The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known to mediate toxic responses to dioxin. However, the role of the AhR in the regulation of cellular physiology has only recently been appreciated, including its ability to control cell cycle progression and apoptosis by unknown mechanisms. We hypothesized that the AhR enhances the activation of the AKT serine/threonine kinase (Akt) pathway to promote cell survival. Utilizing AhR knock-out ( Ahr −/− ) and wild-type ( Ahr +/+ ) mouse lung fibroblasts (MLFs), we found that Ahr −/− MLFs have significantly higher basal Akt phosphorylation but that AhR did not affect Akt phosphorylation in MLFs exposed to growth factors or AhR ligands. Basal Akt phosphorylation was dependent on PI3K but was unaffected by changes in intracellular glutathione (GSH) or p85α. There was no significant decrease in cell viability in Ahr −/− MLFs treated with LY294002—a PI3K inhibitor—although LY294002 did attenuate MTT reduction, indicating an affect on mitochondrial function. Using a mass spectrometry (MS)-based approach, we identified several proteins that were differentially phosphorylated in the Ahr −/− MLFs compared to control cells, including proteins involved in the regulation of extracellular matrix (ECM), focal adhesion, cytoskeleton remodeling and mitochondrial function. In conclusion, Ahr ablation increased basal Akt phosphorylation in MLFs. Our results indicate that AhR may modulate the phosphorylation of a variety of novel proteins not previously identified as AhR targets, findings that help advance our understanding of the endogenous functions of AhR.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor whose physiological function is poorly understood. The AhR is highly expressed in barrier organs such as the skin, intestine, and lung. The lungs are continuously exposed to environmental pollutants such as cigarette smoke (CS) that can induce cell death mechanisms such as apoptosis, autophagy, and endoplasmic reticulum (ER) stress. CS also contains toxicants that are AhR ligands. We have previously shown that the AhR protects against apoptosis, but whether the AhR also protects against autophagy or ER stress is not known. Using cigarette smoke extract (CSE) as our in vitro surrogate of environmental tobacco exposure, we first assessed the conversion of LC3I to LC3II, a classic feature of both autophagic and ER stress-mediated cell death pathways. LC3II was elevated in CSE-exposed lung structural cells [mouse lung fibroblasts (MLFs), MLE12 and A549 cells] when AhR was absent. However, this heightened LC3II expression could not be explained by increased expression of key autophagy genes ( Gabarapl1, Becn1, Map1lc3b), upregulation of upstream autophagic machinery (Atg5–12, Atg3), or impaired autophagic flux, suggesting that LC3II may be autophagy independent. This was further supported by the absence of autophagosomes in Ahr−/− lung cells. However, Ahr−/− lung cells had widespread ER dilation, elevated expression of the ER stress markers CHOP and GADD34, and an accumulation of ubiquitinated proteins. These findings collectively illustrate a novel role for the AhR in attenuating ER stress by a mechanism that may be autophagy independent.

Emphysema, a component of chronic obstructive pulmonary disease (COPD), is characterized by irreversible alveolar destruction that results in a progressive decline in lung function. This alveolar destruction is caused by cigarette smoke, the most important risk factor for COPD. Only 15%-20% of smokers develop COPD, suggesting that unknown factors contribute to disease pathogenesis. We postulate that the aryl hydrocarbon receptor (AHR), a receptor/transcription factor highly expressed in the lungs, may be a new susceptibility factor whose expression protects against COPD. Here, we report that Ahr-deficient mice chronically exposed to cigarette smoke develop airspace enlargement concomitant with a decline in lung function. Chronic cigarette smoke exposure also increased cleaved caspase-3, lowered SOD2 expression, and altered MMP9 and TIMP-1 levels in Ahr-deficient mice. We also show that people with COPD have reduced expression of pulmonary and systemic AHR, with systemic AHR mRNA levels positively correlating with lung function. Systemic AHR was also lower in never-smokers with COPD. Thus, AHR expression protects against the development of COPD by controlling interrelated mechanisms involved in the pathogenesis of this disease. This study identifies the AHR as a new, central player in the homeostatic maintenance of lung health, providing a foundation for the AHR as a novel therapeutic target and/or predictive biomarker in chronic lung disease.

Almost 200 years ago, a general hospital and a medical school were established with the goal of addressing the needs of the growing community of immigrants who came to Montreal in search of prosperity in furs and timber. Within a few years, the school became the Faculty of Medicine of the newly created McGill University, named for the fur trader who gave the land and the initial endowment to establish it.

Background Epithelial-to-mesenchymal transition (EMT), which involves changes in cellular morphology of highly polarized epithelial cells and the gain of mesenchymal cell phenotype with migratory and invasive capacities, is implicated in smoking-related chronic obstructive pulmonary disease (COPD). However, the interactions of fibroblasts and epithelial cells and the participation of fibroblasts in the EMT processes in COPD are poorly understood. Here, we investigated the hypothesis that EMT is active in human bronchial epithelial (HBE) cells of COPD patients, and that mediators secreted by lung fibroblasts from COPD patients induce EMT. Methods Primary HBE cells from normal subjects and COPD patients were purchased from LONZA. HLFs were derived from resected lung obtained from normal (N) and COPD (D) subjects and their conditioned medium (CM) was collected after 2-day culture in serum-free medium. The expression of epithelial and mesenchymal markers as well as EMT-related transcription factors in lung biopsies, and in HBE cells following stimulation with CM from both normal human lung fibroblasts (NHLF) and COPD human lung fibroblasts (DHLF) was evaluated by immunohistochemistry, qRT-PCR and western blot. Results Basal mRNA expression of mesenchymal markers and EMT-related transcription factors were increased in DHBE cells compared to normal human bronchial epithelial cells (NHBE) cells as well as in COPD lungs. CM from NHLF significantly induced vimentin expression in both NHBE and COPD human bronchial epithelial cells (DHBE) cells, but only increased N-cadherin expression in DHBE cells. CM from NHLF significantly induced Twist1 and Twist2 expression in NHBE cells and increased Snai2 (Slug) expression in DHBE cells. While CM from NHLF had no effect on such EMT markers, CM from DHLF significantly increased the protein expression of E-cadherin and vimentin in NHBE cells compared to control. N-cadherin expression was upregulated to a greater degree in NHBE cells than DHBE cells. Only CM from DHLF significantly increased E-/N-cadherin ratio in DHBE cells. Conclusions Our results suggest that DHBE cells have partially undergone EMT under baseline conditions. DHLF-CM promoted EMT in NHBE, suggesting that interactions between fibroblast and epithelial cells may play an important role in the EMT process in COPD.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that responds to man-made environmental toxicants, has emerged as an endogenous regulator of cyclooxygenase-2 (Cox-2) by a mechanism that is poorly understood. In this study, we first used AhR-deficient (AhR(-/-) ) primary pulmonary cells, together with pharmacological tools to inhibit new RNA synthesis, to show that the AhR is a prominent factor in the destabilization of Cox-2 mRNA. The destabilization of Cox-2 mRNA and subsequent suppression of cigarette smoke-induced COX-2 protein expression by the AhR was independent of its ability to bind the dioxin response element (DRE), thereby differentiating the DRE-driven toxicological AhR pathway from its anti-inflammatory abilities. We further describe that the AhR destabilizes Cox-2 mRNA by sequestering HuR within the nucleus. The role of HuR in AhR stabilization of Cox-2 mRNA was confirmed by knockdown of HuR, which resulted in rapid Cox-2 mRNA degradation. Finally, in the lungs of AhR(-/-) mice exposed to cigarette smoke, there was little Cox-2 mRNA despite robust COX-2 protein expression, a finding that correlates with almost exclusive cytoplasmic HuR within the lungs of AhR(-/-) mice. Therefore, we propose that the AhR plays an important role in suppressing the expression of inflammatory proteins, a function that extends beyond the ability of the AhR to respond to man-made toxicants. These findings open the possibility that a DRE-independent AhR pathway may be exploited therapeutically as an anti-inflammatory target.

