous exposure to tobacco smoke (TS) is a key cause of chronic obstructive pulmonary disease (COPD), a complex multifactorial disease that is difficult to model in rodents. The spontaneously hypertensive (SH) rat exhibits several COPD-associated co-morbidities such as hypertension and increased coagulation. We have investigated whether SH rats are a more appropriate animal paradigm of COPD., Methods: SH rats were exposed to TS for 6 hours/day, 3 days/week for 14 weeks, and the lung tissues examined by immunohistochemistry., Results: TS induced a CK13-positive squamous metaplasia in proximal airways, which also stained for Ki67 and p63. We hypothesise that this lesion arises by basal cell proliferation, which differentiates to a squamous cell phenotype. Differences in staining profiles for the functional markers CC10 and surfactant D, but not phospho-p38, indicated loss of ability to function appropriately as secretory cells. Within the parenchyma, there were also differences in the staining profiles for CC10 and surfactant D, indicating a possible attempt to compensate for losses in proximal airways. In human COPD sections, areas of CK13-positive squamous metaplasia showed sporadic p63 staining, suggesting that unlike the rat, this is not a basal cell-driven lesion., Conclusion: This study demonstrates that although proximal airway metaplasia in rat and human are both CK13+ and therefore squamous, they potentially arise by different mechanisms.

Background: Human embryonic stem cells (hESC) have the capacity to differentiate in vivo and in vitro into cells from all three germ lineages. The aim of the present study was to investigate the effect of specific culture conditions on the differentiation of hESC into lung epithelial cells., Methods: Undifferentiated hESC, grown on a porous membrane in hESC medium for four days, were switched to a differentiation medium for four days; this was followed by culture in air-liquid interface conditions during another 20 days. Expression of several lung markers was measured by immunohistochemistry and by quantitative real-time RT-PCR at four different time points throughout the differentiation and compared to appropriate controls., Results: Expression of CC16 and NKX2.1 showed a 1,000- and 10,000- fold increase at day 10 of differentiation. Other lung markers such as SP-C and Aquaporin 5 had the highest expression after twenty days of culture, as well as two markers for ciliated cells, FOXJ1 and beta-tubulin IV. The results from qRT-PCR were confirmed by immunohistochemistry on paraffin-embedded samples. Antibodies against CC16, SP-A and SP-C were chosen as specific markers for Clara Cells and alveolar type II cells. The functionality was tested by measuring the secretion of CC16 in the medium using an enzyme immunoassay., Conclusion: These results suggest that by using our novel culture protocol hESC can be differentiated into the major cell types of lung epithelial tissue.

Background: The study of small airway diseases such as post-transplant bronchiolitis obliterans syndrome (BOS) is hampered by the difficulty in assessing peripheral airway function either physiologically or directly. Our aims were to develop robust methods for sampling small airway epithelial cells (SAEC) and to establish submerged SAEC cultures for downstream experimentation., Methods: SAEC were obtained at 62 post-transplant bronchoscopies in 26 patients using radiologically guided bronchial brushings. Submerged cell cultures were established and SAEC lineage was confirmed using expression of clara cell secretory protein (CCSP)., Results: The cell yield for SAEC (0.956 +/- 0.063 x 106) was lower than for large airway cells (1.306 +/- 0.077 x 106) but did not significantly impact on the culture establishment rate (79.0 +/- 5.2% vs. 83.8 +/- 4.7% p = 0.49). The presence of BOS significantly compromised culture success (independent of cell yield) for SAEC (odds ratio (95%CI) 0.067 (0.01-0.40)) but not LAEC (0.3 (0.05-1.9)). Established cultures were successfully passaged and expanded., Conclusion: Primary SAEC can be successfully obtained from human lung transplant recipients and maintained in culture for downstream experimentation. This technique will facilitate the development of primary in vitro models for BOS and other diseases with a small airway component such as asthma, cystic fibrosis and COPD.

