Clara Cell Secretory Protein Research Articles

Repair of naphthalene-induced acute tracheal injury by basal cells depends on β-catenin

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. 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 invitro expansion. 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. 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.

Serum concentrations of club cell secretory protein (Clara) and cancer mortality in adults: a population-based, prospective cohort study

Club cell secretory protein (Clara) (CC16) is produced mainly by bronchiolar club cells and has been shown to have protective effects against airway inflammation and oxidative stress from cigarette smoking and related carcinogens. The goal of this study was to establish whether serum CC16 concentrations predict all-cause and cancer-specific mortality in adults. We used data from the population-based Tucson Epidemiological Study of Airway Obstructive Diseases (TESAOD), a prospective cohort study of respiratory health initiated in Tucson, AZ, USA, in 1972, that recruited a multistage stratified cluster sample of non-Hispanic white households. We measured serum CC16 concentrations in cryopreserved serum samples and reviewed vital status up to Jan 1, 2011, through contact with next of kin, collection of death certificates, and searches of the National Death Index. Our primary analysis was the relation of baseline serum CC16 to all-cause mortality or cause-specific mortality risk, analysed by adjusted Cox proportional hazards models. 1086 TESAOD participants aged 21-70 years at enrolment were eligible for inclusion. Of these, 653 (60%) had died by 2011, and cause of death was ascertained for 649 (99%). When adjusted for sex, age, education, body-mass index, smoking and pack-years, and baseline levels of lung function, serum CC16 concentrations at baseline were inversely associated with mortality risk over the study follow-up. Mortality risk increased for each 1-SD decrease in CC16 (adjusted hazard ratio [HR] 1·16 [95% CI 1·06-1·26]; p=0·0007). For cause-specific mortality, each 1-SD decrease in serum CC16 was associated with an increased risk of dying of cancer (adjusted HR 1·41 [1·19-1·67]; p<0·0001). In the subset of smokers, the corresponding adjusted HR for mortality by lung cancer was 1·52 (1·14-2·03; p=0·004). Serum CC16 concentrations can predict mortality risk in the general adult population. The excess risk associated with lower CC16 concentrations is predominantly driven by cancer, particularly lung cancer. National Heart, Lung, and Blood Institute.

IL-1β expression in the distal lung epithelium disrupts lung morphogenesis and epithelial cell differentiation in fetal mice

Perinatal inflammation and the inflammatory cytokine IL-1 can modify lung morphogenesis. To examine the effects of antenatal expression of IL-1β in the distal airway epithelium on fetal lung morphogenesis, we studied lung development and surfactant expression in fetal mice expressing human IL-1β under the control of the surfactant protein (SP)-C promoter. IL-1β-expressing pups suffered respiratory failure and died shortly after birth. IL-1β caused fetal lung inflammation and enhanced the expression of keratinocyte-derived chemokine (KC/CXCL1) and monocyte chemoattractant protein 3 (MCP-3/CCL7), the calgranulins S100A8 and S100A9, the acute-phase protein serum amyloid A3, the chitinase-like proteins Ym1 and Ym2, and pendrin. IL-1β decreased the percentage of the total distal lung area made up of air saccules and the number of air saccules in the lungs of fetal mice. IL-1β inhibited the expression of VEGF-A and its receptors VEGFR-1 and VEGFR-2. The percentage of the cellular area of the distal lung made up of capillaries was decreased in IL-1β-expressing fetal mice. IL-1β suppressed the production of SP-B and pro-SP-C and decreased the amount of phosphatidylcholine and the percentage of palmitic acid in the phosphatidylcholine fraction of lung phospholipids, indicating that IL-1β prevented the differentiation of type II epithelial cells. The production of Clara cell secretory protein in the nonciliated bronchiolar (Clara) cells was likewise suppressed by IL-1β. In conclusion, expression of IL-1β in the epithelium of the distal airways disrupted the development of the airspaces and capillaries in the fetal lung and caused fatal respiratory failure at birth.

