Bcl-2, a prosurvival protein, regulates programmed cell death during development and repair processes, and can be oncogenic when cell proliferation is deregulated. The present study investigated what factors modulate Bcl-2 expression in airway epithelial cells and identified the pathways involved. Microarray analysis of mRNA from airway epithelial cells captured by laser microdissection showed that increased expression of IL-1β and IGF-1 coincided with induced Bcl-2 expression compared to controls. Treatment of cultured airway epithelial cells with IL-1β and IGF-1 induced Bcl-2 expression by increasing Bcl-2 mRNA stability with no discernible changes in promoter activity. Silencing the IGF-1 expression using shRNA showed that intracellular (IC)-IGF-1 was increasing Bcl-2 expression. Blocking EGFR or IGF-1R activation also suppressed IC-IGF-1, and abolished the Bcl-2 induction. Induced expression and co-localization of IC-IGF-1 and Bcl-2 were observed in airway epithelial cells of mice exposed to LPS or cigarette smoke and of patients with cystic fibrosis and chronic bronchitis but not in the respective controls. These studies demonstrate that IC-IGF-1 induces Bcl-2 expression in epithelial cells via IGF-1R and EGFR pathways, and targeting IC-IGF-1 could be beneficial to treat chronic airway diseases.
Smoking-related respiratory diseases are a major cause of morbidity and mortality. However, the relationship between smoking and respiratory disease has not been well-studied among ethnic minorities in general and among women in particular.
The objective of this cross-sectional study was to evaluate the risk of airflow obstruction and to assess lung function among Hispanic and non-Hispanic white (NHW) female smokers in a New Mexico cohort.
Participants completed a questionnaire detailing smoking history and underwent spirometry testing. Outcomes studied included airflow obstruction, selected lung function parameters, and chronic mucus hyper-secretion. Chi square, logistic, and linear regression techniques were utilized.
Of the 1,433 eligible women participants, 248 (17.3%) were Hispanic; and 319 had airflow obstruction (22.3%). Hispanic smokers were more likely to be current smokers, and report lower pack-years of smoking, compared to NHW smokers (p < 0.05 for all analyses). Further, Hispanic smokers were at a reduced risk of airflow obstruction compared to NHW smokers, with an O.R. of 0.51, 95% C.I. 0.34, 0.78 (p = 0.002) after adjustment for age, BMI, pack-years and duration of smoking, and current smoking status. Following adjustment for covariates, Hispanic smokers also had a higher mean absolute and percent predicted post-bronchodilator FEV1/FVC ratio, as well as higher mean percent predicted FEV1 (p < 0.05 for all analyses).
Hispanic female smokers in this New Mexico-based cohort had lower risk of airflow obstruction and better lung function than NHW female smokers. Further, smoking history did not completely explain these associations.
Hispanic ethnicity; Smokers; Airflow obstruction; Pulmonary function; Chronic mucus hyper-secretion; Women
The detection of tumor suppressor gene promoter methylation in sputum-derived exfoliated cells predicts early lung cancer. Here we identified genetic determinants for this epigenetic process and examined their biological effects on gene regulation. A two-stage approach involving discovery and replication was employed to assess the association between promoter hypermethylation of a 12-gene panel and common variation in 40 genes involved in carcinogen metabolism, regulation of methylation, and DNA damage response in members of the Lovelace Smokers Cohort (n=1434). Molecular validation of three identified variants was conducted using primary bronchial epithelial cells. Association of study-wide significance (P<8.2×10−5) was identified for rs1641511, rs3730859, and rs1883264 in TP53, LIG1, and BIK, respectively. These SNPs were significantly associated with altered expression of the corresponding genes in primary bronchial epithelial cells. In addition, rs3730859 in LIG1 was also moderately associated with increased risk for lung cancer among Caucasian smokers. Together, our findings suggest that genetic variation in DNA replication and apoptosis pathways impacts the propensity for gene promoter hypermethylation in the aerodigestive tract of smokers. The incorporation of genetic biomarkers for gene promoter hypermethylation with clinical and somatic markers may improve risk assessment models for lung cancer.
