Research Items (81)
Much of what is known about the Aryl Hydrocarbon Receptor (AhR) centers on its ability to mediate the deleterious effects of the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin). However, the AhR is both ubiquitously-expressed and evolutionarily-conserved, suggesting that it evolved for purposes beyond strictly mediating responses to man-made environmental toxicants. There is growing evidence that the AhR is required for the maintenance of health, as it is implicated in physiological processes such as xenobiotic metabolism, organ development and immunity. Dysregulation of AhR expression and activity is also associated with a variety of disease states, particularly those at barrier organs such as the skin, gut and lungs. The lungs are particularly vulnerable to inhaled toxicants such as cigarette smoke. However, the role of the AhR in diseases such as chronic obstructive pulmonary disease (COPD)—a respiratory illness caused predominately by cigarette smoking—and lung cancer remains largely unexplored. This review will discuss the growing body of literature that provides evidence that the AhR protects the lungs against the damaging effects of cigarette smoke.
- Dec 2018
Links between solar UV exposure and immunity date back to the ancient Greeks with the development of heliotherapy. Skin contains several UV-sensitive chromophores and exposure to sunlight can produce molecules, such as vitamin D3, that act in an endocrine manner. We investigated whether the aryl hydrocarbon receptor (AHR), an environmental sensor and ligand-regulated transcription factor activated by numerous planar compounds of dietary or environmental origin, could be the target of endocrine photoproducts produced by cutaneous UV exposure. 15- to 30-minute exposure of cells to a minimal erythemal dose of UVB irradiation in vitro induced translocation of the AHR to the nucleus, rapidly inducing site-specific DNA binding and target gene regulation. Importantly, ex vivo studies with Ahr wild-type or null fibroblasts showed that serum from mice whose skin was exposed to a 15 min UVB dose, but not control serum, contained agonist activity within 30 min of UV irradiation, inducing AHR-dependent gene expression. Moreover, a 15-min cutaneous UVB exposure induced AHR site-specific DNA binding and target gene regulation in vivo within 3-6 hr post-irradiation in blood and in peripheral tissues, including intestine. These results show that cutaneous exposure of mice to a single minimal erythemic dose of UVB induces rapid AHR signaling in multiple peripheral organs, providing compelling evidence that moderate sun exposure can exert endocrine control of immunity through the AHR.
BACKGROUND: Osteogenesis imperfecta (OI) is most often caused by mutations in type I collagen genes. Respiratory complications have been largely attributed to spine and ribcage deformities. We hypothesized that direct involvement of the pulmonary parenchyma and/or diaphragm by the disease may occur. METHODS: In Col1a1Jrt/+ mice, a model of severe dominant OI, mean linear intercept length (Lm) was used to assess distal airspace size. Cross-sectional area (CSA) and myosin heavy chain (MyHC) phenotype of diaphragm muscle fibers, as well as contractile properties, were determined. OI mice were also treated with neutralizing antibodies against TGF-beta. RESULTS: Distal airspace enlargement occurred in OI mice (Lm +27%). Diaphragmatic thickness and fiber number were reduced, with increases in fast-twitch type IIx/IIb MyHC fibers. Ex vivo force generation (normalized for CSA) of the diaphragm was also significantly reduced. The increased Lm values found in OI mice were not prevented by anti-TGF-beta antibody treatment. CONCLUSIONS: The Col1a1Jrt/+ mouse model of OI demonstrates: 1) pulmonary airspace enlargement not driven by TGF-beta; and 2) reduced muscle mass and intrinsic contractile weakness of the diaphragm. These results suggest a complex and multi-faceted basis for respiratory complications in OI that cannot be solely attributed to bone manifestations.
