Search

In asthma, goblet cell numbers are increased within the airway epithelium, perpetuating the production of mucus that is more difficult to clear and results in airway mucus plugging. Notch1, Notch2, or Notch3, or a combination of these has been shown to influence the differentiation of airway epithelial cells. How the expression of specific Notch isoforms differs in fully differentiated adult asthmatic epithelium and whether Notch influences mucin production after differentiation is currently unknown. We aimed to quantify different Notch isoforms in the airway epithelium of individuals with severe asthma and to examine the impact of Notch signaling on mucin MUC5AC. Human lung sections and primary bronchial epithelial cells from individuals with and without asthma were used in this study. Primary bronchial epithelial cells were differentiated at the air-liquid interface for 28 days. Notch isoform expression was analyzed by Taqman quantitative PCR. Immunohistochemistry was used to localize and quantify Notch isoforms in human airway sections. Notch signaling was inhibited in vitro using dibenzazepine or Notch3-specific siRNA, followed by analysis of MUC5AC. NOTCH3 was highly expressed in asthmatic airway epithelium compared with nonasthmatic epithelium. Dibenzazepine significantly reduced MUC5AC production in air-liquid interface cultures of primary bronchial epithelial cells concomitantly with suppression of NOTCH3 intracellular domain protein. Specific knockdown using NOTCH3 siRNA recapitulated the dibenzazepine-induced reduction in MUC5AC. We demonstrate that NOTCH3 is a regulator of MUC5AC production. Increased NOTCH3 signaling in the asthmatic airway epithelium may therefore be an underlying driver of excess MUC5AC production.

Background: Allergic inflammation is commonly observed in a number of conditions that are associated with atopy including asthma, eczema and rhinitis. However, the genetic, environmental or epigenetic factors involved in these conditions are likely to be different. Epigenetic modifications, such as DNA methylation, can be influenced by the environment and result in changes to gene expression., Objectives: To characterize the DNA methylation pattern of airway epithelial cells (AECs) compared to peripheral blood mononuclear cells (PBMCs) and to discern differences in methylation within each cell type amongst healthy, atopic and asthmatic subjects., Methods: PBMCs and AECs from bronchial brushings were obtained from children undergoing elective surgery for non-respiratory conditions. The children were categorized as atopic, atopic asthmatic, non-atopic asthmatic or healthy controls. Extracted DNA was bisulfite treated and 1505 CpG loci across 807 genes were analyzed using the Illumina GoldenGate Methylation Cancer Panel I. Gene expression for a subset of genes was performed using RT-PCR., Results: We demonstrate a signature set of CpG sites that are differentially methylated in AECs as compared to PBMCs regardless of disease phenotype. Of these, 13 CpG sites were specific to healthy controls, 8 sites were only found in atopics, and 6 CpGs were unique to asthmatics. We found no differences in the methylation status of PBMCs between disease phenotypes. In AECs derived from asthmatics compared to atopics, 8 differentially methylated sites were identified including CpGs in STAT5A and CRIP1. We demonstrate STAT5A gene expression is decreased whereas CRIP1 gene expression is elevated in the AECs from asthmatic compared to both healthy and atopic subjects., Discussion: We characterized a cell specific DNA methylation signature for AECs compared to PBMCs regardless of asthmatic or atopic status. Our data highlight the importance of understanding DNA methylation in the epithelium when studying the epithelial contribution to asthma.

Objective: Dysregulated repair following epithelial injury is a key forerunner of disease in many organs, and the acquisition of a mesenchymal phenotype by the injured epithelial cells (epithelial to mesenchymal transition, EMT) may serve as a source of fibrosis. The macrolide antibiotic azithromycin and the DNA synthesis inhibitor mycophenolate are in clinical use but their mechanism of action remains unknown in post-transplant bronchiolitis obliterans syndrome (BOS). Here we determined if regional variation in the EMT response to TGFβ1 underlies the bronchiolocentric fibrosis leading to BOS and whether EMT could be inhibited by azithromycin or mycophenolate., Methods/results: We found that small and large airway epithelial cells from stable lung transplant patients underwent EMT when stimulated with TGFβ1, however mesenchymal protein expression was higher and loss of epithelial protein expression more complete in small airway epithelial cells. This regional difference was not mediated by changes in expression of the TGFβRII or Smad3 activation. Azithromycin potentially inhibited EMT in both small and large airway epithelial cells by inhibiting Smad3 expression, but not activation., Conclusion: Collectively, these observations provide a biologic basis for a previously unexplained but widely observed clinical phenomena, and a platform for the development of new approaches to fibrotic diseases.