DNA damage response; promoter hypermethylation; single nucleotide polymorphism; sputum; smoker
The estimation of genetic ancestry in human populations has important applications in medical genetic studies. Genetic ancestry is used to control for population stratification in genetic association studies, and is used to understand the genetic basis for ethnic differences in disease susceptibility. In this review, we present an overview of genetic ancestry estimation in human disease studies, followed by a review of popular softwares and methods used for this estimation.
Ancestry; Genetic; Polymorphism; Structure
The National Health and Nutrition Examination Survey (NHANES) is one example of cross-sectional datasets that have been used to draw causal inferences regarding environmental chemical exposures and adverse health outcomes. Our objectives were to analyze four NHANES datasets using consistent a priori selected methods to address the following questions: Is there a consistent association between urinary bisphenol A (BPA) measures and diabetes, coronary heart disease (CHD), and/or heart attack across surveys? Is NHANES an appropriate dataset for investigating associations between chemicals with short physiologic half-lives such as BPA and chronic diseases with multi-factorial etiologies? Data on urinary BPA and health outcomes from 2003–2004, 2005–2006, 2007–2008, and 2009–2010 were available.
Methodology and Findings
Regression models were adjusted for creatinine, age, gender, race/ethnicity, education, income, smoking, heavy drinking, BMI, waist circumference, calorie intake, family history of heart attack, hypertension, sedentary time, and total cholesterol. Urinary BPA was not significantly associated with adverse health outcomes for any of the NHANES surveys, with ORs (95% CIs) ranging from 0.996 (0.951–1.04) to 1.03 (0.978–1.09) for CHD, 0.987 (0.941–1.04) to 1.04 (0.996–1.09) for heart attack, and 0.957 (0.899–1.02) to 1.01 (0.980–1.05) for diabetes.
Using scientifically and clinically supportable exclusion criteria and outcome definitions, we consistently found no associations between urinary BPA and heart disease or diabetes. These results do not support associations and causal inferences reported in previous studies that used different criteria and definitions. We are not drawing conclusions regarding whether BPA is a risk factor for these diseases. We are stating the opposite–that using cross-sectional datasets like NHANES to draw such conclusions about short-lived environmental chemicals and chronic complex diseases is inappropriate. We need to expend resources on appropriately designed epidemiologic studies and toxicological explorations to understand whether these types of chemicals play a causal role in chronic diseases.
Rationale: The epidemiology of cigarette smoking–related chronic obstructive pulmonary disease (COPD) is not well characterized in Hispanics in the United States. Understanding how ethnicity influences COPD is important for a number of reasons, from informing public health policies to dissecting the genetic and environmental effects that contribute to disease.
Objectives: The present study assessed differences in risk between Hispanics and non-Hispanic whites for longitudinal and cross-sectional COPD phenotypes. Genetic ancestry was used to verify findings based on self-reported ethnicity. Hispanics in New Mexico are primarily differentiated from non-Hispanic whites by their proportion of Native American ancestry.
Methods: The study was performed in a New Mexican cohort of current and former smokers. Self-reported Hispanic and non-Hispanic white ethnicity was validated by defining genetic ancestry proportions at the individual level using 48 single-nucleotide polymorphism markers. Self-reported ethnicity and genetic ancestry were independently used to assess associations with cross-sectional and longitudinal measures of lung function. Multivariable models were adjusted for indicators of smoking behavior.
Measurements and Main Results: Self-reported Hispanic ethnicity was significantly associated with lower odds of COPD (odds ratio, 0.49; 95% confidence interval, 0.35–0.71; P = 0.007), and this protection was validated by the observation that Hispanic smokers have reduced risk of rapid decline in lung function (odds ratio, 0.48; 95% confidence interval, 0.30–0.78; P = 0.003). Similar findings were noted when Native American genetic ancestry proportions were used as predictors instead of self-report of Hispanic ethnicity.
Conclusions: Hispanic ethnicity is inversely associated with cross-sectional and longitudinal spirometric COPD phenotypes even after adjustment for smoking. Native American genetic ancestry may account for this “Hispanic protection.”