- Jan 2018
Background: Bronchial vascular remodeling may contribute to the severity of airway narrowing through mucosal congestion. Interleukin (IL)-17A is associated with the most severe asthmatic phenotype but whether it might contribute to vascular remodeling is uncertain. Objective: To assess vascular remodeling in severe asthma and whether IL-17A directly or indirectly may cause endothelial cell activation and angiogenesis. Methods: Bronchial vascularization was quantified in asthmatic subjects, COPD and healthy subjects together with the number of IL-17A+ cells as well as the concentration of angiogenic factors in the sputum. The effect of IL-17A on in vitro angiogenesis, cell migration and endothelial permeability was assessed directly on primary human lung microvascular endothelial cells (HMVEC-L) or indirectly with conditioned medium derived from normal bronchial epithelial cells (NHBEC), fibroblasts (NHBF) and airway smooth muscle cells (ASMC) after IL-17A stimulation. Results: Severe asthmatics have increased vascularity compared to the other groups, which correlates positively with the concentrations of angiogenic factors in sputum. Interestingly we demonstrated that increased bronchial vascularity correlates positively with the number of subepithelial IL-17A+ cells. However IL-17A had no direct effect on HMVEC-L function but it enhanced endothelial tube formation and cell migration through the production of angiogenic factors by NHBE and ASMC. Conclusions & clinical relevance: Our results shed light on the role of IL-17A in vascular remodeling, most likely through stimulating the synthesis of other angiogenic factors. Knowledge of these pathways may aid in the identification of new therapeutic targets. This article is protected by copyright. All rights reserved.
Background Chronic obstructive pulmonary disease (COPD) is a progressive, irreversible chronic inflammatory disorder typified by increased recruitment of monocytes, lymphocytes and neutrophils. Because of this, as well as the convenience of peripheral blood nuclear cells (PBMCs) assessments, miRNA profiling of PBMCs has drawn increasing attention in recent years for various disease. Therefore, we analyzed miRNA and mRNA profiles to understand their regulatory network between COPD subjects versus smokers without airflow limitation. Methods miRNA and mRNA profiling of PBMCs from pooled 17 smokers and 14 COPD subjects was detected by high-throughput microarray. The expression of dysregulated miRNAs were validated by q-PCR. The miRNA targets in dysregulated mRNAs were predicted and the pathway enrichment was analyzed. Results miRNA microarray showed that 8 miRNAs were up-regulated and 3 miRNAs were down-regulated in COPD subjects compared with smokers; the upregulation of miR-24-3p, miR-93-5p, miR-320a and miR-320b and the downregulation of miR-1273 g-3p were then validated. Bioinformatic analysis of regulatory network between miRNA and mRNA showed that NOD and TLR were the most enriched pathways. miR-24-3p was predicted to regulate IL-18, IL-1β, TNF, CCL3 and CCL4 and miR-93-5p to regulate IκBα. Conclusions The expression of miRNA and mRNA were dysregulated in PBMCs of COPD patients compared with smokers without airflow limitation. The regulation network between the dysregulated miRNA and mRNA may provide potential therapeutic targets for COPD.
Heightened inflammation, including expression of COX-2, is associated with chronic obstructive pulmonary disease (COPD) pathogenesis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is reduced in COPD-derived lung fibroblasts. The AhR also suppresses COX-2 in response to cigarette smoke, the main risk factor for COPD, by destabilizing the Cox-2 transcript by mechanisms that may involve the regulation of microRNA (miRNA). Whether reduced AhR expression is responsible for heightened COX-2 in COPD is not known. Here, we investigated the expression of COX-2 as well as the expression of miR-146a, a miRNA known to regulate COX-2 levels, in primary lung fibroblasts derived from non-smokers (Normal) and smokers (At Risk) with and without COPD. To confirm the involvement of the AhR, AhR knock-down via siRNA in Normal lung fibroblasts and MLE-12 cells was employed as were A549-AhRko cells. Basal expression of COX-2 protein was higher in COPD lung fibroblasts compared to Normal or Smoker fibroblasts but there was no difference in Cox-2 mRNA. Knockdown of AhR in lung structural cells increased COX-2 protein by stabilizing the Cox-2 transcript. There was less induction of miR-146a in COPD-derived lung fibroblasts but this was not due to the AhR. Instead, we found that RelB, an NF-κB protein, was required for transcriptional induction of both Cox-2 and miR-146a. Therefore, we conclude that the AhR controls COX-2 protein via mRNA stability by a mechanism independent of miR-146a. Low levels of the AhR may therefore contribute to the heightened inflammation common in COPD patients.