Given the contribution various fibroblast subsets make to wound healing and tissue remodeling, the concept of lung fibroblast heterogeneity is of great interest. However, the mechanisms contributing to this heterogeneity are unknown. To this aim, we compared molecular and biophysical characteristics of fibroblasts concurrently isolated from normal human proximal bronchi (B-FBR) and distal lung parenchyma (P-FBR). Using quantitative RT-PCR, spontaneous expression of more than 30 genes related to repair and remodeling was analyzed. All P-FBR lines demonstrated significantly increased basal α-smooth muscle actin (α-SMA) mRNA and protein expression levels when compared with donor-matched B-FBR. These differences were not associated with sex, age, or disease history of lung tissue donors. In contrast to B-FBR, P-FBR displayed enhanced transforming growth factor (TGF)-β/Smad signaling at baseline, and inhibition of either ALK-5 or neutralization of endogenously produced and activated TGF-β substantially decreased basal α-SMA protein in P-FBR. Both B-FBR and P-FBR up-regulated α-SMA after stimulation with TGF-β1, and basal expression levels of TGF-β1, TGF-βRI, and TGF-βRII were not significantly different between fibroblast pairs. Blockade of metalloproteinase-dependent activation of endogenous TGF-β did not significantly modify α-SMA expression in P-FBR. However, resistance to mechanical tension of these cells was significantly higher in comparison with B-FBR, and added TGF-β1 significantly increased stiffness of both cell monolayers. Our data suggest that in contrast with human normal bronchial tissue explants, lung parenchyma produces mesenchymal cells with a myofibroblastic phenotype by intrinsic mechanisms of TGF-β activation in feed-forward manner. These results also offer a new insight into mechanisms of human fibroblast heterogeneity and their function in the airway and lung tissue repair and remodeling.

Background: Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus which reproduces asexually by releasing abundant airborne conidia (spores), which are easily respirable. In allergic and immunocompromised individuals A. fumigatus can cause a wide spectrum of diseases, including allergic bronchopulmonary aspergillosis, aspergilloma and invasive aspergillosis. Previous studies have demonstrated that A. fumigatus conidia are internalized by macrophages and lung epithelial cells; however the exact transcriptional responses of airway epithelial cells to conidia are currently unknown. Thus, the aim of this study was to determine the transcriptomic response of the human bronchial epithelial cell line (16HBE14o-) following interaction with A. fumigatus conidia. We used fluorescence-activated cell sorting (FACS) to separate 16HBE14o- cells having bound and/or internalized A. fumigatus conidia expressing green fluorescent protein from cells without spores. Total RNA was then isolated and the transcriptome of 16HBE14o- cells was evaluated using Agilent Whole Human Genome microarrays., Results: Immunofluorescent staining and nystatin protection assays demonstrated that 16HBE14o- cells internalized 30-50% of bound conidia within six hrs of co-incubation. After FAC-sorting of the same cell culture to separate cells associated with conidia from those without conidia, genome-wide analysis revealed a set of 889 genes showing differential expression in cells with conidia. Specifically, these 16HBE14o- cells had increased levels of transcripts from genes associated with repair and inflammatory processes (e.g., matrix metalloproteinases, chemokines, and glutathione S-transferase). In addition, the differentially expressed genes were significantly enriched for Gene Ontology terms including: chromatin assembly, G-protein-coupled receptor binding, chemokine activity, and glutathione metabolic process (up-regulated); cell cycle phase, mitosis, and intracellular organelle (down-regulated)., Conclusions: We demonstrate a methodology using FACs for analyzing the transcriptome of infected and uninfected cells from the same cell population that will provide a framework for future characterization of the specific interactions between pathogens such as A. fumigatus with human cells derived from individuals with or without underlying disease susceptibility.