Our previous studies have characterized the inflammatory response of intratracheally instilled lipopolysaccharides (LPS) in F344/N rats. To better reflect the environmentally relevant form of LPS exposure, the present study evaluated the inflammatory response of F344/N rats exposed to LPS by inhalation. Rats were exposed by nose-only inhalation to aerosolized LPS at a median particle diameter of 1 μm and a dose range from 0.08 to 480 μg. Animals were sacrificed 72 h post exposure and the inflammatory cell counts and differentials, the cytokine/chemokine levels in the bronchoalveolar lavage fluid (BALF), and the changes in intraepithelial stored mucosubstances, mucous cells per mm basal lamina, and Bcl-2-positive mucous cells were quantified. We observed a dose-dependent increase reaching maximum values at the 75μg LPS dose for the numbers of neutrophils, macrophages and lymphocytes, for the levels of IL-6, IL-1α, IL-1β, TNFα, MCP-1 and GRO-KC. In addition, mucous cell metaplasia and the percentage of Bcl-2-positive mucous cells were increased with increasing deposited LPS dose. When rats were treated with the phosphodiesterase-4 (PDE4) inhibitor, rolipram (10 mg/kg), prior to exposure to aerosolized LPS neutrophil numbers in the BAL were reduced at 8 h but not at 24 or 72 h post LPS exposure. These results demonstrate that exposure to aerosolized LPS resulted in a more potent inflammatory response at lower doses and that inflammation was more uniformly distributed throughout the lung compared to inflammation caused by intratracheal LPS instillation. Therefore, this animal model will be useful for screening efficacy of anti-inflammatory drugs.
pulmonary inflammation; airway epithelial hyperplasia; mucous cell metaplasia; apoptosis; phosphodiesterase inhibitor
Rationale: Aberrant regulation of airway epithelial cell numbers in airways leads to increased mucous secretions in chronic lung diseases such as chronic bronchitis. Because the Bcl-2 family of proteins is crucial for airway epithelial homeostasis, identifying the players that reduce cigarette smoke (CS)-induced mucous cell metaplasia can help to develop effective therapies.
Objectives: To identify the Bcl-2 family of proteins that play a role in reducing CS-induced mucous cell metaplasia.
Methods: We screened for dysregulated expression of the Bcl-2 family members.
Measurements and Main Results: We identified Bik to be significantly reduced in bronchial brushings of patients with chronic epithelial cell hyperplasia compared with nondiseased control subjects. Reduced Bik but increased MUC5AC mRNA levels were also detected when normal human airway epithelial cells (HAECs) were exposed to CS or when autopsy tissues from former smokers with and without chronic bronchitis were compared. Similarly, exposure of C57Bl/6 mice to CS resulted in increased numbers of epithelial and mucous cells per millimeter of basal lamina, along with reduced Bik but increased Muc5ac expression, and this change was sustained even when mice were allowed to recover in filtered air for 8 weeks. Restoring Bik expression significantly suppressed CS-induced mucous cell metaplasia in differentiated primary HAEC cultures and in airways of mice in vivo. Bik blocked nuclear translocation of phospho-ERK1/2 to induce apoptosis of HAECs. The conserved Leu61 within Bik and ERK1/2 activation were essential to induce cell death in hyperplastic mucous cells.
Conclusions: These studies show that CS suppresses Bik expression to block airway epithelia cell death and thereby increases epithelial cell hyperplasia in chronic bronchitis.
Bcl-2 family of proteins; chronic bronchitis; differentiated human airway cultures; former smokers; MUC5AC
Oxidative stress plays an important role in cigarette smoke-induced lung inflammation and emphysema. We produced an enriched diet by adding freeze-dried fruits and vegetables and additional supplements to the 8604 Teklad Rodent Diet, a standard rodent diet. In this study, we examined the effects of the antioxidant-enriched diet on cigarette smoke-induced lung inflammation and emphysema. CH3/HeN mice were fed either a regular diet or an antioxidant diet. These mice were exposed to filtered air, a low concentration of cigarette smoke (total particulate matter: 100 mg/m3) or a high concentration of cigarette smoke (total particulate matter: 250mg/m3) for 6 hours/day, 5 days/week for total 16 weeks. Surprisingly, increased mortality (53%) was observed in the high concentration of cigarette smoke-exposed mice fed the antioxidant diet compared to the high concentration of cigarette smoke-exposed mice that were fed a regular diet (13%). The necropsy analysis revealed nasal passage obstruction due to mucous plugging in cigarette smoke-exposed mice on the antioxidant diet. However, the antioxidant diet significantly reduced neutrophilic inflammation and emphysema in the high concentration of cigarette smoke-exposed mice as compared to the regular diet /high concentration of cigarette smoke controls. The antioxidant capacity in the bronchoalveolar fluid or oxidative damage to the lung tissue was not affected by the antioxidant diet. Pro-MMP-2, MMP-2, and MMP-9 activity did not correlate with the protective effects of AOD on cigarette smoke-induced emphysema. These data suggest that the antioxidant diet reduced cigarette smoke -induced inflammation and emphysema, but increased mortality in the obligate nose-breathing mice.