Club cell secretory protein improves survival in a murine obliterative bronchiolitis model

Club cell secretory protein (CCSP) is an indirect phospholipase A2 inhibitor with some immunosuppressive and antiproliferative properties that is expressed in bronchiolar Club cells. In our murine bone marrow transplant (BMT) model of obliterative bronchiolitis (OB), CCSP is diminished; however, its role is unknown. To determine the role of CCSP, B6 wild-type (WT) or CCSP-deficient (CCSP(-/-)) mice were lethally conditioned and given allogeneic bone marrow with a sublethal dose of allogeneic splenic T cells to induce OB. We found that CCSP(-/-) mice demonstrated a higher mortality following BMT-induced OB compared with WT mice. Mice were analyzed 60 days post-BMT for protein expression, pulmonary function, and histology. CCSP levels were reduced in WT mice with BMT-induced OB, and lower levels correlated to decreased lung compliance. CCSP(-/-) had a higher degree of injury and fibrosis as measured by hydroxy proline, along with an increased lung resistance and the inflammatory markers, leukotriene B4 and CXCL1. Replacement with recombinant intravenous CCSP partially reversed the weight loss and improved survival in the CCSP(-/-) mice. In addition, CCSP replacement improved histology and decreased inflammatory cells and markers. These findings indicate that CCSP has a regulatory role in OB and may have potential as a preventive therapy.

Bone marrow-derived progenitor cells in end-stage lung disease patients

BackgroundChronic lung diseases are marked by progressive inflammation, tissue damage and remodelling. Bone marrow-derived progenitor cells may contribute to these processes. The objectives of this study were to (1) to quantify CD45+Collagen-1+ fibrocytes and a novel epithelial-like population of bone marrow-derived cells, which express Clara Cell Secretory Protein, in patients at the time of lung transplant and (2) to evaluate mediators that may act to recruit these cells during injury.MethodsUsing an observational design, progenitor cells were quantified by flow cytometry from both bone marrow (BM) and peripheral blood (PB). Migration was tested using in vitro transwell assays. Multiplex bead-based assays were used to quantify plasma cytokines.ResultsAn increase in CD45+Collagen-1+ fibrocytes was found in pulmonary fibrosis and bronchiolitis obliterans patients. Cystic fibrosis patients had an increase in CCSP+ cells in both the BM and PB. The proportion of CCSP+ cells in the BM and PB was correlated. CCSP+ cells express the chemokine receptors CCR2, CCR4, CXCR3, and CXCR4, and significantly migrated in vitro toward Stromal Derived Factor-1 (SDF-1) and Stem Cell Growth Factor-β (SCGF-β). Plasma cytokine levels differed between disease groups, with a significant correlation between SCGF-β and CCSP+ cells and between Monocyte Chemotactic Protein-1 and fibrocytes.ConclusionsDifferent bone marrow-derived cells are found in various lung diseases. Increased fibrocytes were associated with fibrotic lung diseases. An increase in the novel CCSP+ epithelial-like progenitors in cystic fibrosis patients was found. These differences may be mediated by alterations in plasma cytokines responsible for cell recruitment.

BPA and Altered Airway Cells: Association Seen in Rhesus Macaques after Third-Trimester Exposure