Background: Asthma is associated with structural changes to airways such as extracellular matrix deposition and epithelial damage. Evidence suggests that asthmatic airway epithelial repair is abnormal and that elevated plasminogen activator inhibitor-1 levels observed in asthma may be involved in the epithelial repair process and in excessive matrix accumulation., Objective: To assess the ability of asthmatic airway epithelial cells (AECs) to repair mechanically induced wounds and to investigate the role that plasminogen activator inhibitor-1 plays in the repair process., Methods: AECs were isolated from atopic asthmatic and healthy non-atopic children by bronchial brushing, subcultured and wound repair experiments were performed. Plasminogen activator inhibitor-1 gene expression was assessed using real-time PCR while protein activity was measured in cell lysates as well as plasma. The role of plasminogen activator inhibitor-1 in epithelial proliferation and wound repair was investigated using siRNA., Results: Cells from asthmatic children have a significantly longer repair time in comparison with cells from otherwise healthy donors. Plasminogen activator inhibitor-1 mRNA expression was up-regulated 68-fold in freshly isolated asthmatic cells compared with normal cells, and protein levels were also significantly elevated in the asthmatic cell lysates, but plasma levels were similar in both groups. Plasminogen activator inhibitor-1 cells expression increased in both cohorts during culture. Gene silencing substantially reduced the rate of proliferation in asthmatic and healthy cells. Mechanical wounding of epithelial monolayers induced plasminogen activator inhibitor-1 expression in asthmatic and non-asthmatic cohorts, while gene silencing delayed wound repair of healthy cell, with minimal effect on those from asthmatics., Conclusion: Asthmatic AECs are inherently dysfunctional in their ability to repair wounds; plasminogen activator inhibitor-1 mRNA and protein activity are constitutively up-regulated in asthmatic epithelium and play functional roles in both proliferation and repair of healthy cells. In asthmatic cells, elevated plasminogen activator inhibitors-1 levels fail to stimulate epithelial repair.

Inappropriate repair after injury to the epithelium generates persistent activation, which may contribute to airway remodeling. In the present study we hypothesized that IL-13 is a normal mediator of airway epithelial repair. Mechanical injury of confluent airway epithelial cell (AEC) monolayers induced expression and release of IL-13 in a time-dependent manner coordinate with repair. Neutralizing of IL-13 secreted from injured epithelial cells by shIL-13Ralpha2.FC significantly reduced epithelial repair. Moreover, exogenous IL-13 enhanced epithelial repair and induced epidermal growth factor receptor (EGFR) phosphorylation. We examined secretion of two EGFR ligands, epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), after mechanical injury. Our data showed a sequential release of the EGF and HB-EGF by AEC after injury. Interestingly, we found that IL-13 induces HB-EGF, but not EGF, synthesis and release from AEC. IL-13-induced EGFR phosphorylation and the IL-13-reparative effect on AEC are mediated via HB-EGF. Finally, we demonstrated that inhibition of EGFR tyrosine kinase activity by tyrphostin AG1478 increases IL-13 release after injury, suggesting negative feedback between EGFR and IL-13 during repair. Our data, for the first time, showed that IL-13 plays an important role in epithelial repair, and that its effect is mediated through the autocrine release of HB-EGF and activation of EGFR. Dysregulation of EGFR phosphorylation may contribute to a persistent repair phenotype and chronically increased IL-13 release, and in turn result in airway remodeling.

Endothelin-1 (ET-1), a G protein-coupled receptor-activating peptide, is increased in airway epithelium, plasma, and bronchoalveolar lavage fluid of asthmatic patients. We hypothesized that ET-1 may contribute to the increased airway smooth muscle mass found in severe asthma by inducing hypertrophy and inhibiting apoptosis of smooth muscle cells. To investigate this hypothesis, we determined that treatment of primary human bronchial smooth muscle cells with ET-1 dose dependently [10(-11)-10(-7) M] inhibited the apoptosis induced by serum withdrawal. ET-1 treatment also resulted in a significant increase in total protein synthesis, mediated through both ET(A) and ET(B) receptors, cell size, as well as increased expression of myosin heavy chain, alpha-smooth muscle actin, and calponin. ET-1-induced hypertrophy was accompanied by activation of JAK1/STAT-3 and MAPK1/2 (ERK1/2) cell signaling pathways. Inhibition of JAK1/STAT-3 pathways by piceatannol or ERK1/2 by the MAPK/ERK kinase 1/2 inhibitor U0126 blunted the increase in total protein synthesis. The hypertrophic effect of ET-1 was equivalent to that of the gp130 cytokine oncostatin M and greater than that induced by cardiotrophin-1. ET-1 induced release of IL-6 but not IL-11, leukemia inhibitory factor, oncostatin M, or cardiotrophin-1, although treatment of cells with IL-6 alone did not induce hypertrophy. These results suggest that ET-1 is a candidate mediator for the induction of increased smooth muscle mass in asthma and identify signaling pathways activated by this mediator.