cigarette smoke; emphysema; oxidative stress; lung inflammation; adverse effects; lipid peroxidation; metalloproteinase
Both atopy and smoking are known to be associated with increased bronchial responsiveness. Fraction of nitric oxide (NO) in the exhaled air (FENO), a marker of airways inflammation, is decreased by smoking and increased by atopy. NO has also a physiological bronchodilating and bronchoprotective role.
To investigate how the relation between FENO and bronchial responsiveness is modulated by atopy and smoking habits.
Exhaled NO measurements and methacholine challenge were performed in 468 subjects from the random sample of three European Community Respiratory Health Survey II centers: Turin (Italy), Gothenburg and Uppsala (both Sweden). Atopy status was defined by using specific IgE measurements while smoking status was questionnaire-assessed.
Increased bronchial responsiveness was associated with increased FENO levels in non-smokers (p = 0.02) and decreased FENO levels in current smokers (p = 0.03). The negative association between bronchial responsiveness and FENO was seen only in the group smoking less <10 cigarettes/day (p = 0.008). Increased bronchial responsiveness was associated with increased FENO in atopic subjects (p = 0.04) while no significant association was found in non-atopic participants. The reported interaction between FENO and smoking and atopy, respectively were maintained after adjusting for possible confounders (p-values<0.05).
The present study highlights the interactions of the relationship between FENO and bronchial responsiveness with smoking and atopy, suggesting different mechanisms behind atopy- and smoking-related increases of bronchial responsiveness.
Epigenetic therapy for solid tumors could benefit from an in vivo model that defines tumor characteristics of responsiveness and resistance to facilitate patient selection. Here we report that combining the histone deacetylase inhibitor entinostat with the demethylating agent vidaza profoundly affected growth of K-ras/p53 mutant lung adenocarcinomas engrafted orthotopically in immunocompromised nude rats by targeting and ablating pleomorphic cells that occupied up to 75% of the tumor masses. A similar reduction in tumor burden was seen with epigenetic therapy in K-ras or EGFR mutant tumors growing orthotopically. Increased expression of pro-apoptotic genes and the cyclin dependent kinase inhibitor p21 was seen. Hundreds of genes were demethylated highlighted by the re-expression of polycomb-regulated genes coding for transcription factor binding proteins and the p16 gene, a key regulator of the cell cycle. Highly significant gene expression changes were seen in key regulatory pathways involved in cell cycle, DNA damage, apoptosis, and tissue remodeling. These findings demonstrate the promise for epigenetic therapy in cancer management and provide an orthotopic lung cancer model that can assess therapeutic efficacy and reprogramming of the epigenome in tumors harboring different genetic and epigenetic profiles to guide use of these drugs.