- May 2017
Silica nanoparticles are widely used for biomedical purposes, but also in cosmetic products, food, the car industry, paints, etc. Considering their mega production, one should not ignore their potential hazardous effects on humans, flora and fauna. Human exposure to nanosilica can occur unintentionally in daily life and in industrial settings. Here, we review the common methods of silica nanoparticle production and its applications in biomedical investigations and nanotoxicology. The use of silica nanoparticles in biomedicine is discussed in terms of drug delivery, their responsiveness to different stimuli, theranostic applications and their uses in the food and cosmetic industries. Advantages and limitations of silica nanoparticles are presented and the effects of these nanoparticles are discussed in relation to their route of entry and impact on biochemical and epigenetic processes in human and animal cells.
- Feb 2017
Chronic obstructive pulmonary disease (COPD) is a chronic and prevalent respiratory disease caused primarily by long term inhalation of cigarette smoke. A major hallmark of COPD is elevated apoptosis of structural lung cells including fibroblasts. The NF-κB member RelB may suppress apoptosis in response to cigarette smoke, but its role in lung cell survival is not known. RelB may act as a pro-survival factor by controlling the expression of superoxide dismutase 2 (SOD2). SOD2 is also regulated by the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that suppresses cigarette smoke-induced apoptosis. As the AhR is also a binding partner for RelB, we speculate that RelB suppresses cigarette smoke-induced apoptosis by regulating the AhR. Using an in vitro model of cigarette smoke exposure (cigarette smoke extract [CSE]), we found that CSE down-regulated RelB expression in mouse lung fibroblasts, which was associated with elevated levels of cleaved PARP. Genetic ablation of RelB elevated CSE-induced apoptosis, including chromatin condensation, and reduced mitochondrial function. There was also more reactive oxygen species production in RelB-/- cells exposed to CSE. While there was no alteration in Nrf2 expression or localization between RelB-/- and wild type cells in response to CSE, RelB-/- cells displayed significantly decreased AhR mRNA and protein expression, concomitant with loss of AhR target gene expression (Cyp1a1, Cyp1b1, Nqo1). Finally, we found that RelB binds to the Ahr gene at 3 sites to potentially increase its expression via transcriptional induction. These data support that RelB suppresses cigarette smoke-induced apoptosis, potentially by increasing the AhR. Together, these two proteins may comprise an important cell survival signalling pathway that reduces apoptosis upon cigarette smoke exposure.
Air pollution of anthropogenic origin is largely from the combustion of biomass (e.g., wood), fossil fuels (e.g., cars and trucks), incinerators, landfills, agricultural activities and tobacco smoke. Air pollution is a complex mixture that varies in space and time, and contains hundreds of compounds including volatile organic compounds (e.g., benzene), metals, sulphur and nitrogen oxides, ozone and particulate matter (PM). PM0.1 (ultrafine particles (UFP)), those particles with a diameter less than 100 nm (includes nanoparticles (NP)) are considered especially dangerous to human health and may contribute significantly to the development of numerous respiratory and cardiovascular diseases such as chronic obstructive pulmonary disease (COPD) and atherosclerosis. Some of the pathogenic mechanisms through which PM0.1 may contribute to chronic disease is their ability to induce inflammation, oxidative stress and cell death by molecular mechanisms that include transcription factors such as nuclear factor κB (NF-κB) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Epigenetic mechanisms including non-coding RNA (ncRNA) may also contribute towards the development of chronic disease associated with exposure to PM0.1. This paper highlights emerging molecular concepts associated with inhalational exposure to PM0.1 and their ability to contribute to chronic respiratory and systemic disease.