Clara cell secretory protein (CCSP) is one of the most abundant proteins in the airway surface fluid, and has many putative functions. Recent advances in the field of stem cells and lung regeneration have identified potentially new roles of CCSP and CCSP-expressing cell populations in airway maintenance, repair and regeneration. This review focuses on the airway regenerative potential of CCSP and the cells that express this protein. The use of this protein or CCSP-expressing cells as an indication of biologic processes that contribute to lung injury or repair is highlighted.

Background: Although prior studies have demonstrated a smoking-induced field of molecular injury throughout the lung and airway, the impact of smoking on the airway epithelial proteome and its relationship to smoking-related changes in the airway transcriptome are unclear., Methodology/principal Findings: Airway epithelial cells were obtained from never (n = 5) and current (n = 5) smokers by brushing the mainstem bronchus. Proteins were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE). After in-gel digestion, tryptic peptides were processed via liquid chromatography/ tandem mass spectrometry (LC-MS/MS) and proteins identified. RNA from the same samples was hybridized to HG-U133A microarrays. Protein detection was compared to RNA expression in the current study and a previously published airway dataset. The functional properties of many of the 197 proteins detected in a majority of never smokers were similar to those observed in the never smoker airway transcriptome. LC-MS/MS identified 23 proteins that differed between never and current smokers. Western blotting confirmed the smoking-related changes of PLUNC, P4HB1, and uteroglobin protein levels. Many of the proteins differentially detected between never and current smokers were also altered at the level of gene expression in this cohort and the prior airway transcriptome study. There was a strong association between protein detection and expression of its corresponding transcript within the same sample, with 86% of the proteins detected by LC-MS/MS having a detectable corresponding probeset by microarray in the same sample. Forty-one proteins identified by LC-MS/MS lacked detectable expression of a corresponding transcript and were detected in

It has been suggested that some adult bone marrow cells (BMC) can localize to the lung and develop tissue-specific characteristics including those of pulmonary epithelial cells. Here, we show that the combination of mild airway injury (naphthalene-induced) as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype. Confocal analysis of airway and alveolar-localized BMC (fluorescently labeled) with epithelial markers shows expression of the pulmonary epithelial proteins, Clara cell secretory protein, and surfactant protein C. To confirm epithelial gene expression by BMC, we generated transgenic mice expressing green fluorescent protein (GFP) driven by the epithelial-specific cytokeratin-18 promoter and injected BMC from these mice transtracheally into wild-type recipients after naphthalene-induced airway injury. BMC retention in the lung was observed for at least 120 days following cell delivery with increasing GFP transgene expression over time. Some BMC cultured in vitro over time also expressed GFP transgene, suggesting epithelial transdifferentiation of the BMC. The results indicate that targeted delivery of BMC can promote airway regeneration.

Background/aim: In congenital diaphragmatic hernia (CDH), there is pulmonary neuroendocrine cell (PNEC) hyperplasia and Clara (nonendocrine) cell hypoplasia, the meaning of which remains unknown. In embryonic/fetal lung, an intricate cross talk between Notch pathway and basic helix-loop-helix transcription factors Mash1 and Hes1 determines the balance between endocrine and nonendocrine epithelial cell fate. Differences at the molecular level in pulmonary epithelial cell differentiation between control and CDH hypoplastic lungs were investigated., Material and Methods: The nitrofen-induced CDH rat model was used. At 15.5 days postconception (dpc), fetuses were assigned to 2 experimental groups: control and nitrofen (exposed to nitrofen, without CDH), whereas at 17.5, 19.5, and 21.5 dpc, fetuses were assigned to 3 experimental groups: control, nitrofen, and CDH (exposed to nitrofen, with CDH). The fetal lungs were processed for expression quantification of CC10, Hes1, Mash1, and Dll1 by real-time polymerase chain reaction., Results: In control fetuses, expression of all studied genes increased with gestational age. In nitrofen-exposed fetal lungs, endocrine cell marker Mash1 was downregulated only at the earliest studied gestational age, whereas Dll1 expression levels were significantly increased in the CDH group at 19.5 and 21.5 dpc. Regarding nonendocrine markers, Hes1 presented increased expression at 15.5 and 19.5 dpc, whereas CC10 was downregulated at 17.5 and 19.5 dpc but not at term., Conclusions: This study suggests that PNEC hyperplasia in CDH fetal lung is likely because of Notch signaling deregulation, whereas Clara cell hypoplasia in CDH lungs could be a consequence of protein synthesis delay, reflecting a functional maturation hindrance and not a cell fate commitment problem.