Nitric oxide (NO) plays an important role in innate host defense and inflammation. In response to infection, NO is generated by inducible nitric oxide synthase (iNOS), a gene product whose expression is highly modulated by different stimuli, including lipopolysaccharide (LPS) from gram-negative bacteria. We reported recently that LPS from Pseudomonas aeruginosa altered Na⁺ transport in alveolar epithelial cells via a suramin-dependent process, indicating that LPS activated a purinergic response in these cells. To further study this question, in the present work, we tested whether iNOS mRNA and protein expression were modulated in response to LPS in alveolar epithelial cells. We found that LPS induced a 12-fold increase in iNOS mRNA expression via a transcription-dependent process in these cells. iNOS protein, NO, and nitrotyrosine were also significantly elevated in LPS-treated cells. Ca²⁺ chelation and protein kinase C (PKCα-β1) inhibition suppressed iNOS mRNA induction by LPS, implicating Ca²⁺-dependent PKC signaling in this process. LPS evoked a significant increase of extracellular ATP. Because PKC activation is one of the signaling pathways known to mediate purinergic signaling, we evaluated the hypothesis that iNOS induction was ATP dependent. Although high suramin concentration inhibited iNOS mRNA induction, the process was not ATP dependent, since specific purinergic receptor antagonists could not inhibit the process. Altogether, these findings demonstrate that iNOS expression is highly modulated in alveolar epithelial cells by LPS via a Ca²⁺/PKCα-β1 pathway independent of ATP signaling.

Asthma is a chronic inflammatory disease characterized by episodic and reversible airflow obstruction, airway hyperresponsiveness, and airway wall remodeling. It is common among older adults, and it is estimated that 4% to 13% of adults older than 65 years have asthma,1McHugh M.K. Symanski E. Pompeii L.A. Declos G.L. Prevalence of asthma among adult females and males in the United States: results from the National Health and Nutrition Examination Survey (NHANES), 2001-2004.J Asthma. 2009; 46: 759-766PubMed Google Scholar suggesting that aging could be a risk factor and contribute to the clinical outcome of asthma in the elderly. The normal aging process involves cellular senescence, a state of permanent growth arrest that limits tissue renewal. Cellular senescence can be characterized as either replicative senescence or stress-induced premature senescence and involves the shortening of telomeres.2Kirkwood T.B. Understanding the odd science of aging.Cell. 2005; 120: 437-447Abstract Full Text Full Text PDF PubMed Scopus (1310) Google Scholar Telomeres are terminal regions of chromosomes containing repeats of TTAGGG that protect DNA from damage.3Martínez P. Blasco M.A. Telomeric and extra-telomeric roles for telomerase and the telomere-binding proteins.Nat Rev Cancer. 2011; 11: 161-176Crossref PubMed Scopus (384) Google Scholar When telomeres critically shorten, cells become susceptible to senescence or apoptosis, indicating that telomere length is a feature of cellular aging. Telomerase plays an important role in telomere maintenance, cell proliferation, and immortality by preventing the shortening of telomeres.4Calado R.T. Young N.S. Telomere diseases.N Engl J Med. 2009; 361: 2353-2365Crossref PubMed Scopus (626) Google Scholar Telomerase contains 2 main subunits: a telomerase RNA component (TERC) and the catalytic subunit of telomerase reverse transcriptase (TERT). TERT is a core functional component of telomerase activity, and the potential roles of TERT expression and/or activity in disease pathogenesis have become a focus of active investigation in cancer, aging, and metabolic and cardiovascular diseases.4Calado R.T. Young N.S. Telomere diseases.N Engl J Med. 2009; 361: 2353-2365Crossref PubMed Scopus (626) Google Scholar Studies investigating telomere length in respiratory diseases have demonstrated correlations between telomere shortening and disease outcome. Patients with chronic obstructive pulmonary disease (COPD) have shorter telomeres in circulating leukocytes than do age-matched healthy control subjects.5Savale L. Chaouat A. Bastuji-Garin S. Marcos E. Boyer L. Maitre B. et al.Shortened telomeres in circulating leukocytes of patients with chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 2009; 179: 566-571Crossref PubMed Scopus (241) Google Scholar Other studies reported a significant relationship between telomere length and airflow obstruction in patients with COPD6Mui T.S. Man J.M. McElhaney J.E. Sandford A.J. Coxson H.J. Birmingham C.L. et al.Telomere length and chronic obstructive pulmonary disease: evidence of accelerated aging.J Am Geriatr Soc. 2009; 57: 2372-2374Crossref PubMed Scopus (52) Google Scholar and as a risk factor for idiopathic pulmonary fibrosis.7Diaz de Leon A. Cronkhite J.T. Katzenstein A.L. Godwin J.D. Raghu G. Glazer C.S. et al.Telomere lengths, pulmonary fibrosis and telomerase (TERT) mutations.PLoS One. 2010; 5: e10680Crossref PubMed Scopus (283) Google Scholar While these studies suggest that analysis of telomere length is a predictor of disease progression in COPD and idiopathic pulmonary fibrosis, telomere length and telomerase expression in asthma remain unexplored. We hypothesized that cellular senescence is a marker for disease outcome in asthma and is related to asthma severity. Our aim was to investigate whether differences exist in telomere length and telomerase expression between asthmatic adults and healthy control subjects. We studied 15 healthy and 14 asthmatic adults aged 25 to 60 years who were nonsmokers. Asthmatic adults consisted of 6 with mild and 8 with severe asthma. For comparison, we also studied 7 patients with COPD aged 58 to 77 years. All patients with COPD were classified as Global Initiative for Chronic Obstructive Lung Disease (GOLD) II according to the current GOLD criteria (GOLD 2011). The mean age of study subjects was as follows: adults with severe asthma, 52.63 ± 2.12 years; adults with mild asthma, 40.83 ± 4.42 years; control subjects, 37.80 ± 2.07 years; patients with COPD, 68.14 ± 3.08 years. We used blood and bronchial biopsy tissues stored at the Tissue Bank of the Respiratory Health Network of the Fonds de la Recherche en Santé du Québec. A hospital research ethics committee approved the study protocol, and written consent was obtained from all subjects. To evaluate telomere length, genomic DNA of peripheral leukocytes was isolated from peripheral blood by using the FlexiGene DNA kit (Qiagen, Toronto, Ontario, Canada). Absolute telomere length was measured by determining the number of TTAGGG hexamer repeats by using quantitative real-time polymerase chain reaction.8O'Callaghan N.J. Fenech M. A quantitative PCR method for measuring absolute telomere length.Biol Proced Online. 