Prenatal bisphenol A (BPA) exposure has been shown to alter the development of reproductive organs in animal models,1 although the impacts on development of other organ systems remain largely unknown. Researchers at the University of California, Davis, now report in EHP that BPA exposure late in gestation alters airway cell development in rhesus macaques.2 Previous studies have associated BPA exposure with an experimental model of asthma in mice.3 Epidemiological studies have found evidence of an association between prenatal BPA exposure and wheeze in young children,4 and between postnatal exposure and childhood asthma.5 “This study sheds light on the possible mechanisms by which BPA may affect lung health,” says Kathleen Donohue, an assistant professor of medicine at Columbia University. Donohue was not involved in the current study. BPA exposure is widespread. One report from the National Health and Nutrition Examination Survey found that more than 90% of urine samples collected from U.S. males and females over age 6 years contained detectable levels of the chemical.6 For the current study, pregnant rhesus macaques received BPA via subcutaneous implant for 50 days during gestational days 50–100 or 100–150 (roughly comparable to the second and third trimesters, respectively, in humans). Control macaques received a corn oil implant or ate corn oil–treated fruit. Treatment groups included at least 6 animals, with histopathologic analyses conducted on smaller subgroups. “Our goal was to model constant serum levels of BPA that have been measured in humans,” says first author Laura Van Winkle, a toxicologist at the university. “Lung development patterns and cellular abundance in the airways of these animals match humans much more closely than rodent models.” At the end of each group’s exposure period the researchers collected fetal airway tissue samples. They used only female fetuses, as the study originated from a project designed to examine the effects of BPA on female reproductive development. They found that BPA exposure in late pregnancy was associated with increased expression of secretory proteins in fetal tissue. The cells that produce these proteins mature late in gestation; the proteins themselves—Clara cell secretory protein (CCSP) and the mucins MUC5AC and MUC5B—are key components of airway mucous secretions.2 Expression of the Muc5B gene was approximately six times higher at 150 days’ gestation in fetuses whose mothers were exposed to BPA late in pregnancy compared with those whose mothers received no BPA exposure. Expression of the Muc5AC gene also was increased. Histological staining of the lung tissue indicated there were more mucous cells in the airway epithelium of exposed fetuses and suggested an increase in the amount of mucous production (however, the researchers did not have a large enough sample size to run statistical analyses).2 Late-pregnancy exposure to BPA (B) was associated with a higher number of mucous cells in the airway epithelium of exposed fetuses, compared with exposure in mid-pregnancy (D) and controls (A and C). There were no significant changes in protein expression after exposure during mid-pregnancy, suggesting that late pregnancy may be a critical period in which BPA exposure may alter airway cell development. This critical window may also apply to human exposures to BPA in the third trimester of pregnancy due to similarities in the timing of cellular development and airway structure between rhesus macaques and humans.2 “Taken together with earlier studies in humans, this study in monkeys is important because it is another link in the chain of evidence [potentially] connecting BPA exposure to lung disease,” says Donohue. An increase in mucous cell abundance is one of the hallmarks of asthma, the authors report. However, they say, the clinical relevance of this particular study remains to be seen. The researchers examined only fetal airway tissue samples, and it is unknown whether an increase in mucous cell abundance would have resulted in airway disease after birth. The potential effects of environmental estrogens such as BPA on lung development also are not well known, says Van Winkle. Although estrogen is known to increase the expression of MUC5B in cultured airway epithelium cells from humans,7 and lung tissue does have estrogen receptors,8 it’s not clear how this affects the development of secretory proteins. Recent experimental studies have suggested that BPA may also disrupt fetal development through epigenetic and nonestrogenic pathways.9,10 Van Winkle suspects a combination of hormonal and nonhormonal effects may be at play. “A third possibility is that BPA affects something else entirely in the body that in turn alters lung development,” she says.

Significance of changes of serum Clara cell secretory protein 16, pulmonary surfactant protein A in newborn infants with acute lung injury/acute respiratory distress syndrome

Objective To investigate the effect of serum Clara cell secretory protein 16 (CC16), pulmonary surfactant protein A(SP-A) in diagnosis of acute lung injury (ALI)/acute respiratory distress syndrome(ARDS) in newborn infants. Methods Fifty ALI/ARDS newborn infants in Zhuhai People's Hospital between 2011 Sep.and Dec.2012 were selected as treatment group(ALI group including 35 cases, ARDS group including 15 cases), 50 healthy newborns were selected as healthy control group.The changes of serum CC16, SP-A were detected by enzyme-linked immuno sorbent assay and were compared in the 3 groups. Results 1.Serum CC16 in ALI group and ARDS group were (34.19±1.09) mg/L, (55.28±0.82) mg/L, respectively, which were significantly higher than that in healthy control group [(12.67±1.10) mg/L](all P<0.05), and there was significant difference between ALI group and ARDS group(P<0.05).2.Serum SP-A in ALI group and ARDS group were(56.78±3.48) mg/L, (70.23±5.14) mg/L respectively, which were significantly higher than that in healthy control group [(22.10±3.32) mg/L](all P<0.05), and there was significant difference between ALI group and ARDS group(P<0.05). Conclusion Serum CC16, SP-A can be effective parameter for early monitoring ALI/ARDS in newborn infants. Key words: Acute lung injury; Acute respiratory distress syndrome; Newborn infant