lung cancer; DNA methylation; polycomb; epigenetic therapy; nude rat
Mucin depleted foci (MDF) are precancerous lesions of the colon in carcinogen-treated rodents and humans at high risk. Since MDF show signs of inflammation we hypothesized that the defective mucous production would expose them to the risk of being penetrated by intestinal bacteria, which can be sensed by Toll-like receptors (Tlrs) and activate inflammatory pathways. To verify this hypothesis we tested the expression of 84 genes coding for Tlrs and associated pathways using RT-qPCR in MDF (n = 7) from 1,2-dimethylhydrazine (DMH)-treated rats. Among the 84 tested genes, 26 were differentially expressed in MDF with 5 genes significantly up-regulated and 21 down-regulated when compared to the normal mucosa. Tlr2, as well as other downstream genes (Map4k4, Hspd1, Irak1, Ube2n), was significantly up-regulated. Among the genes regulating the NFkB pathway, only Map4k4 was significantly up-regulated, while 19 genes were not varied and 6 were down-regulated. Tlr2 protein was weakly expressed both in normal mucosa and MDF. To determine whether inflammation observed in MDF could be caused by bacteria contacting or infiltrating crypts, we performed fluorescence in situ hybridization (FISH) experiments with a rRNA universal bacterial probe. None of the 21 MDF tested, showed bacteria inside the crypts, while among the colonic tumors (n = 15), only one had very few bacteria on the surface and on the surrounding normal mucosa. In conclusion, the up-regulation of Tlr2 in MDF, suggests a link between this receptor and carcinogenesis, possibly related to a defective barrier function of these lesions. The data of FISH experiments do not support the hypothesis that inflammation in MDF and tumors is stimulated by bacterial infiltration.
Rationale: Wood smoke–associated chronic obstructive pulmonary disease (COPD) is common in women in developing countries but has not been adequately described in developed countries.
Objectives: Our objective was to determine whether wood smoke exposure was a risk factor for COPD in a population of smokers in the United States and whether aberrant gene promoter methylation in sputum may modify this association.
Methods: For this cross-sectional study, 1,827 subjects were drawn from the Lovelace Smokers' Cohort, a predominantly female cohort of smokers. Wood smoke exposure was self-reported. Postbronchodilator spirometry was obtained, and COPD outcomes studied included percent predicted FEV1, airflow obstruction, and chronic bronchitis. Effect modification of wood smoke exposure with current cigarette smoke, ethnicity, sex, and promoter methylation of lung cancer-related genes in sputum on COPD outcomes were separately explored. Multivariable logistic and poisson regression models were used for binary and rate-based outcomes, respectively.
Measurements and Main Results: Self-reported wood smoke exposure was independently associated with a lower percent predicted FEV1 (point estimate [± SE] −0.03 ± 0.01) and a higher prevalence of airflow obstruction and chronic bronchitis (odds ratio, 1.96; 95% confidence interval, 1.52–2.52 and 1.64 (95% confidence interval, 1.31–2.06, respectively). These associations were stronger among current cigarette smokers, non-Hispanic whites, and men. Wood smoke exposure interacted in a multiplicative manner with aberrant promoter methylation of the p16 or GATA4 genes on lower percent predicted FEV1.
Conclusions: These studies identify a novel link between wood smoke exposure and gene promoter methylation that synergistically increases the risk for reduced lung function in cigarette smokers.
wood smoke; cigarette smokers; airflow obstruction; gene promoter methylation in sputum DNA
Horses develop recurrent airway obstruction (RAO) that resembles human bronchial asthma. Differentiated primary equine bronchial epithelial cells (EBEC) in culture that closely mimic the airway cells in vivo would be useful to investigate the contribution of bronchial epithelium in inflammation of airway diseases. However, because isolation and characterization of EBEC cultures has been limited, we modified and optimized techniques of generating and culturing EBECs from healthy horses to mimic in vivo conditions.
Large numbers of EBEC were obtained by trypsin digestion and successfully grown for up to 2 passages with or without serum. However, serum or ultroser G proved to be essential for EBEC differentiation on membrane inserts at ALI. A pseudo-stratified muco-ciliary epithelium with basal cells was observed at differentiation. Further, transepithelial resistance (TEER) was more consistent and higher in P1 cultures compared to P0 cultures while ciliation was delayed in P1 cultures.
This study provides an efficient method for obtaining a high-yield of EBECs and for generating highly differentiated cultures. These EBEC cultures can be used to study the formation of tight junction or to identify epithelial-derived inflammatory factors that contribute to lung diseases such as asthma.
Anti-IgE treatment of intermittent to mild-persistent asthma in a cohort of seven volunteers resulted in improved allergen-induced airway hyperreactivity and a significant reduction in the number of airway myeloid dendritic cells.
Anti-IgE treatment; Allergic Asthma; Dendritic cells (DCs); Myeloid DCs (mDCs); Plasmacytoid DCs (pDCs)
COPD; Genetics; Association analysis; Consortium
Genetic variants influencing lung function in children and adults may ultimately lead to the development of chronic obstructive pulmonary disease (COPD), particularly in high-risk groups.