2011; 13: 3Crossref PubMed Scopus (324) Google Scholar We performed immunohistochemistry on paraffin-embedded bronchial biopsies to evaluate the localization and expression of human TERT (hTERT) protein by using a rabbit polyclonal antibody to hTERT (Santa Cruz Biotechnology, Paso Robles, Calif; sc-7212). Telomere length measurements in peripheral blood cells can provide information about the replicative history of cells and the clinical value of telomere length assessment in asthmatic patients. This appears to be reflected in patients with severe asthma whose peripheral blood cells had significantly shorter telomeres than those of control subjects (P < .05, multiple comparison test after Kruskal-Wallis) (Fig 1). The mean telomere length (kb telomere/genome) was 64.3 ± 8.9 in patients with severe asthma, 76.4 ± 12.4 in patients with mild asthma, 77.9 ± 10.2 in patients with COPD, versus 96.3 ± 7.5 in control subjects. Telomere length decreased with age in asthmatic patients (data not shown). A decrease in absolute telomere length in circulating leukocytes in asthmatic patients could reflect increased inflammatory/immune cell turnover leading to a progressive decline in telomere reserves, and thus telomere shortening. As total leukocytes are a diverse cell population, it would be of interest in future studies to investigate whether telomere length was equally affected among all leukocyte cell types. We also analyzed the telomere length of peripheral blood cells and PBMCs. No statistical difference in telomere length was found between peripheral blood cells and PBMCs (data not shown). As telomerase modulates telomere length, we next performed immunohistochemistry for the evaluation of hTERT protein expression. Fig 2 shows representative immunohistochemical staining of hTERT in the submucosa of bronchial biopsies of patients with severe asthma, patients with mild asthma, control subjects, and patients with COPD. Biopsies from patients with severe or mild asthma revealed a fewer number of immunostained cells positive for hTERT than did biopsies from control subjects. In contrast, hTERT expression was minimal in biopsies from patients with COPD. The mean number of hTERT immunopositive cells in bronchial biopsies from control subjects (30.83 ± 7.83) was significantly higher when compared with biopsies from patients with mild (4.62 ± 1.43) or severe (2.01 ± 0.41) asthma or patients with COPD (1.59 ± 0.57) (P < .001, Kruskal-Wallis multiple comparison test with Bonferroni correction) (see Fig E1 in this article's Online Repository at www.jacionline.org). Next, double immunocytochemistry was used to identify whether T lymphocytes (CD4+), monocytes/macrophages (CD68+), neutrophils (elastase+), fibroblasts (vimentin+), eosinophils (EG2+), or smooth muscle cells (alpha-smooth muscle actin [SMA]+) express TERT protein. Results (see Fig E2 in this article's Online Repository at www.jacionline.org) demonstrated that lymphocytes, macrophages, neutrophils, fibroblasts, eosinophils, and smooth muscle cells expressed TERT protein. hTERT protein was expressed both in the nuclei and in the cytoplasm of cells distributed in the bronchial submucosa, showing the usefulness of immunohistochemistry to study the localization of hTERT protein at the cellular level in normal or asthmatic airways. Low expression of hTERT protein in the airway wall paralleled telomere shortening in circulating leukocytes of asthmatic patients. Asthmatic patients displaying telomere shortening in peripheral leukocytes and reduced hTERT expression may have worse outcomes. Telomere shortening could prompt exhaustion of immune/inflammatory cells, possibly reflecting excessive proliferative stress of these cells in asthma. It is reasonable to speculate that although telomerase expression was detected in asthmatic patients, it might be inadequate to offset end replication losses during the progression of the disease. While the cause of telomerase shortening in asthmatic patients remains to be identified, one potential mechanism linking telomere loss could be oxidative stress.9Houben J.M. Moonen H.J. van Schooten F.J. Hageman G.J. Telomere length assessment: biomarker of chronic oxidative stress?.Free Radic Biol Med. 2008; 44: 235-246Crossref PubMed Scopus (406) Google Scholar Given that asthma is characterized by systemic and chronic inflammation and oxidative stress, increased levels of reactive oxygen species may play a role in enhancing telomere loss. Furthermore, environmental and/or genetic factors may contribute to telomere erosion and telomere shortening in asthmatic patients. Future mechanistic and association studies are needed to advance our understanding of the relevance of telomere shortening for cellular aging in asthmatic patients. In summary, we have shown that accelerated aging of peripheral blood leukocytes, indicated by short telomeres, may play a role in asthma severity. We thank Andrea Karen Mogas and Cathy Fugere for their assistance in the conduct of the study. Fig E2Representative photomicrographs showing immunohistochemical double staining of bronchial mucosa for the purpose of identifying the cellular phenotype and their expression for hTERT. A, CD4+ T lymphocytes. B, CD68+ monocytes/macrophages. C, Elastase+ neutrophils. D, Vimentin+ fibroblasts. E, EG2 (eosinophil granule)+ eosinophils. F, Alpha-SMA+ smooth muscle cells. CD4-, CD68-, elastase-, vimentin-, EG2-, and α-SMA-positive cells are stained red (with the fast red complex [alkaline phosphatase-antialkaline phosphatase]), hTERT-positive cells are stained brown (avidin-biotin complex technique), and nucleus is stained blue. Cells showing colocalization are stained brown-red-orange (white arrows). Double immunostaining revealed that lymphocytes, monocytes/macrophages, neutrophils, fibroblasts eosinophils, and smooth muscle cells expressed TERT protein. Original magnification ×1000. SMA, Smooth muscle actin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Asthma affects all age groups and