Respiratory Function and Changes in Lung Epithelium Biomarkers after a Short-Training Intervention in Chlorinated vs. Ozone Indoor Pools

BackgroundSwimming in indoor pools treated with combined chemical treatments (e.g. ozone) may reduce direct exposure to disinfection byproducts and thus have less negative effects on respiratory function compared to chlorinated pools. The aim of this study is to analyze the effects of a short-term training intervention on respiratory function and lung epithelial damage in adults exercising in indoor swimming pool waters treated with different disinfection methods (chlorine vs. ozone with bromine).MethodsLung permeability biomakers [surfactant protein D (SP-D) and Clara cell secretory protein (CC16) in plasma] and forced expiratory volumes and flow (FEV1, FVC and FEF25–75) were measured in 39 healthy adults. Thirteen participants swam during 20 sessions in a chlorinated pool (CP), 13 performed and equivolumic intervention in an ozone pool (OP) and 13 were included in a control group (CG) without exposition.ResultsMedian plasma CC16 levels increased in CP swimmers (4.27±3.29 and 6.62±5.51 µg/L, p = 0.01, pre and post intervention respectively) while no significant changes in OP and CG participants were found. No significant changes in median plasma SP-D levels were found in any of the groups after the training period. FVC increased in OP (4.26±0.86 and 4.43±0.92 L, p<0.01) and CP swimmers (4.25±0.86 and 4.35±0.85 L, p<0.01). FEV1 only increased in OP swimmers (3.50±0.65 and 3.59±0.67, p = 0.02) and FEF25–75 decreased in CP swimmers (3.70±0.87 and 3.37±0.67, p = 0.02).ConclusionDespite lung function being similar in both groups, a higher lung permeability in CP compared to OP swimmers was found after a short-term swimming program. Combined chemical treatments for swimming pools such as ozone seem to have less impact on lung epithelial of swimmers compared to chlorinated treated pools.

Vitamin D, vitamin D binding protein, lung function and structure in COPD

Vitamin D and vitamin D binding protein (DBP) have been associated with COPD and FEV1. There are limited data regarding emphysema and vitamin D and DBP. This is a pilot study of a portion of the subjects in the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) study designed to examine the relationship between vitamin D status, DBP, FEV1 and emphysema in COPD patients. We measured serum 25(OH)D and DBP in 498 ECLIPSE subjects. Subjects were distributed amongst smoker controls, non-smoker controls, and GOLD stages 2, 3 and 4. Within each GOLD stage, the subjects were equally divided amongst high and low emphysema burden. The associations between 25(OH)D, DBP, and free vitamin D with FEV1, CT-defined emphysema, biomarkers and clinical data including CT-measured bone attenuation were assessed. 25(OH)D and DBP were measured using tandem mass spectroscopy and competitive enzyme-linked immunosorbent assay, respectively, 25(OH)D was correlated with FEV1 (p=0.01) and with severity of emphysema (p<0.01). 25(OH)D was also associated with six-minute walk (p=0.02), bronchodilator response (p=0.04), and Clara cell secretory protein (CC-16) (p=0.01). 25(OH)D levels were not associated with CT-measured bone attenuation, however DBP was associated with bone attenuation in subjects with emphysema. DBP was not associated with FEV1 or emphysema. 25(OH)D and DBP were inversely associated (p=0.01). This is the first study to demonstrate a relationship between emphysema and vitamin D. We also provide further evidence for a relationship between vitamin D and FEV1.