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor historically known for its toxic responses to man-made pollutants such as dioxin. More recently, the AhR has emerged as a suppressor of inflammation, oxidative stress and apoptosis from cigarette smoke by mechanisms that may involve the regulation of microRNA. However, little is known about the AhR regulation of miRNA expression in the lung in response to inhaled toxicants. Therefore, we exposed Ahr−/− and Ahr+/− mice to cigarette smoke for 4 weeks and evaluated lung miRNA expression by PCR array. There was a dramatic regulation of lung miRNA by the AhR in the absence of exogenous ligand. In response to cigarette smoke, there were more up-regulated miRNA in Ahr−/− mice compared to Ahr+/− mice, including the cancer-associated miRNA miR-96. There was no significant change in the expression of the AhR regulated proteins HuR and cyclooxygenase-2 (COX-2). There were significant increases in the anti-oxidant gene sulfiredoxin 1 (Srxn1) and FOXO3a- predicted targets of miR-96. Collectively, these data support a prominent role for the AhR in regulating lung miRNA expression. Further studies to elucidate a role for these miRNA may further uncover novel biological function for the AhR in respiratory health and disease.
Pseudomonas aeruginosa are gram-negative bacteria that frequently infect the lungs of cystic fibrosis (CF) patients. This bacterium is highly responsive to changes in its environment, resulting in the expression of a diverse array of genes that may contribute to the host inflammatory response. P. aeruginosa is well-known to induce neutrophilic inflammation via the activation of Toll-Like Receptors (TLRs). Recently, it was shown that pyocyanin, a phenazine produced by P. aeruginosa, binds to the aryl hydrocarbon receptor (AhR), leading to neutrophilic inflammation as part of the host defense response. In this study, we have investigated the contribution of the TLR and AhR signaling pathways to the expression of the neutrophil chemoattractant IL-8 in response to P. aeruginosa diffusible material. Although both pathways are involved in IL-8 synthesis, the AhR played a greater role when planktonic P. aeruginosa was grown in a media favoring phenazine synthesis. However, when P. aeruginosa was grown in a media that mimics the nutritional composition of CF sputa, both pathways contributed similarly to IL-8 synthesis. Finally, when P. aeruginosa was grown as a biofilm, the TLR pathway did not contribute to biofilm-driven IL-8 synthesis and AhR was found to only partially contribute to IL-8 synthesis, suggesting the contribution of another unknown signaling pathway. Therefore, the interaction between P. aeruginosa and airway epithelial cells is very dynamic, and sensor engagement is variable according to the adaptation of P. aeruginosa to the CF lung environment.
- Apr 2016
Background: Chronic inflammation, typified by increased expression of IL-17A, together with airway and parenchymal remodeling are features of chronic lung diseases. Emerging evidence suggests that phenotypic heterogeneity of repair and inflammatory capacities of fibroblasts may contribute to the differential structural changes observed in different regions of the lung. Objective: To investigate phenotypic differences in parenchymal and bronchial fibroblasts, either in terms of inflammation and remodeling or the ability of these fibroblasts to respond to IL-17A. Methods: Four groups of primary fibroblasts were used: normal human bronchial fibroblast (NHBF), normal human parenchymal fibroblast (NHPF), COPD human bronchial fibroblast (CHBF) and COPD human parenchymal fibroblast (CHPF). Cytokine and extracellular matrix (ECM) expression were measured at baseline and after stimulation with IL-17A. Actinomycin D was used to measure cytokine mRNA stability. Results: At baseline, we observed higher protein production of IL-6 in NHPF than NHBF, but higher levels of IL-8 and GRO-α in NHBF. IL-17A induced a higher expression of GRO-α (CXCL1) and IL-6 in NHPF than in NHBF, and a higher level of IL-8 expression in NHBF. IL-17A treatment decreased the mRNA stability of IL-6 in NHBF when compared with NHPF. CHPF expressed higher protein levels of fibronectin, collagen-I and collagen-III than CHBF, NHBF and NHPF. IL-17A increased fibronectin and collagen-III protein only in NHPF and collagen-III protein production in CHBF and CHPF. Conclusions and clinical relevance: These findings provide insight into the inflammatory and remodeling processes that may be related to the phenotypic heterogeneity of fibroblasts from airway and parenchymal regions and in their response to IL-17A. This article is protected by copyright. All rights reserved.