presents itself as a spectrum of severity and symptoms. Reactive oxygen species (ROS) play a pivotal role in asthma pathogenesis. Exhaled levels of mediators associated with ROS positively correlate with asthma severity.1Horvath I. Donnelly L.E. Kiss A. Kharitonov S.A. Lim S. Chung K.F. et al.Combined use of exhaled hydrogen peroxide and nitric oxide in monitoring asthma.Am J Respir Crit Care Med. 1998; 158: 1042-1046Crossref PubMed Scopus (232) Google Scholar Autophagy, the process of cellular waste disposal through lysosome-dependent pathways, is induced by ROS to remove oxidized proteins or organelles to minimize tissue damage.2Szumiel I. Autophagy, reactive oxygen species and the fate of mammalian cells.Free Radic Res. 2011; 45: 253-265Crossref PubMed Scopus (49) Google Scholar Although autophagy is augmented in the lungs of patients with chronic obstructive pulmonary disease compared with healthy control subjects,3Chen Z.H. Kim H.P. Sciurba F.C. Lee S.J. Feghali-Bostwick C. Stolz D.B. et al.Egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease.PLoS One. 2008; 3: e3316Crossref PubMed Scopus (365) Google Scholar evidence for autophagy in asthmatic patients, particularly those with moderate-to-severe asthma, has not been reported. We hypothesize that autophagy is associated with asthma pathogenesis and sought to detect its presence using both genetic and histologic approaches.We conducted a genetic association study to investigate whether single nucleotide polymorphisms (SNPs) in genes of the autophagy pathway are associated with asthma. We selected 5 genes of the autophagy pathway (unc-51-like kinase 1 [ULK1], sequestosome 1 [SQSTM1], microtubule-associated protein 1 light chain 3β [MAP1LC3B], beclin 1 [BECN1], and autophagy-related 5 homolog [ATG5]).2Szumiel I. Autophagy, reactive oxygen species and the fate of mammalian cells.Free Radic Res. 2011; 45: 253-265Crossref PubMed Scopus (49) Google Scholar We tested for genetic associations in an asthma family-based study from northeastern Quebec, Canada (the Saguenay-Lac-Saint-Jean [SLSJ] asthma study), by using the family-based association test statistic and UNPHASED software for an odds ratio estimate.4Dudbridge F. Likelihood-based association analysis for nuclear families and unrelated subjects with missing genotype data.Hum Hered. 2008; 66: 87-98Crossref PubMed Scopus (542) Google Scholar, 5Lake S.L. Blacker D. Laird N.M. Family-based tests of association in the presence of linkage.Am J Hum Genet. 2000; 67: 1515-1525Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar Patient recruitment has previously been described.6Moffatt M.F. Gut I.G. Demenais F. Strachan D.P. Bouzigon E. Heath S. et al.A large-scale, consortium-based genomewide association study of asthma.N Engl J Med. 2010; 363: 1211-1221Crossref PubMed Scopus (1450) Google Scholar These SNPs have been genotyped previously in a genome-wide association study.6Moffatt M.F. Gut I.G. Demenais F. Strachan D.P. Bouzigon E. Heath S. et al.A large-scale, consortium-based genomewide association study of asthma.N Engl J Med. 2010; 363: 1211-1221Crossref PubMed Scopus (1450) Google Scholar To reduce the likelihood of false-positive findings, we reset the statistical significance threshold from a P value of .05 to a P value of .001, according to the Bonferroni method. To confirm our positive findings, we tested the association in a second family-based population, the non-Hispanic white participants of the Childhood Asthma Management Program (CAMP), and patient recruitment has previously been described.7The Childhood Asthma Management Program (CAMP): design, rationale, and methods. Childhood Asthma Management Program Research Group.Control Clin Trials. 1999; 20: 91-120Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar The SLSJ local ethics committee and the Institutional Review Board for CAMP approved the study, and all subjects provided informed consent.In the SLSJ asthma study a total of 1338 subjects (483 nuclear families) with known asthma status were included in the analysis, and 336 subjects were either probands or affected siblings. Of this group, the male/female ratio was 0.83. The mean age was 16.45 years (SD, ±9.43 years), and 77.1% were atopic. The mean FEV1 percent predicted value was 94.1% (SD, 20.1%). A total of 39 SNPs were tested, and after Bonferroni correction, SNP rs12212740 G>A of ATG5 remained statistically significant (Table I). Allele G, with an allele frequency of 0.88, is overtransmitted to asthmatic offspring (P = .0002; odds ratio, 1.35; 95% CI, 1.01-1.89). SNP rs12212740 was not associated with asthma in CAMP; however, it was associated with prebronchodilator FEV1 (adjusted for age, sex, and height; P = .04). In the SLSJ cohort rs12212740 was associated with prebronchodilator FEV1 (percent predicted; P = .007). In both populations allele G was negatively associated with adjusted prebronchodilator FEV1. SNP rs12212740 is located in intron 3 of ATG5, which is located 7 kb downstream and 8 kb upstream of exons 3 and 4, respectively. At present, the functional consequence of SNP rs12212740 is unknown, and it is probable that the association is due to the linkage disequilibrium between SNP rs12212740 and the true causative variant. Nevertheless, the association between a genetic variant of ATG5 and prebronchodilator FEV1 in both study populations suggests that autophagy is associated with reduced lung function in asthmatic patients.Table IAssociation of SNPs and asthma in the SLSJ asthma studyGeneSNPAlleleAllele frequencyNo. of informative familiesP valueBECN1rs10512488A0.82180.1019MAP1LC3Brs7204722T0.82167.3834rs8051218T0.9564.141rs2873702A0.9919−.9351rs2241617T0.87118.8613rs3748400T0.75196.8694SQSTM1rs166624G0.86131−.5718rs10516140C0.8168.9025rs565280G0.59224.9447rs155788T0.9197−.1876rs10277C0.6221.8631rs1065154T0.6222.8886rs248244C0.9560−.7241rs248247G0.62232−.7656rs248248C0.78198.4782rs2303677C0.78180−.2451rs155793C0.9191−.6668rs748197G0.55232.3494ULK1rs11246867G0.9568.0187rs9652059C0.85148.0126rs10902472C0.9564.0387rs7487166G0.87132.036ATG5rs9398071T0.77176−.0694rs6920944C0.9177.9309rs10484575C0.9287−.8106rs9386514T0.8164−.1043rs9486306T0.87117−.2731rs1769972T0.9561.739rs3804332T0.9665−.4294rs3851210C0.9482.2755rs3804333C0.78174−.1332rs633724C0.66222.0145rs9486314C0.82143−.1427rs12529626A0.9107.0627rs10485352A0.9665−.4297rs12212740∗After Bonferroni correction, SNP rs12212740 of ATG5 remained statistically significant.G0.88130.0002rs1766208C0.88132−.5169rs3761796A0.9915.3656rs2355380T0.7220.2373Thirty-nine SNPs were tested for association with asthma in the SLSJ asthma study. The gene symbols, rs numbers for each SNP, major alleles of each SNP, numbers of informative families that contributed to the test statistic, and unadjusted P values are listed. Positive P values represent overtransmission of the major allele to affected offspring, and negative P values represent undertransmission.