Classical tissue recombination experiments have reported that at early gestation both tracheal and distal lung epithelium have the plasticity to respond to mesenchymal signals. Herein we examined the role of epithelial-mesenchymal interactions in maintaining epithelial differentiation at late (E19-E21, term = 22 days) fetal gestation in the rat. Isolated distal lung epithelial cells were recombined with mesenchymal cells from lung, skin, and intestine, and the homotypic or heterotypic recombinant cell aggregates were cultured for up to 5 days. Recombining lung epithelial cells with mesenchyme from various sources induced a morphological pattern that was specific to the type of inducing mesenchyme. In situ analysis of surfactant protein (SP)-C, SP-B, and Clara cell secretory protein (CCSP) expression, as well as SP-C and CCSP promoter transactivation experiments, revealed that distal lung epithelium requires lung mesenchyme to maintain the alveolar, but not bronchiolar, phenotype. Incubation of lung recombinants with an anti-FGF7 antibody resulted in a partial inhibition of mesenchyme-induced SP-C promoter transactivation. Immunoreactivity for Delta and Lunatic fringe, components of the Notch pathway that regulates cell differentiation, was downregulated in the heterotypic recombinants. In contrast, Hes1 mRNA expression was increased in these recombinants. Cumulatively, these results suggest that at late fetal gestation, distal lung epithelial cells are not fully committed to a specific phenotype and still have the plasticity to respond to various signals. Their alveolar phenotype is likely maintained by Notch/Notch ligand interactions and mesenchymal factors, including FGF7.

It is well established that exposure to high levels of oxygen (hyperoxia) injures and kills microvascular endothelial and alveolar type I epithelial cells. In contrast, significant death of airway and type II epithelial cells is not observed at mortality, suggesting that these cell types may express genes that protect against oxidative stress and damage. During a search for genes induced by hyperoxia, we previously reported that airway and alveolar type II epithelial cells uniquely express the growth arrest and DNA damage (Gadd)45a gene. Because Gadd45a has been implicated in protection against genotoxic stress, adult Gadd45a (+/+) and Gadd45a (-/-) mice were exposed to hyperoxia to investigate whether it protected epithelial cells against oxidative stress. During hyperoxia, Gadd45a deficiency did not affect loss of airway epithelial expression of Clara cell secretory protein or type II epithelial cell expression of pro-surfactant protein C. Likewise, Gadd45a deficiency did not alter recruitment of inflammatory cells, edema, or overall mortality. Consistent with Gadd45a not affecting the oxidative stress response, p21(Cip1/WAF1) and heme oxygenase-1 were comparably induced in Gadd45a (+/+) and Gadd45a (-/-) mice. Additionally, Gadd45a deficiency did not affect oxidative DNA damage or apoptosis as assessed by oxidized guanine and terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling staining. Overexpression of Gadd45a in human lung adenocarcinoma cells did not affect viability or survival during exposure, whereas it was protective against UV-radiation. We conclude that increased tolerance of airway and type II epithelial cells to hyperoxia is not attributed solely to expression of Gadd45a.