Background: Exacerbations of chronic obstructive pulmonary disease (COPD) are acute events of worsened respiratory symptoms that may increase the risk of cardiovascular disease (CVD), a leading cause of mortality amongst COPD patients. The utility of lung-specific inflammatory mediators such as club cell protein-16 (CC-16) and surfactant protein D (SPD) and that of a novel marker of CV outcomes in COPD- RelB- in predicting adverse cardiovascular events during exacerbation is not known. Methods: Thirty-eight subjects with COPD admitted to the hospital for severe exacerbation were included in this analysis. Clinical, physiological and arterial stiffness measurements were performed within 72 hours of admission; this was followed by measurements taken every 3 days until hospital discharge, then once a week until 30 days after discharge, and then again at 90 and 180 days. Plasma concentrations of inflammatory mediators were measured from peripheral venous blood taken at admission, and at days 15, 30, 90 and 180. Results: CC-16 and RelB concentrations were increased at day 15 of exacerbations whereas SPD concentrations were decreased. The course of change in CC-16 and RelB levels over time was inversely associated with that of carotid-femoral pulse wave velocity, the gold-standard measure of arterial stiffness. Increases in CC-16 could predict a decreased number of subsequent exacerbations during follow-up. Conclusions: Lung-specific (CC-16) and novel (RelB) biomarkers are associated with systemic cardiovascular changes over time. CC-16 can predict subsequent exacerbations in subjects with severe COPD and may be an important biomarker of pulmonary and systemic stress in COPD.
- Sep 2015
The aryl hydrocarbon receptor (AhR) is a ubiquitously expressed receptor/transcription factor that mediates toxicological responses of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Emerging evidence indicates that the AhR suppresses apoptosis and proliferation independent of exogenous ligands, including suppression of apoptosis by cigarette smoke, a key risk factor for chronic obstructive pulmonary disease (COPD). As cigarette smoke is a potent inducer of oxidative stress, a feature that may contribute to the development of COPD, we hypothesized that the AhR prevents smoke-induced apoptosis by regulating oxidative stress. Utilizing primary lung fibroblasts derived from AhR(+/+) and AhR(-/-) mice as well as A549 human lung adenocarcinoma cells deficient in AhR expression (A549-AhR(ko)), we first show that AhR(-/-) fibroblasts and A549-AhR(ko) epithelial cells have a significant increase in cigarette smoke extract (CSE)-induced oxidative stress compared to wild type. CSE induced a significant increase in the mRNA expression of key antioxidant genes, including Nqo1 and Srxn1, predominantly in AhR(+/+) fibroblasts, with significantly less induction in AhR(-/-) cells. The induction of Srxn1, but not Nqo1, was independent of dioxin-responsive element (DRE) binding as AhR(DBD/DBD) lung fibroblasts, which express an AhR incapable of binding the DRE, increased Srxn1 to a degree similar to wild-type cells in response to CSE. There was no difference in Nrf2 expression or activation based on AhR expression. Lung fibroblasts derived from COPD subjects have significantly less AhR protein expression compared with both never-smokers (Normal) and smokers (At Risk). Consequently, COPD-derived fibroblasts were less robust in their induction of both Nqo1 and Srxn1 mRNA after exposure to CSE, which also failed to activate the AhR in the COPD fibroblasts. Taken together, these results support a new role for the AhR in regulating antioxidant defense in lung structural cells, such that low AhR expression may facilitate the development or progression of COPD.
Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology. © The Author 2015. Published by Oxford University Press.
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. 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. 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. 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.
An emerging area in environmental toxicology is the role that chemicals and chemical mixtures have on the cells of the human immune system. This is an important area of research that has been most widely pursued in relation to autoimmune diseases and allergy/asthma as opposed to cancer causation. This is despite the well-recognized role that innate and adaptive immunity play as essential factors in tumorigenesis. Here, we review the role that the innate immune cells of inflammatory responses play in tumorigenesis. Focus is placed on the molecules and pathways that have been mechanistically linked with tumor-associated inflammation. Within the context of chemically induced disturbances in immune function as co-factors in carcinogenesis, the evidence linking environmental toxicant exposures with perturbation in the balance between pro- and anti-inflammatory responses is reviewed. Reported effects of bisphenol A, atrazine, phthalates and other common toxicants on molecular and cellular targets involved in tumor-associated inflammation (e.g. cyclooxygenase/prostaglandin E2, nuclear factor kappa B, nitric oxide synthesis, cytokines and chemokines) are presented as example chemically mediated target molecule perturbations relevant to cancer. Commentary on areas of additional research including the need for innovation and integration of systems biology approaches to the study of environmental exposures and cancer causation are presented.
Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.
Heightened inflammation, including expression of COX-2, is associated with COPD pathogenesis. RelB is an NF-κB family member that attenuates COX-2 in response to cigarette smoke by a mechanism that may involve the miRNA miR-146a. There is no information on the expression of RelB in COPD or if RelB prevents COX-2 expression through miR-146a. RelB, Cox-2 and miR-146a levels were evaluated in lung fibroblasts and blood samples derived from non-smokers (Normal) and smokers (At Risk) with and without COPD by qRT-PCR. RelB and COX-2 protein levels were evaluated by western blot. Human lung fibroblasts from Normal subjects and smokers with and without COPD, along with RelB knock-down (siRNA) in Normal cells, were exposed to cigarette smoke extract (CSE) in vitro and COX-2 mRNA/protein and miR-146a levels assessed. Basal expression of RelB mRNA and protein were significantly lower in lung cells derived from smokers with and without COPD, the latter of which expressed more Cox-2 mRNA and protein in response to CSE. Knock-down of RelB in Normal fibroblasts increased Cox-2 mRNA and protein induction by CSE. Basal miR-146a levels were not different between the three groups, and only Normal fibroblasts increased miR-146a expression in response to smoke. There was a positive correlation between systemic RelB and Cox-2 mRNA levels and circulating miR-146a levels were higher only in GOLD stage I subjects. Our data indicate that RelB attenuates COX-2 expression in lung structural cells, such that loss of pulmonary RelB may be an important determinant in the aberrant, heightened inflammation associated with COPD pathogenesis.
Background Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder marked by relative resistance to steroids. The IL-17 superfamily, which mediates cross-talk between the adaptive and innate immune systems, has been associated with diminished responses to steroids. Increasing evidence supports elevated IL-17 expression in the lung of COPD subjects. However, whether cells of the immune system (systemic) and/or local lung cells are contributing to the elevated IL-17 remains unclear. To address this issue, we utilized a human parenchymal lung tissue explant culture system with cigarette smoke exposure to investigate the expression of IL-17 and the mechanisms involved.Methods Parenchymal lung tissue removed from 10 non-COPD and 8 COPD patients was sectioned and cultured with different concentrations of cigarette smoke extract (CSE) for 3 or 6 hours. Tissue viability was evaluated by LDH (lactate dehydrogenase) in culture supernatants. Western blot and real-time PCR were performed to evaluate IL-17A/F expression. To investigate the mechanisms, pharmacological inhibitors for MAPK p38, ERK1/2, NF-¿B and PI3K pathways were added into the culture media.ResultsNo tissue damage was observed after the cigarette smoke exposure for 3 h or 6 h compared with the control media. At the protein level, the expression of both IL-17A (2.4¿±¿0.6 fold) and IL-17 F (3.7¿±¿0.7 fold) in the tissue from non-COPD subjects was significantly increased by 5% of CSE at 3 h. For COPD subjects, IL-17A/F expression were significantly increased only at 6 h with 10% of CSE (IL-17A: 4.2¿±¿0.8 fold; IL-17 F: 3.3¿±¿0.8 fold). The increased expression of IL-17A/F is also regulated at the mRNA level. The inhibitors for NF-¿B and PI3K pathways significantly inhibited CSE-induced IL-17A/F expression from lung tissue of non-COPD subjects.Conclusions We found the evidence that the expression of both IL-17A and IL-17 F is increased by the cigarette smoke exposure in explants from both non-COPD and COPD subjects, supporting that local lung cells contribute IL-17 production. The elevated IL-17A/F expression is dependent on NF-¿B and PI3K pathways. These observations add to the growing evidence which suggests that Th17 cytokines play a significant role in COPD.