∗ After Bonferroni correction, SNP rs12212740 of ATG5 remained statistically significant. Open table in a new tab Bronchial biopsy specimens stored at the Tissue Bank of the Respiratory Health Network of the Fonds de la Recherché en Santé du Québec (McGill University Health Centre site) were obtained to determine whether autophagy is present in the airways of asthmatic patients. Patient recruitment and bronchoscopy have previously been described.8Shannon J. Ernst P. Yamauchi Y. Olivenstein R. Lemiere C. Foley S. et al.Differences in airway cytokine profile in severe asthma compared to moderate asthma.Chest. 2008; 133: 420-426Crossref PubMed Scopus (180) Google Scholar Bronchial biopsy tissue from a patient with moderately severe asthma and a healthy control subject were viewed by means of electron microscopy (EM) for double-membrane autophagosomes.Here we demonstrated evidence of autophagy in asthma pathogenesis using EM in a tissue sample from a patient with moderately severe asthma. By using EM, double-membrane autophagosomes were detected in fibroblasts and epithelial cells. A representative fibroblast from a bronchial biopsy tissue of a patient with moderate asthma is depicted in Fig 1, A to C, and epithelial cells are depicted in Fig 2, A to C. Corresponding fibroblast and epithelial cells from a healthy control subject are depicted in Fig 1, D to F, and Fig 2, D to F, respectively, where fewer or no autophagosome were detected.Fig 2Autophagosomes were detected in bronchial epithelial cells from a patient with moderate asthma. Tissue was viewed at magnifications of ×4030 (A), ×9760 (B), and ×37,000 (C), respectively. Corresponding epithelial cells of a healthy subject were viewed at magnifications of ×6,390 (D), ×16,100 (E), and ×37,000 (F), respectively. Arrows indicate double-membrane autophagosomes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)This is the first report to present genetic and histologic evidence of autophagy in asthma pathogenesis. ATG5 is involved in the elongation step of the autophagosome formation. ATG5 forms a complex with ATG12 and ATG16L1, and the complexes are found on the outer membrane of the forming autophagosome.2Szumiel I. Autophagy, reactive oxygen species and the fate of mammalian cells.Free Radic Res. 2011; 45: 253-265Crossref PubMed Scopus (49) Google Scholar We speculate that the positive association of allele G with asthma and the negative association with prebronchodilator FEV1 in asthmatic patients might be due to the inverse relationship between prebronchodilator FEV1 and asthma severity.9Lange P. Parner J. Vestbo J. Schnohr P. Jensen G.A. 15-year follow-up study of ventilatory function in adults with asthma.N Engl J Med. 1998; 339: 1194-1200Crossref PubMed Scopus (1077) Google Scholar If allele G is a risk factor for low prebronchodilator FEV1, given that prebronchodilator FEV1 tends to be less in those with more severe forms of asthma, the allele would also increase the risk of moderate-to-severe asthma. The genetic association of ATG5 with prebronchodilator FEV1 in asthmatic patients and the detection of autophagosomes in fibroblasts and epithelial cells in tissues from a patient with moderately severe asthma suggest an association between autophagy and reduced lung function in asthmatic patients. At present, the mechanistic pathway of autophagy in asthma is unclear, but it opens up a new avenue to explore the mechanism of the chronic nature of asthma pathogenesis. Asthma affects all age groups and presents itself as a spectrum of severity and symptoms. Reactive oxygen species (ROS) play a pivotal role in asthma pathogenesis. Exhaled levels of mediators associated with ROS positively correlate with asthma severity.1Horvath I. Donnelly L.E. Kiss A. Kharitonov S.A. Lim S. Chung K.F. et al.Combined use of exhaled hydrogen peroxide and nitric oxide in monitoring asthma.Am J Respir Crit Care Med. 1998; 158: 1042-1046Crossref PubMed Scopus (232) Google Scholar Autophagy, the process of cellular waste disposal through lysosome-dependent pathways, is induced by ROS to remove oxidized proteins or organelles to minimize tissue damage.2Szumiel I. Autophagy, reactive oxygen species and the fate of mammalian cells.Free Radic Res. 2011; 45: 253-265Crossref PubMed Scopus (49) Google Scholar Although autophagy is augmented in the lungs of patients with chronic obstructive pulmonary disease compared with healthy control subjects,3Chen Z.H. Kim H.P. Sciurba F.C. Lee S.J. Feghali-Bostwick C. Stolz D.B. et al.Egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease.PLoS One. 2008; 3: e3316Crossref PubMed Scopus (365) Google Scholar evidence for autophagy in asthmatic patients, particularly those with moderate-to-severe asthma, has not been reported. We hypothesize that autophagy is associated with asthma pathogenesis and sought to detect its presence using both genetic and histologic approaches. We conducted a genetic association study to investigate whether single nucleotide polymorphisms (SNPs) in genes of the autophagy pathway are associated with asthma. We selected 5 genes of the autophagy pathway (unc-51-like kinase 1 [ULK1], sequestosome 1 [SQSTM1], microtubule-associated protein 1 light chain 3β [MAP1LC3B], beclin 1 [BECN1], and autophagy-related 5 homolog [ATG5]).2Szumiel I. Autophagy, reactive oxygen species and the fate of mammalian cells.Free Radic Res. 2011; 45: 253-265Crossref PubMed Scopus (49) Google Scholar We tested for genetic associations in an asthma family-based study from northeastern Quebec, Canada (the Saguenay-Lac-Saint-Jean [SLSJ] asthma study), by using the family-based association test statistic and UNPHASED software for an odds ratio estimate.4Dudbridge F. Likelihood-based association analysis for nuclear families and unrelated subjects with missing genotype data.Hum Hered. 2008; 66: 87-98Crossref PubMed Scopus (542) Google Scholar, 5Lake S.L. Blacker D. Laird N.M. Family-based tests of association in the presence of linkage.Am J Hum Genet. 