Gestational Exposure of Mice to Secondhand Cigarette Smoke Causes Bronchopulmonary Dysplasia Blocked by the Nicotinic Receptor Antagonist Mecamylamine

Background: Cigarette smoke (CS) exposure during gestation may increase the risk of bronchopulmonary dysplasia (BPD)—a developmental lung condition primarily seen in neonates that is characterized by hypoalveolarization, decreased angiogenesis, and diminished surfactant protein production and may increase the risk of chronic obstructive pulmonary disease.Objective: We investigated whether gestational exposure to secondhand CS (SS) induced BPD and sought to ascertain the role of nicotinic acetylcholine receptors (nAChRs) in this response.Methods: We exposed BALB/c and C57BL/6 mice to filtered air (control) or SS throughout the gestation period or postnatally up to 10 weeks. Lungs were examined at 7 days, 10 weeks, and 8 months after birth.Results: Gestational but not postnatal exposure to SS caused a typical BPD-like condition: suppressed angiogenesis [decreased vascular endothelial growth factor (VEGF), VEGF receptor, and CD34/CD31 (hematopoietic progenitor cell marker/endothelial cell marker)], irreversible hypoalveolarization, and significantly decreased levels of Clara cells, Clara cell secretory protein, and surfactant proteins B and C, without affecting airway ciliated cells. Importantly, concomitant exposure to SS and the nAChR antagonist mecamylamine during gestation blocked the development of BPD.Conclusions: Gestational exposure to SS irreversibly disrupts lung development leading to a BPD-like condition with hypoalveolarization, decreased angiogenesis, and diminished lung secretory function. Nicotinic receptors are critical in the induction of gestational SS–induced BPD, and the use of nAChR antagonists during pregnancy may block CS-induced BPD.

Fetal Exposure of Rhesus Macaques to Bisphenol A Alters Cellular Development of the Conducting Airway by Changing Epithelial Secretory Product Expression

Background: Bisphenol A (BPA) exposure early in life results in organizational changes in reproductive organs, but the effect of BPA on conducting airway cellular maturation has not been studied. Late gestation is characterized by active differentiation of secretory cells in the lung epithelium.Objective: We evaluated the hypothesis that BPA exposure disrupts epithelial secretory cell development in the fetal conducting airway of the rhesus macaque.Methods: We exposed animals to BPA during either the second (early term) or the third (late term) trimester. There were four treatment groups: a) sham control early term, b) sham control late term, c) BPA early term (BPA-early), and d) BPA late term (BPA-late). Because cellular maturation occurs nonuniformly in the lung, we defined mRNA and protein expression by airway level using microdissection.Results: BPA exposure of the dam during late term significantly accelerated secretory cell maturation in the proximal airways of the fetus; both Clara cell secretory protein (CCSP) and MUC5AC/5B mRNA and protein expression increased.Conclusions: BPA exposure during late gestation accelerates secretory cell maturation in the proximal conducting airways. We identified a critical window of fetal susceptibility for BPA effects on lung epithelial cell maturation in the third trimester. This is of environmental health importance because increases in airway mucins are hallmarks of a number of childhood lung diseases that may be affected by BPA exposure.

A change in the number of CCSPpos/SPCpos cells in mouse lung during development, growth, and repair

Putative resident stem/progenitor cells have been identified in the bronchoalveolar duct junction (BADJ) of the murine lung. However, the contribution of stem cells expressing both Clara cell secretory protein (CCSP) and pro-surfactant protein C (SP-C) to the repair and maintenance of normal homeostasis is still unclear. In this study, we identified and then quantified CD45(neg)/CCSP(pos)/SP-C(pos) cell numbers in normal and lung-injured mice. Normal lung tissues of fetal, newborn, and adult mice were used to evaluate lung progenitor cells during development and growth. Mice treated with naphthalene were used for the bronchiolar epithelium injury model, and mice treated with bleomycin were used for the alveolar epithelium injury model. These lung tissues were stained with CD45, CCSP, and SP-C antibodies by immunofluorescence. The number of lung progenitor cells was counted as CD45(neg)/CCSP(pos)/SP-C(pos) cells by flow cytometry. CCSP(pos)/SP-C(pos) epithelial cells in the BADJ were identified from E18 to 7 months after birth. The percentage of CD45(neg)/CCSP(pos)/SP-C(pos) cells was relatively stable to 7 months (between 0.3±0.04% and 1.28±0.11%). When lungs were treated with naphthalene, the proliferation of CCSP(pos)/SP-C(pos) cells was observed as patches of double-positive cells and preceded the recovery of bronchioles. In contrast, when lungs were treated with bleomycin, the proliferation of CCSP(pos)/SP-C(pos) cells was observed, but the type II alveolar epithelial cells never recovered to baseline. CCSP(pos)/SP-C(pos) lung cells were stable until 7 months after birth. These cells in the BADJ primarily regenerate bronchiolar epithelial cells and not alveolar epithelial cells.