We tested for an association between single-nucleotide polymorphisms (SNPs) in the gene encoding matrix metalloproteinase 12 (MMP12) and a measure of lung function (prebronchodilator forced expiratory volume in 1 second [FEV1]) in more than 8300 subjects in seven cohorts that included children and adults. Within the Normative Aging Study (NAS), a cohort of initially healthy adult men, we tested for an association between SNPs that were associated with FEV1 and the time to the onset of COPD. We then examined the relationship between MMP12 SNPs and COPD in two cohorts of adults with COPD or at risk for COPD.
The minor allele (G) of a functional variant in the promoter region of MMP12 (rs2276109 [−82A→G]) was positively associated with FEV1 in a combined analysis of children with asthma and adult former and current smokers in all cohorts (P=2×10−6). This allele was also associated with a reduced risk of the onset of COPD in the NAS cohort (hazard ratio, 0.65; 95% confidence interval [CI], 0.46 to 0.92; P = 0.02) and with a reduced risk of COPD in a cohort of smokers (odds ratio, 0.63; 95% CI, 0.45 to 0.88; P = 0.005) and among participants in a family-based study of early-onset COPD (P = 0.006).
The minor allele of a SNP in MMP12 (rs2276109) is associated with a positive effect on lung function in children with asthma and in adults who smoke. This allele is also associated with a reduced risk of COPD in adult smokers.
Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase super family that can mediate cell proliferation and apoptosis. The Ras–Raf–MEK–ERK signaling cascade controlling cell proliferation has been well studied but the mechanisms involved in ERK1/2-mediated cell death are largely unknown. This review focuses on recent papers that define ERK1/2 translocation to the nucleus and the proteins involved in the cytosolic retention of activated ERK1/2. Cytosolic retention of ERK1/2 denies access to the transcription factor substrates that are responsible for the mitogenic response. In addition, cytosolic ERK1/2, besides inhibiting survival and proliferative signals in the nucleus, potentiates the catalytic activity of some proapoptotic proteins such as DAP kinase in the cytoplasm. Studies that further define the function of cytosolic ERK1/2 and its cytosolic substrates that enhance cell death will be essential to harness this pathway for developing effective treatments for cancer and chronic inflammatory diseases.
MAP kinases; activation; epithelial cells; nuclear translocation; cancer; lung diseases
TNF-related apoptosis-inducing ligand (TRAIL) is a potential anticancer agent due to its selectivity in killing transformed cells. However, TRAIL can also stimulate TRAIL-resistant cancer cells’ proliferation and metastasis. Thus, acquired TRAIL resistance during TRAIL therapy would shift the patient’s treatment from beneficial to detrimental. In this study we focused on the acquired TRAIL resistance mechanism and demonstrated that the elevated expression of the anti-apoptotic factor cellular FLICE-like inhibitory protein (c-FLIP) and the pro-survival Bcl-2 family member myeloid cell leukemia 1 (Mcl-1) underlie the main mechanism of this type of TRAIL resistance in lung cancer cells. Chronic exposure to TRAIL resulted in lung cancer cell resistance to TRAIL-induced cytotoxicity, and this resistance was associated with the increase in the cellular levels of c-FLIP L and Mcl-1L. Overexpresssion of c-FLIPL suppressed recruitment of caspase-8 to the death-inducing signaling complex (DISC) while increased Mcl-1L expression blunted the mitochondrial apoptosis pathway. The elevation of c-FLIP L and Mcl-1L expression was due to Akt-mediated stabilization of these proteins in TRAIL-resistant cells. Importantly, suppressing c-FLIPL and Mcl-1L expression by RNA interference collectively alleviated acquired TRAIL resistance. Taken together, these results identify c-FLIPL and Mcl-1L as the major determinants of acquired TRAIL resistance and could be molecular targets for improving TRAIL’s therapeutic value against lung cancer.