Rationale: Convincing evidence of epithelial damage and aberrant repair exists in adult asthmatic airways, even in the absence of inflammation. However, comparable studies in children have been limited by access and availability of clinical samples., Objectives: To determine whether bronchial epithelial cells from children with asthma are inherently distinct from those obtained from children without asthma., Methods: Epithelial cells were obtained by nonbronchoscopic bronchial brushing of children with mild asthma (n = 7), atopic children without asthma (n = 9), and healthy children (n = 12). Cells were subject to morphologic, biochemical, molecular, and functional assessment. Responses were also compared with commercially available epithelial cultures and the transformed cell line 16HBE140., Results: All epithelial cells exhibited a "cobblestone" morphology, which was maintained throughout culture and repeated passage. Expression of cytokeratin 19 varied, with disease phenotype being greatest in healthy nonatopics and lowest in asthmatics. In contrast, expression of cytokeratin 5/14 was greatest in asthmatic samples and least in healthy nonatopic samples. Asthmatic epithelial cells also spontaneously produced significantly greater amounts of interleukin (IL)-6, prostaglandin E2, and epidermal growth factor, and equivalent amounts of IL-1beta and soluble intracellular adhesion molecule-1, but significantly lower amounts of transforming growth factor beta1. This profile was maintained through successive passages. Asthmatic epithelial cells also exhibited greater rates of proliferation than nonasthmatic cells., Conclusions: This study has shown that epithelial cells from children with mild asthma are intrinsically different both biochemically and functionally compared with epithelial cells from children without asthma. Importantly, these differences are maintained over successive passages, suggesting that they are not dependent on an in vivo environment.

Background: Pulmonary neuroendocrine cells (PNEC) are specialized epithelial cells that are thought to play important roles in lung development and airway function. PNEC occur either singly or in clusters called neuroepithelial bodies. Our aim was to characterize the three dimensional morphology of PNEC, their distribution, and their relationship to the epithelial nerves in whole mounts of adult human bronchi using confocal microscopy., Methods: Bronchi were resected from non-diseased portions of a lobe of human lung obtained from 8 thoracotomy patients (Table 1) undergoing surgery for the removal of lung tumors. Whole mounts were stained with antibodies to reveal all nerves (PGP 9.5), sensory nerves (calcitonin gene related peptide, CGRP), and PNEC (PGP 9.5, CGRP and gastrin releasing peptide, GRP). The analysis and rendition of the resulting three-dimensional data sets, including side-projections, was performed using NIH-Image software. Images were colorized and super-imposed using Adobe Photoshop., Results: PNEC were abundant but not homogenously distributed within the epithelium, with densities ranging from 65/mm2 to denser patches of 250/mm2, depending on the individual wholemount. Rotation of 3-D images revealed a complex morphology; flask-like with the cell body near the basement membrane and a thick stem extending to the lumen. Long processes issued laterally from its base, some lumenal and others with feet-like processes. Calcitonin gene-related peptide (CGRP) was present in about 20% of PNEC, mainly in the processes. CGRP-positive nerves were sparse, with some associated with the apical part of the PNEC., Conclusion: Our 3D-data demonstrates that PNEC are numerous and exhibit a heterogeneous peptide content suggesting an active and diverse PNEC population.

The effect of histamine on the production of prostaglandin F2 alpha and the actions of prostaglandin F2 alpha on the responsiveness of human isolated bronchial smooth muscle were examined by organ bath techniques using bronchi from lung tissue resected from 18 patients. Following exposure to histamine, epithelium-intact bronchi generated 34.26 +/- 16.3 pg of prostaglandin F2 alpha/mg of tissue and epithelium-denuded preparations produced 32.62 +/- 11.83 pg/mg, suggesting that histamine-induced release of prostaglandin F2 alpha was from non-epithelial sources, presumably smooth muscle. The histamine H2 receptor antagonist ranitidine did not affect the release of prostaglandin F2 alpha, suggesting that its generation may have resulted from histamine H1 receptor activation. Carbachol did not influence prostaglandin F2 alpha generation. Contractile responses to histamine, prostaglandin F2 alpha and carbachol were measured in the presence and absence of the prostaglandin TP receptor antagonist SQ 29,548 ([1 S-[1 alpha,2 beta(5Z),3 beta,4 alpha]]-7-[3[[2-[9-phenylamino)carbonyl]hydrazino] methyl-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid) (0.4 microM). SQ 29,548 abolished responses to prostaglandin F2 alpha suggesting that contractions were mediated via TP receptors. Exposure to SQ 29,548 also produced a 3-fold rightward shift in the concentration-effect curve for histamine (P = 0.01) without influencing the maximum response. SQ 29,548 did not affect responses to carbachol. These results suggest that histamine selectively stimulates the generation of prostaglandin F2 alpha from epithelium-denuded human airway tissue (presumably from the smooth muscle), which in turn, amplifies the contractile responses of human airway smooth muscle to histamine.