2000; 67: 1515-1525Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar Patient recruitment has previously been described.6Moffatt M.F. Gut I.G. Demenais F. Strachan D.P. Bouzigon E. Heath S. et al.A large-scale, consortium-based genomewide association study of asthma.N Engl J Med. 2010; 363: 1211-1221Crossref PubMed Scopus (1450) Google Scholar These SNPs have been genotyped previously in a genome-wide association study.6Moffatt M.F. Gut I.G. Demenais F. Strachan D.P. Bouzigon E. Heath S. et al.A large-scale, consortium-based genomewide association study of asthma.N Engl J Med. 2010; 363: 1211-1221Crossref PubMed Scopus (1450) Google Scholar To reduce the likelihood of false-positive findings, we reset the statistical significance threshold from a P value of .05 to a P value of .001, according to the Bonferroni method. To confirm our positive findings, we tested the association in a second family-based population, the non-Hispanic white participants of the Childhood Asthma Management Program (CAMP), and patient recruitment has previously been described.7The Childhood Asthma Management Program (CAMP): design, rationale, and methods. Childhood Asthma Management Program Research Group.Control Clin Trials. 1999; 20: 91-120Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar The SLSJ local ethics committee and the Institutional Review Board for CAMP approved the study, and all subjects provided informed consent. In the SLSJ asthma study a total of 1338 subjects (483 nuclear families) with known asthma status were included in the analysis, and 336 subjects were either probands or affected siblings. Of this group, the male/female ratio was 0.83. The mean age was 16.45 years (SD, ±9.43 years), and 77.1% were atopic. The mean FEV1 percent predicted value was 94.1% (SD, 20.1%). A total of 39 SNPs were tested, and after Bonferroni correction, SNP rs12212740 G>A of ATG5 remained statistically significant (Table I). Allele G, with an allele frequency of 0.88, is overtransmitted to asthmatic offspring (P = .0002; odds ratio, 1.35; 95% CI, 1.01-1.89). SNP rs12212740 was not associated with asthma in CAMP; however, it was associated with prebronchodilator FEV1 (adjusted for age, sex, and height; P = .04). In the SLSJ cohort rs12212740 was associated with prebronchodilator FEV1 (percent predicted; P = .007). In both populations allele G was negatively associated with adjusted prebronchodilator FEV1. SNP rs12212740 is located in intron 3 of ATG5, which is located 7 kb downstream and 8 kb upstream of exons 3 and 4, respectively. At present, the functional consequence of SNP rs12212740 is unknown, and it is probable that the association is due to the linkage disequilibrium between SNP rs12212740 and the true causative variant. Nevertheless, the association between a genetic variant of ATG5 and prebronchodilator FEV1 in both study populations suggests that autophagy is associated with reduced lung function in asthmatic patients. Thirty-nine SNPs were tested for association with asthma in the SLSJ asthma study. The gene symbols, rs numbers for each SNP, major alleles of each SNP, numbers of informative families that contributed to the test statistic, and unadjusted P values are listed. Positive P values represent overtransmission of the major allele to affected offspring, and negative P values represent undertransmission. Bronchial biopsy specimens stored at the Tissue Bank of the Respiratory Health Network of the Fonds de la Recherché en Santé du Québec (McGill University Health Centre site) were obtained to determine whether autophagy is present in the airways of asthmatic patients. Patient recruitment and bronchoscopy have previously been described.8Shannon J. Ernst P. Yamauchi Y. Olivenstein R. Lemiere C. Foley S. et al.Differences in airway cytokine profile in severe asthma compared to moderate asthma.Chest. 2008; 133: 420-426Crossref PubMed Scopus (180) Google Scholar Bronchial biopsy tissue from a patient with moderately severe asthma and a healthy control subject were viewed by means of electron microscopy (EM) for double-membrane autophagosomes. Here we demonstrated evidence of autophagy in asthma pathogenesis using EM in a tissue sample from a patient with moderately severe asthma. By using EM, double-membrane autophagosomes were detected in fibroblasts and epithelial cells. A representative fibroblast from a bronchial biopsy tissue of a patient with moderate asthma is depicted in Fig 1, A to C, and epithelial cells are depicted in Fig 2, A to C. Corresponding fibroblast and epithelial cells from a healthy control subject are depicted in Fig 1, D to F, and Fig 2, D to F, respectively, where fewer or no autophagosome were detected. This is the first report to present genetic and histologic evidence of autophagy in asthma pathogenesis. ATG5 is involved in the elongation step of the autophagosome formation. ATG5 forms a complex with ATG12 and ATG16L1, and the complexes are found on the outer membrane of the forming autophagosome.2Szumiel I. Autophagy, reactive oxygen species and the fate of mammalian cells.Free Radic Res. 2011; 45: 253-265Crossref PubMed Scopus (49) Google Scholar We speculate that the positive association of allele G with asthma and the negative association with prebronchodilator FEV1 in asthmatic patients might be due to the inverse relationship between prebronchodilator FEV1 and asthma severity.9Lange P. Parner J. Vestbo J. Schnohr P. Jensen G.A. 15-year follow-up study of ventilatory function in adults with asthma.N Engl J Med. 1998; 339: 1194-1200Crossref PubMed Scopus (1077) Google Scholar If allele G is a risk factor for low prebronchodilator FEV1, given that prebronchodilator FEV1 tends to be less in those with more severe forms of asthma, the allele would also increase the risk of moderate-to-severe asthma. The genetic association of ATG5 with prebronchodilator FEV1 in asthmatic patients and the detection of autophagosomes in fibroblasts and epithelial cells in tissues from a patient with moderately severe asthma suggest an association between autophagy and reduced lung function in asthmatic patients. At present, the mechanistic pathway of autophagy in asthma is unclear, but it opens up a new avenue to explore the mechanism of the chronic nature of asthma pathogenesis. We thank Glenda Wright, Louise Pelletier, and Anja Geitmann for their assistance in EM.