TRAIL; c-FLIP; Mcl-1; Akt; apoptosis; lung cancer
Allergic asthma, an inflammatory disease characterized by infiltration and activation of various leukocytes, production of Th2 cytokines and leukotrienes, and atopy, also affects the function of other cell types, causing goblet cell hyperplasia/hypertrophy, increased mucus production/secretion, and airway hyperreactivity. Eosinophilic inflammation is a characteristic feature of human asthma, and recent evidence suggests that eosinophils also play a critical role in T cell trafficking in animal models of asthma. Nicotine is an anti-inflammatory, but the association between smoking and asthma is highly contentious, and some report that smoking cessation increases the risk of asthma in ex-smokers. To ascertain the effects of nicotine on allergy/asthma, Brown Norway rats were treated with nicotine, and sensitized and challenged with allergens. Results unequivocally show that, even after multiple allergen sensitizations, nicotine dramatically suppresses inflammatory/allergic parameters in the lung, including eosinophilic/lymphocytic emigration; mRNA and/or protein expression of Th2 cytokines/chemokines IL-4, IL-5, IL-13, IL-25, and eotaxin; leukotriene C4; and total as well as allergen-specific IgE. While nicotine did not significantly affect hexosaminidase release, IgG, or methacholine-induced airway resistance, it significantly decreased mucus content in bronchoalveolar lavage; interestingly, however, in spite of the strong suppression of IL-4/IL-13, nicotine significantly increased the intraepithelial stored mucosubstances, and Muc5ac mRNA expression. These results suggest that nicotine modulates allergy/asthma primarily by suppressing eosinophil trafficking and suppressing Th2 cytokine/chemokine responses without reducing goblet cell metaplasia, mucous production, and may explain the lower risk of allergic diseases in smokers. To our knowledge this is the first direct evidence that nicotine modulates allergic responses.
IFNγ induces cell death in epithelial cells, but the mediator for this death pathway has not been identified. In this study, we find that expression of Bik/Blk/Nbk is increased in human airway epithelial cells (AECs [HAECs]) in response to IFNγ. Expression of Bik but not mutant BikL61G induces and loss of Bik suppresses IFNγ-induced cell death in HAECs. IFNγ treatment and Bik expression increase cathepsin B and D messenger RNA levels and reduce levels of phospho–extracellular regulated kinase 1/2 (ERK1/2) in the nuclei of bik+/+ compared with bik−/− murine AECs. Bik but not BikL61G interacts with and suppresses nuclear translocation of phospho-ERK1/2, and suppression of ERK1/2 activation inhibits IFNγ- and Bik-induced cell death. Furthermore, after prolonged exposure to allergen, hyperplastic epithelial cells persist longer, and nuclear phospho-ERK is more prevalent in airways of IFNγ−/− or bik−/− compared with wild-type mice. These results demonstrate that IFNγ requires Bik to suppress nuclear localization of phospho-ERK1/2 to channel cell death in AECs.
One of the major aspects of airway remodeling in asthma is the development of mucous cell metaplasia (MCM). The role of cytokines in the generation and resolution of MCM has been studied in mice and in isolated airway epithelial cells in culture. However, studies using organ cultures that keep the tubular structure of the airways intact and allow studies in the absence of inflammatory cells have not been reported. We established an organ culture system that replicates the allergen-induced MCM in mice and analyzed the role of Bax in the IFN-γ–induced resolution of MCM. IL-9 or IL-13 induced MCM independently, but a combined IL-9/IL-13 treatment enhanced MCM synergistically. Addition of IFN-γ at 0.1 ng/ml concentration further increased MCM to levels observed in allergen-exposed mice in vivo. However, MCM was reduced when explants were treated with 50 ng/ml IFN-γ after MCM was established. While IL-9/IL-13 induced MCM in bronchioles microdissected from bax+/+ and bax−/− mice to a similar extent, IFN-γ treatment reduced MCM only in bronchioles from bax+/+ but not in bax−/− bronchioles. Restoration of Bax expression in bax−/− bronchioles using an adenoviral expression system reduced IL-9/IL-13–induced MCM while MCM was similar in noninfected or adenoviral green fluorescent protein–infected bax−/− bronchioles. Furthermore, expressing Bax using an adenoviral expression system reduced allergen-induced MCM in mice. These studies show that allergen-induced MCM is a response to a combination of various cytokines at defined concentrations and that IFN-γ requires Bax for the resolution of MCM.