The effects of exogenous prostaglandin E2 and prostacyclin on the function of epithelium-intact and epithelium-denuded human bronchial smooth muscle and the role of these mediators in the inhibition of histamine-induced contraction was examined using bronchi obtained from 22 patients undergoing thoracotomy. Under resting tension, a variable biphasic contraction-relaxation or monophasic relaxation was observed following the cumulative addition of exogenous prostaglandin E2 or prostacyclin. Cumulative addition of these mediators to pre-contracted bronchi produced incomplete relaxation, irrespective of the presence of epithelium. Addition of prostaglandin E2, at a concentration equating to that produced after histamine stimulation (1.2 x 10(-9)M), produced a reduction (24%) in the maximum contractile response (Emax) to a subsequent histamine challenge (P < 0.03). However, a similar response was not observed after the addition of prostacyclin at a concentration similar to that produced endogenously (6.5 x 10(-10)M). The combined addition of both mediators resulted in a significant reduction (26%) in the Emax to histamine (P < 0.02) but this effect was not statistically different to that of prostaglandin E2 alone. The addition of supramaximal concentrations (1 microM) of each prostanoid, either alone or in combination, did not inhibit responses to histamine. These data suggest that whilst prostaglandin E2 does not act as a direct acting relaxant agonist, it may inhibit histamine-induced muscle contraction and thereby contribute to the observed tachyphylaxis to this mediator. In contrast, prostacyclin appears to be of little importance in modulating human bronchial smooth muscle responses to histamine either directly or by enhancing responses to prostaglandin E2. The inhibitory effect of prostaglandin E2 appears to be concentration-dependent and suggests a bimodal action of this mediator in human airways.

The role of the bronchial epithelium and inhibitory prostaglandins in the induction of histamine tachyphylaxis in human isolated bronchial smooth muscle was investigated using bronchi obtained from 14 patients who had been treated with non-steroidal anti-inflammatory drugs (NSAID) for > 2 months and from 14 untreated patients. Epithelium-intact bronchial strips from untreated patients demonstrated tachyphylaxis to histamine with the maximum response (Emax) reduced by 30 +/- 5% (P < 0.02) and the EC50 increased 1.86-fold (P < 0.02). Tachyphylaxis was not observed in epithelium-denuded strips. In epithelium-intact bronchial preparations from NSAID treated patients, the mean initial maximum tension generated in response to histamine was significantly greater than that for bronchial preparations from untreated patients (P < 0.05). In NSAID-treated patients, both epithelium-intact and denuded preparations failed to demonstrate tachyphylaxis. The generation of prostaglandin E2 and prostacyclin was assessed by radio-immunoassay using bronchi from untreated patients (n = 8). In epithelium-intact bronchial preparations, the generation of both prostaglandin E2 and prostacyclin was significantly increased by histamine exposure (P < 0.05) and was completely inhibited by indomethacin. However, the selective histamine H2 receptor antagonist, ranitidine, selectively inhibited the synthesis of prostaglandin E2 alone. Production of both prostaglandins was not altered by exposure to acetylcholine. These results suggest that prostaglandin E2 and prostacyclin are released primarily from the epithelium in response to histamine and may be specifically involved in inhibiting human bronchial smooth muscle responsiveness to this mediator. Significantly, the release of prostaglandin E2 appears to be selectively controlled by histamine H2 receptors, resident on the epithelium.

The role of airway epithelium and H2-receptors in the development of histamine tachyphylaxis was studied using human isolated bronchial smooth muscle strips obtained from 18 patients undergoing thoracotomy. In epithelium-intact strips, a 38% reduction in the maximal contractile response (Emax) (p less than 0.002) and a 2.14-fold increase in the EC50 (p less than 0.02; n = 18) was observed after three separate histamine cumulative concentration effect curves (CCEC). In contrast, significant differences were not seen for either Emax (p greater than 0.4; n = 10) or EC50 (p greater than 0.26; n = 10) in epithelium-denuded strips. In separate experiments, both intact and denuded muscle strips were treated with the H2-receptor antagonist ranitidine (60 microM), either 30 min prior to the first or 30 min prior to the second histamine CCEC. In epithelium-intact strips, pretreatment with ranitidine caused a 1.8-fold increase in Emax in the initial CCEC (p less than 0.02), and both ranitidine schedules prevented tachyphylaxis (n = 8). In epithelium-denuded preparations, ranitidine did not enhance the responsiveness to histamine beyond that seen in untreated epithelium-denuded strips (n = 6). These data suggest that histamine-induced tachyphylaxis occurs in human airway smooth muscle and is mediated, at least in part, via H2-receptors resident on airway epithelium. In vivo, this may function as a protective mechanism, but damage to the epithelium and loss of H2-receptors may be significant in the development of histamine bronchial hyperreactivity as seen in asthma.