Reactive oxidative species (ROS) are essential in cellular survival; however, excessive production and chronic exposure to ROS pose serious health threats. Excessive production of ROS is thought to play a pivotal role in the pathogenesis of asthma, where exhaled levels of ROS have been found to positively correlate with disease severity. Autophagy is induced by ROS to remove oxidized proteins or organelles to minimize tissue damage, and presents itself as a good candidate pathway for investigation in asthma pathogenesis. Given the role of oxidative stress in the pathogenesis of asthma and disease severity, we hypothesized that autophagy is associated with asthma pathogenesis, and sought to detect its presence using both genetic and histological approaches. We found variant rs12212740, an intronic SNP of ATG5, to be associated with asthma and forced expiratory volume in 1 second (FEV1) percent predicted in the French Canadian population and with FEV1 in an American Caucasian cohort. Furthermore, double-membrane autophagosomes were more easily detected in fibroblast and epithelial cells from a bronchial biopsy tissue of a moderately severe asthma patient compared with corresponding cells of a healthy subject. Asthma is associated with a cytokine milieu [e.g., interleukin (IL)-13] that promotes transforming growth factor-β1 (TGFβ1) affiliated airway remodeling, and agonistic relationships existed among these cytokines and ROS. Hence, autophagy may be a cellular mechanism that promotes TGFβ1 airway remodeling and loss of lung function in asthma.

Background Cigarette smoke is a major risk factor for chronic obstructive pulmonary disease (COPD), an inflammatory lung disorder. COPD is characterized by an increase in CD8 + T cells within the central and peripheral airways. We hypothesized that the CD8 + T cells in COPD patients have increased Toll-like receptor (TLR) expression compared to control subjects due to the exposure of cigarette smoke in the airways. Methods Endobronchial biopsies and peripheral blood were obtained from COPD patients and control subjects. TLR4 and TLR9 expression was assessed by immunostaining of lung tissue and flow cytometry of the peripheral blood. CD8 + T cells isolated from peripheral blood were treated with or without cigarette smoke condensate (CSC) as well as TLR4 and TLR9 inhibitors. PCR and western blotting were used to determine TLR4 and TLR9 expression, while cytokine secretion from these cells was detected using electrochemiluminescence technology. Results No difference was observed in the overall expression of TLR4 and TLR9 in the lung tissue and peripheral blood of COPD patients compared to control subjects. However, COPD patients had increased TLR4 and TLR9 expression on lung CD8 + T cells. Exposure of CD8 + T cells to CSC resulted in an increase of TLR4 and TLR9 protein expression. CSC exposure also caused the activation of CD8 + T cells, resulting in the production of IL-1β, IL-6, IL-10, IL-12p70, TNFα and IFNγ. Furthermore, inhibition of TLR4 or TLR9 significantly attenuated the production of TNFα and IL-10. Conclusions Our results demonstrate increased expression of TLR4 and TLR9 on lung CD8 + T cells in COPD. CD8 + T cells exposed to CSC increased TLR4 and TLR9 levels and increased cytokine production. These results provide a new perspective on the role of CD8 + T cells in COPD.

To investigate peroxidase induced 3′-nitrotyrosine (3NT) formation, neutrophil derived myeloperoxidase (MPO) (0.025μM) was directly administered to A549 epithelial cells with or without H2O2 (150μM). Little evidence of 3NT was found. In contrast, there was a dose dependent increase in intracellular NO (p<0.001, n=8) following MPO (0.025μM) treatment, which was further enhanced (p<0.0003, n=8) by addition of H2O2. Extracellular NO also increased after MPO (p<0.002, n=5) and with MPO and H2O2 (p<0.004, n=5). Substantial 3NT formation was only detected following addition of nitrite (NO2−, ⩾100μM), which induced a dose dependent increase in epithelial 3NT. In contrast, protein carbonyl formation and increased GSSG/GSH ratios were associated with MPO treatment even in the absence of NO2−. Co-culture of A549 epithelial cells with polymorphonuclear leukocytes (PMN) (106/ml) led to immunocytochemical detection of epithelial 3NT and induction of nitric oxide synthase (NOS2). However, in a Transwell system direct contact between PMN and A549 cells was necessary for immunodetection of 3NT but not of NOS2 consistent with a role for high local nitrite concentrations. These findings demonstrate dissociation between epithelial endogenous NO production and 3NT formation. Although MPO can influence cellular oxidative stress, particularly in the presence of H2O2, 3NT formation requires the presence of high concentrations of NO2− in the milieu.