lung airway cells; Th1 and Th2 cytokines; apoptosis; bronchiolar organ culture; adenoviral expression
Expression of the anti-apoptotic proto-oncogene bcl-2 is negatively affected by the pro-apoptotic p53. To understand the regulation of bcl-2 expression by p53, we studied the bcl-2 promoter regions individually and in the context of the full-length promoter. While the P1 promoter displayed the highest p53-independent activity, the P2 promoter activity was suppressed in p53-sufficient cancer cell lines. In addition, P2 activity was higher in primary airway epithelial cells from p53-/- mice compared to those from p53+/+ mice. Chromatin immunoprecipitation assays confirmed that p53 interacts within a 140 bp sequence of P2 that contained the CCAAT- and TATA-elements. However, when P1 and P2 are linked in one construct, P2 suppressed P1 activity independent of p53. A potential novel promoter with a p53-dependent activity was identified located between P1 and P2, and was designated M. In the context of the full-length bcl-2 promoter, M counteracted in a p53-dependent manner the suppressive activity of P2 on P1. Collectively, these data suggest that P1 promoter is the main driving force for transcribing the bcl-2 gene and P1 activity is modulated by M and P2 in a p53-dependent and -independent manner. These findings may have implications for therapies that are geared towards inhibiting bcl-2 gene expression and inducing cell death.
bcl-2 gene transcription; promoter activity; negative regulatory element; lung; airway epithelium
The airway epithelium functions primarily as a barrier to foreign particles and as a modulator of inflammation. Apoptosis is induced in airway epithelial cells (AECs) by viral and bacterial infections, destruction of the cytoskeleton, or by exposure to toxins such as high oxygen and polycyclic hydrocarbons. Various growth factors and cytokines including TGF-β, IFN-γ, or the activators of the death receptors, TNF-α and FasL, also induce apoptosis in AECs. However, cell death is observed in maximally 15% of AECs after 24 h of treatment. Preincubation with IFN-γ or a zinc deficiency increases the percentage of apoptotic AECs in response to TNF-α or FasL, suggesting that AECs have mechanisms to protect them from cell death. Apoptosis of AECs is a major mechanism in reducing cell numbers after hyperplastic changes in airway epithelia that may arise due to major injuries in response to LPS or allergen exposures. Resolution of hyperplastic changes or changes during prolonged exposure to an allergen is primarily regulated by the Bcl-2 family of proteins. Fas and FasL are both expressed in AECs, and their main function may be to control inflammation by inducing Fas-induced death in inflammatory cells without inducing apoptosis in neighboring cells. Furthermore, AECs engulf dying eosinophils to clear them by phagocytosis. Therefore, in the airway epithelium apoptosis serves three main roles: (1) to eliminate damaged cells; (2) to restore homeostasis following hyperplastic changes; and (3) to control inflammation, and thereby support the barrier and anti-inflammatory functions.
cytokines; infection; inflammation; phagocytosis; mucus cell hyperplasia
The resolution of inflammatory responses in the lung has not been described in detail and the role of specific cytokines influencing the resolution process is largely unknown.
The present study was designed to describe the resolution of inflammation from 3 h through 90 d following an acute injury by a single intratracheal instillation of F344/N rats with LPS. We documented the inflammatory cell types and cytokines found in the bronchoalveolar lavage fluid (BALF), and epithelial changes in the axial airway and investigated whether IL-18 may play a role in the resolution process by reducing its levels with anti-IL-18 antibodies.
Three major stages of inflammation and resolution were observed in the BALF during the resolution. The first stage was characterized by PMNs that increased over 3 h to 1 d and decreased to background levels by d 6–8. The second stage of inflammation was characterized by macrophage influx reaching maximum numbers at d 6 and decreasing to background levels by d 40. A third stage of inflammation was observed for lymphocytes which were elevated over d 3–6. Interestingly, IL-18 and IL-9 levels in the BALF showed a cyclic pattern with peak levels at d 4, 8, and 16 while decreasing to background levels at d 1–2, 6, and 12. Depletion of IL-18 caused decreased PMN numbers at d 2, but no changes in inflammatory cell number or type at later time points.
These data suggest that IL-18 plays a role in enhancing the LPS-induced neutrophilic inflammation of the lung, but does not affect the resolution of inflammation.