Bone Marrow Cells Expressing Clara Cell Secretory Protein Increase Epithelial Repair After Ablation of Pulmonary Clara Cells

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.

Inflammatory response to acute hypoxia in humans

BackgroundIn animal studies hypoxia is known to cause an inflammatory response, inducing multiple transcription factors and activating molecular processes at the cellular level. However, it is not known whether acute hypoxia causes similar inflammatory effects in humans, although such an assumption is commonly made. MethodsThe effects of acute hypoxic exposure were studied in 12 healthy adults: Each subject was studied on 2 different days. Group 1 (mean age 33 ± 5.5 years; 2 females, 4 males) was exposed either to a hypoxic gas mixture or room air for 30 min and Group 2 (mean age 26.5 ± 7.5 years; 3 females, 3 males) for 60 min. Measurements of circulating adhesion molecules (AMs), Clara cell secretory protein (CC16), hypoxia inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and C reactive protein (hsCRP) were made at baseline and at intervals following exposure for 240 min. ResultsNo significant changes were seen in circulating AMs, CC16, TNF-α, IL-6 or hsCRP, although both HIF-1α and VEGF levels increased significantly (p < 0.05) after hypoxic exposure. ConclusionsAcute hypoxic exposure in normal man does not induce a measurable change in inflammatory or epithelial biomarkers, in contrast to studies at the cellular level in animals. However, acute hypoxic exposure does induce the expression of HIF-1α and VEGF. These results indicate that in humans acute hypoxic exposure for up to 60 min does not induce a generalized inflammatory response, indicating that the human response to hypoxia is more complex than inferred from animal/cellular studies.

Exacerbation of Lung Radiation Injury by Viral Infection: The Role of Clara Cells and Clara Cell Secretory Protein

Viral infections have been associated with exacerbation of disease in human cases of idiopathic pulmonary fibrosis. Since pulmonary fibrosis is a common outcome after irradiation to the lung, we hypothesized that viral infection after radiation exposure would exacerbate radiation-induced lung injury. Epithelial injury, a frequent outcome after infection, has been hypothesized to contribute to the pathogenesis of pulmonary fibrosis and bronchiolar epithelial Clara cells participate in epithelial repair. Therefore, it was further hypothesized that altered responses after irradiation involve the bronchiolar epithelial Clara cells. C57BL/6J or CCSP(-/-) mice were irradiated with 0 (sham), 5, 10 or 15 Gy to the whole thorax. At ten weeks post-irradiation, animals were mock infected or infected with influenza A virus and body weight and survival were monitored. Pulmonary function was assessed by whole-body plethysmography. The Clara cell markers, CCSP and Cyp2f2, were measured in the lung by qRT-PCR, and protein expression was visualized in the lung by immunofluorescence. Following pulmonary function tests, mice were sacrificed and tissues were collected for pathological analysis. In 15 Gy irradiated animals infected with influenza A virus, accelerated respiratory rates, reduced pulmonary function, and exacerbated lung pathology occurred earlier post-irradiation than previously observed after irradiation alone, suggesting infection accelerates the development of radiation injury. After irradiation alone, CCSP and Cyp2f2 mRNA levels were reduced, correlating with reductions in the number of Clara cells lining the airways. When combined with infection, these markers further declined and an apparent delay in recovery of mRNA expression was observed, suggesting that radiation injury leads to a chronic reduction in the number of Clara cells that may potentiate the epithelial injury observed after influenza A virus infection. This novel finding may have considerable therapeutic implications with respect to both thoracic tumor patients and recipients of bone marrow transplants.