Primary airway epithelial cell cultures can provide a faithful representation of the in vivo airway while allowing for a controlled nutrient source and isolation from other tissues or immune cells. The methods used have significant differences based on tissue source, cell isolation, culture conditions, and assessment of culture purity. We modified and optimized a method for generating tracheal epithelial cultures from Syrian golden hamsters and characterized the cultures for cell composition and function. Soon after initial plating, the epithelial cells reached a high transepithelial resistance and formed tight junctions. The cells differentiated into a heterogeneous, multicellular culture containing ciliated, secretory, and basal cells after culture at an air-liquid interface (ALI). The secretory cell populations initially consisted of MUC5AC-positive goblet cells and MUC5AC/CCSP double-positive cells, but the makeup changed to predominantly Clara cell secretory protein (CCSP)-positive Clara cells after 14 d. The ciliated cell populations differentiated rapidly after ALI, as judged by the appearance of beta tubulin IV-positive cells. The cultures produced mucus, CCSP, and trypsin-like proteases and were capable of wound repair as judged by increased expression of matrilysin. Our method provides an efficient, high-yield protocol for producing differentiated hamster tracheal epithelial cells that can be used for a variety of in vitro studies including tracheal cell differentiation, airway disease mechanisms, and pathogen-host interactions.

Airway mucus hypersecretion is a prominent feature of many obstructive lung diseases. We thus determined the ontogeny and exocytic phenotype of mouse airway mucous cells. In naive mice, ciliated (approximately 40%) and nonciliated (approximately 60%) epithelial cells line the airways, and > 95% of the nonciliated cells are Clara cells that contain Clara cell secretory protein (CCSP). Mucous cells comprise < 5% of the nonciliated cells. After sensitization and a single aerosol antigen challenge, alcian blue-periodic acid Schiff's positive mucous cell numbers increase dramatically, appearing 6 h after challenge (21% of nonciliated/nonbasal cells), peaking from Days 1-7 (99%), and persisting at Day 28 (65%). Throughout the induction and resolution of mucous metaplasia, ciliated and Clara cell numbers identified immunohistochemically change only slightly. Intracellular mucin content peaks at Day 7, and mucin expression is limited specifically to a Clara cell subset in airway generations 2-4 that continue to express CCSP. Functionally, Clara cells are secretory cells that express the regulated exocytic marker Rab3D and, in antigen-challenged mice, rapidly secrete mucin in response to inhaled ATP in a dose-dependent manner. Thus, Clara cells show great plasticity in structure and secretory products, yet have molecular and functional continuity in their identity as specialized apical secretory cells.

pRb, p107 and p130 are important regulators of cell cycle and have extensive overlapping functions; however, only Rb has been shown to be a bone fide tumor suppressor. Defining the overlapping versus distinct pocket protein functions is therefore an important step to understanding the unique role of Rb. Using lung as a model, the present studies demonstrate that pocket proteins are important not only in regulating cell cycle and survival but also in cell lineage specification. An inducible lung-specific Rb knockout strategy was used to demonstrate that Rb is specifically required for restricting neuroendocrine cell fate despite functional compensation for Rb deficiency in other cell types. Ablation of total Rb family function resulted in opposing effects in specification along distinct cell lineages, providing evidence that pocket proteins inhibit neuroendocrine cell fate while being required for differentiation in other cell types. These findings identify a novel role for pocket proteins in cell fate determination, and establish a unique cell lineage-specific function for Rb that explains, at least in part, why Rb and p16 are inactivated in phenotypically distinct carcinomas.

Ninety percent of all human lung cancers are related to cigarette smoking. Both tobacco smoke and lung tumorigenesis are associated with drastically reduced levels of Clara cell 10-kDa protein (CC10), a multifunctional secreted protein, naturally produced by the airway epithelia of virtually all mammals. We previously reported that the expression of CC10 is markedly reduced in animals exposed to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK, a potent carcinogen in tobacco smoke. Furthermore, it has been reported that CC10 expression, induced in certain tumor cells, reverses the transformed phenotype. We demonstrate here that NNK exposure of CC10-knock-out (CC10-KO) mice causes a significantly higher incidence of airway epithelial hyperplasia and lung adenomas compared with wild type (WT) littermates (30% CC10-KO versus 5% WT, p = 0.041). We also found that compared with NNK-treated WT mice, CC10-KO mice manifest increased frequency of K-ras mutation, elevated level of Fas ligand (FasL) expression, and increased MAPK/Erk phosphorylation, all of which are considered predisposing events in NNK-induced lung tumorigenesis. We propose that CC10 has a protective role against NNK-induced lung tumorigenesis mediated via down-regulation of the above-mentioned predisposing events.