The effect of muscle activation on muscle nitric oxide (NO) production remains controversial. Whereas NO release increases in in vitro activated muscles and in vivo limb muscles, diaphragmatic NO synthase (NOS) activity declines after 3 h of inspiratory resistive loading (IRL). We tested in this study the hypotheses that acute IRL decreases diaphragmatic NO derivatives levels and reduces protein expression of neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) NO synthases, as well as 3-nitrotyrosine formation. Anesthetized, tracheostomized, spontaneously breathing adult rats were subjected to IRL (50% of the maximum inspiratory pressure) for 1, 3, or 6 h. Quietly breathing rats served as controls. After 3 h of IRL, muscle eNOS and nNOS protein levels rose by 80 and 60% of control values, respectively. Whereas eNOS expression did not change any further, nNOS expression reached 550% of control values after 6 h of IRL. Strong iNOS protein expression was detected in the diaphragms after 6 h of IRL. Total NO derivatives levels in the diaphragm declined during IRL as a result of reduction in nitrate, nitrite, and nitrosothiols. Diaphragmatic protein tyrosine nitration decreased in response to IRL, and this reduction was mainly due to reduced tyrosine nitration of enolase and aldolase. We conclude that diaphragmatic NO derivatives levels decline in response to IRL and that the rise in diaphragmatic NOS protein expression may be a compensatory response designed to counterbalance the decline in NOS activity.

Nitric oxide synthase 1 (NOS1) is a major determinant of bronchial responsiveness in mice and has been proposed as an asthma gene in man. Nevertheless, how nitric oxide production by NOS1 contributes to airway responsiveness remains unclear. Although NOS1 is usually closely associated with nerves, it has also been found in a variety of other cell types, particularly epithelium. We sought to better understand the role of NOS1 by determining its major site of expression in murine airways. Using nicotinamide adenine dinucleotide phosphate-diaphorase (diaphorase), which non-selectively detects nitric oxide synthase (NOS), we found strong evidence of NOS in the airways largely restricted to the airway epithelium and trachea glands. In contrast, diaphorase staining of NOS1-deficient mutant mice demonstrated a marked reduction in epithelial cells of the trachea but not bronchioles, suggesting that the epithelium is the major site of NOS1 expression. This was supported by immunohistochemistry, which also demonstrated significant staining in glands and to a lesser degree in airway smooth muscle. Double immunofluorescence staining of tracheas for NOS1 and the nerve marker PGP 9.5 failed to demonstrate co-localization, indicating that nerves are not an important source of NOS1 in the murine airway wall. Finally, removal of the trachea epithelium by digestion resulted in a marked decrease in NOS1 detection by Western blotting, confirming the epithelium as the major site of NOS1 expression in the murine airway. These findings support the notion that the role of NOS1 in murine bronchial responsiveness involves the epithelium of the central airways.

BACKGROUND:Although increased nitric oxide (NO) production in asthma is mediated largely by upregulation of the inducible form of nitric oxide synthase (iNOS, or NOS 2), some studies have suggested an important role for the usually constitutive neural NOS isoform (nNOS, or NOS 1).AIM:To investigate how NOS 1 may influence allergic inflammation, we used NOS 1 knockout mice and their wild-type (WT) controls.METHODS:Mice were sensitized and challenged with ovalbumin (OVA) using a protocol known to upregulate NOS 2 in the airways.RESULTS:In addition to expected increases in NOS 2 activity, OVA challenge led to increases in calcium-dependent NOS activity, which was accounted for by increased expression of NOS 1 at both mRNA (n = 5, p < 0.001) and protein levels (n = 5, p < 0.01). In NOS-1-deficient mice, OVA challenge induced less eosinophilia (n = 7, p < 0.05) and much less NO production (n = 10, p < 0.01) than in WT controls, reflecting not only the expected absence of NOS 1, but also lack of upregulation of NOS 2. This interaction appeared to be stimulus specific as NOS-1-deficient mice did upregulate NOS 2 following exposure to lipopolysaccharide (n = 5, p < 0.001).CONCLUSIONS:These findings underscore the importance of NOS 1 in allergic airway inflammation and suggest a mechanism by which NOS 1 may influence overall NO production in the airways.

Decreased endothelial NO synthase (eNOS)-derived NO bioavailability and impaired vasomotor control are crucial factors in cardiovascular disease pathogenesis. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a vascular disorder associated with ENDOGLIN (ENG) haploinsufficiency and characterized by venous dilatations, focal loss of capillaries, and arteriovenous malformations (AVMs). We report that resistance arteries from Eng+/- mice display an eNOS-dependent enhancement in endothelium-dependent dilatation and impairment in the myogenic response, despite reduced eNOS levels. We have found that eNOS is significantly reduced in endoglin-deficient endothelial cells because of decreased eNOS protein half-life. We demonstrate that endoglin can reside in caveolae and associate with eNOS, suggesting a stabilizing function of endoglin for eNOS. After Ca2+-induced activation, endoglin-deficient endothelial cells have reduced eNOS/Hsp90 association, produce less NO, and generate more eNOS-derived superoxide (O2-), indicating that endoglin also facilitates eNOS/Hsp90 interactions and is an important regulator in the coupling of eNOS activity. Treatment with an O2- scavenger reverses the vasomotor abnormalities in Eng(+/-) arteries, suggesting that uncoupled eNOS and resulting impaired myogenic response represent early events in HHT1 pathogenesis and that the use of antioxidants may provide a novel therapeutic modality.

IL-13 is a mediator of allergen-induced airway hyperresponsiveness (AHR). The aim of this study was to evaluate whether eotaxin and IL-5 were implicated in the effects of IL-13 on allergen-induced AHR or whether IL-13 may exert its effects through direct actions on airway smooth muscle (ASM). To study this question airway inflammation and AHR were induced in mice by sensitization and subsequent challenge on three successive days with ovalbumin. A monoclonal anti-IL-13 antibody administered before each challenge significantly reduced AHR without affecting airway eosinophilia. No changes of mRNA in BAL and lung tissues or protein levels in BAL of IL-5 or eotaxin were found following anti-IL-13 treatment. Combined injection of monoclonal anti-IL-5 and antieotaxin antibodies before each antigen challenge blocked airway eosinophilia but failed to reduce AHR. IL-13 induced calcium transients in cultured murine ASM cells and augmented the calcium and contractile responses of these cells to leukotriene D4. These results suggest that IL-13 plays an important role in allergen-induced AHR and is important in the early phases of the inflammatory process. Its effects on AHR are mediated independently of IL-5 and eotaxin and may involve a direct effect on ASM to augment its responsiveness.