Over‐expression of RAGE by proximal lung epithelial cells causes an inflammatory response in adult mice

Receptors for advanced glycation end‐products (RAGE) are multiligand cell‐surface receptors expressed abundantly by pulmonary epithelium. Our lab has discovered RAGE‐mediated effects in lung inflammation induced by tobacco smoke and environmental pollutants; however, the precise contribution of RAGE to proximal airway inflammation was yet unknown. We generated a Tet‐inducible transgenic mouse that conditionally over‐expresses RAGE using the Clara Cell Secretory Protein (CCSP) promoter active in proximal airway Clara cells. RAGE was induced for 40 days from weaning (20 days of age) until sacrifice date at 60 days. Compared to controls, immunohistochemistry, immunoblotting, and qPCR revealed significant RAGE up‐regulation; however, H&amp;E staining revealed no detectible morphological disturbances. Freshly procured bronchoalveolar lavage fluid (BALF) from RAGE TG mice had significantly greater total protein content, total leukocyte quantity, PMN abundance, and cytokine concentration compared to age‐matched control littermates. These data support the concept that RAGE up‐regulation in lung airways may participate in inflammation of the proximal airway. Supported by the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU MEG (PRR).

Uteroglobin Protective Role in CLAD, from Proteomic to In Vitro Biological Activity

Purpose The role of Uteroglobin, a Clara cell secretory protein, in the development of CLAD, is controversial. A proteomic survey suggested its protective role, while other studies reported BAL-fluid or serum increase of uteroglobin levels in BOS patients. Aim of present work was to clarify the possible role of UG in lung repair and in fibrogenesis underlying CLAD development. Methods and Materials Bronchoalveolar lavage fluid (BAL-f) samples from 24 patients (coupled pre- and post-CLAD samples) were analyzed to identify the whole proteome by Liquid Chromatography-Mass Spectrometry (LC-MS). All analyses were carried out on a HPLC coupled to ESI-ION Trap Mass Spectrometer (LC-MS, Thermo Finningan, San Jose, CA, USA). Mesenchimal cells (MC) cultures from BAL-f of patients at pre- and post-CLAD stages were equipped to assess UG activity. A specific colorimetric assay was applied to quantify collagen synthesis. ELISA was applied for Cytokine release assay. Results BAL-f analysis by LC-MS confirmed the identification of many proteins and showed a frequent presence of inflammatory cytokines, small proteins and peptides with molecular masses below 10 kDa. UG was detectable only in pre-CLAD samples of 14 patients with slowly progressive and late lung dysfunction while it was lacking in all but one POST CLAD samples and in pre-CLAD fluid of those patients who developed an earlier and rapidly progressive CLAD. Furthermore UG was able to inhibit collagen production by MC obtained from CLAD patients (mean inhibition 37,3 % at 48 hs) while this effect was not detectable on skin derived fibroblasts. UG significantly inhibited IL8 release by LPS stimulated Alveolar macrophages from CLAD patients (LPS treated Macr 1,45±0,26 mcg/ml versus UG + LPS 0,76±0,04 mcg/ml). Conclusions Our comparative analysis of BAL-f proteomic profiles strongly suggest that UG may play a protective role in CLAD pathogenesis. Its role is confirmed by its ability to modulate in vitro collagen production by CLAD derived MC and IL8 release by macrophages.

Ozone-Induced Injury and Oxidative Stress in Bronchiolar Epithelium Are Associated with Altered Pulmonary Mechanics

In these studies, we analyzed the effects of ozone on bronchiolar epithelium. Exposure of rats to ozone (2 ppm, 3 h) resulted in rapid (within 3 h) and persistent (up to 72 h) histological changes in the bronchiolar epithelium, including hypercellularity, loss of cilia, and necrotizing bronchiolitis. Perivascular edema and vascular congestion were also evident, along with a decrease in Clara cell secretory protein in bronchoalveolar lavage, which was maximal 24 h post-exposure. Ozone also induced the appearance of 8-hydroxy-2'-deoxyguanosine, Ym1, and heme oxygenase-1 in the bronchiolar epithelium. This was associated with increased expression of cleaved caspase-9 and beclin-1, indicating initiation of apoptosis and autophagy. A rapid and persistent increase in galectin-3, a regulator of epithelial cell apoptosis, was also observed. Following ozone exposure (3-24 h), increased expression of cyclooxygenase-2, inducible nitric oxide synthase, and arginase-1 was noted in bronchiolar epithelium. Ozone-induced injury and oxidative stress in bronchiolar epithelium were linked to methacholine-induced alterations in pulmonary mechanics. Thus, significant increases in lung resistance and elastance, along with decreases in lung compliance and end tidal volume, were observed at higher doses of methacholine. This indicates that ozone causes an increase in effective stiffness of the lung as a consequence of changes in the conducting airways. Collectively, these studies demonstrate that bronchiolar epithelium is highly susceptible to injury and oxidative stress induced by acute exposure to ozone; moreover, this is accompanied by altered lung functioning.

Serum levels of Clara cell secretory protein, asthma, and lung function in the adult general population

a Centre for Research in Environmental Epidemiology (CREAL), Barcelona, Spain b Inserm U1018, CESP Centre for Research in Epidemiology and Population Health, Respiratory and Environmental Epidemiology Team, Villejuif, France c Universite Paris Sud 11, Villejuif, France d IMIM (Hospital del Mar Research Institute), Barcelona, Spain e CIBER Epidemiologia y Salud Publica (CIBERESP), Spain f Pulmonology Department-Muscle and Respiratory System Research Unit (URMAR), Hospital del Mar-IMIM, Parc de Recerca Biomedica de Barcelona (PRBB), Barcelona, and Centro de Investigacion en Red de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III (ISCIII), Bunyola, Majorca, Balearic Islands, Spain g Hospital General de Almansa, Complejo Hospitalario Universitario de Albacete, Spain, and the Unit of Epidemiology and Health Information, Public Health of Bizkaia, Bizkaia, Spain h Department of Health, Basque Government, Bilbao, Basque Country, Spain i Unit of Epidemiology and Medical Statistics, Department of Public Health and Community Medicine, University of Verona, Verona, Italy j Universitat Pompeu Fabra, Barcelona, Spain k Arizona Respiratory Center, University of Arizona, Tucson, Ariz

Functional and biological characteristics of asthma in cleaning workers

Cleaning workers have an increased risk of asthma but the underlying mechanisms are largely unknown. We studied functional and biological characteristics in asthmatic cleaners and compared these to healthy cleaners. Forty-two cleaners with a history of asthma and/or recent respiratory symptoms and 53 symptom-free controls were identified. Fractional exhaled nitric oxide (FeNO) was measured and forced spirometry with reversibility testing was performed. Total IgE, pulmonary surfactant protein D and the 16kDa Clara Cell secretory protein were measured in blood serum. Interleukins and other cytokines, growth factors, cys-leukotrienes and 8-isoprostane were measured in exhaled breath condensate. Information on occupational and domestic use of cleaning products was obtained in an interview. Associations between asthma status, specific characteristics and the use of cleaning products were evaluated using multivariable linear and logistic regression analyses. Asthma was associated with an 8% (95% confidence interval (CI) 1-15%) lower postbronchodilator FEV1, a higher prevalence of atopy (42% vs. 10%) and a 2.9 (CI 1.5-5.6) times higher level of total IgE. Asthma status was not associated with the other respiratory biomarkers. Most irritant products and sprays were more often used by asthmatic cleaners. The use of multiuse products, glass cleaners and polishes at work was associated with higher FeNO, particularly in controls. Asthma in cleaning workers is characterised by non-reversible lung function decrement and increased total IgE. Oxidative stress, altered lung permeability and eosinophilic inflammation are unlikely to play an important underlying role, although the latter may be affected by certain irritant cleaning exposures.