Chronic obstructive pulmonary disease (COPD) exacerbations are acute events of worsened respiratory symptoms and enhanced inflammation partly mediated by NF-κB activation. RelB, an NF-κB family member, suppresses cigarette smoke-induced inflammation but its expression in COPD is unknown. Moreover, there is no information on its association with clinical features of COPD. The objectives of this study were to assess RelB expression relative to markers of inflammation as well as its association with cardiovascular and pulmonary features of COPD patients at stable-state and exacerbation. METHODS: Data from 48 COPD patients were analyzed. Blood samples were collected from stable-state and exacerbating patients. After RNA isolation, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to assess RelB, Cox-2, IL-8 and IL-1β mRNA expression and their associations with measured clinical variables. RESULTS: Of the 48 COPD subjects, 18 were in stable-state and 30 were in exacerbation. RelB mRNA expression was lower than that of Cox-2, IL-8, and IL-1β in all cases (all p<0.001, except for IL-8 at exacerbation (p = 0.22)). Cox-2, IL-8 and IL-1β were significantly associated with clinical features of patients in both stable-state and at exacerbation. There was no association with RelB expression and any clinical features in COPD subjects at stable-state. RelB mRNA levels were significantly associated with cardiovascular events such as systolic blood pressure during exacerbation. CONCLUSIONS: RelB mRNA expression is lower than that of the other inflammatory mediators. Expression of Cox-2, IL-8 and IL-1β were related to clinical features in both stable-state and at exacerbation. However, RelB expression was associated with clinical features of patients only during exacerbation, suggesting that RelB may represent a novel marker of health outcomes, in particular cardiovascular, during exacerbation in COPD.
- Sep 2014
Background Infiltration of fibrocytes (FC) in the airway smooth muscle is a feature of asthma but the pathological significance is unknown.Objective We sought to explore if FC modulate the phenotype of airway smooth muscle cells (ASMC) in asthmatic vs. control subjects.MethodsFC were isolated from CD14+monocytes from asthmatic and normal subjects. Proliferation of ASMC of asthmatic or normal subjects were analyzed by 3H-thymidine incorporation, cell number counting and Ki-67 expression after treatment of ASMC with FC-conditioned medium (FCCM) or co-culture with FC. ASMC-associated cytokines/chemokines implicated in asthma (TGF-β1, eotaxin, IL-6 and IL-8) were measured in co-culture or transwell culture of ASMC+FC by ELISA. Immunofluorescence staining was performed to localize these cytokines in ASMC. Cytokine secretion was measured in the transwell culture of ASMC+FC where NF-κB-p65 or ERK1/2 in ASMC was silenced by siRNA. Contractile phenotype of ASMC in transwell culture was assessed by immunoblotting of α-smooth muscle actin (α-SMA) and myosin light chain kinase (MLCK).ResultsFC did not affect ASMC expression of TGF-β1, eotaxin, α-SMA and MLCK; however, ASMC production of IL-8 and IL-6 was increased in the co-culture and transwell culture by FC. ASMC treated with FCCM were immunopositive for IL-8 / IL-6 and produced more IL-8 / IL-6. Furthermore, siRNA silencing of NF-κB-p65 or ERK1/2 in transwell cultures of asthmatic ASMC with normal subject FC decreased IL-8 and IL-6 production.ConclusionFC promoted IL-8 and IL-6 production by ASMC, demonstrating a proinflammatory role for FC and a possible mechanism of the inflammatory phenotype in asthma.This article is protected by copyright. All rights reserved.
- Aug 2014
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor implicated in the regulation of apoptosis and proliferation. Although activation of the AhR by xenobiotics such as dioxin inhibits the cell cycle and control apoptosis, paradoxically, AhR expression also promotes cell proliferation and survival independent of exogenous ligands. The microRNA (miRNA) miR-196a has also emerged as a regulator of proliferation and apoptosis but a relationship between the AhR and miR-196a is not known. Therefore, we hypothesized that AhR-dependent regulation of endogenous miR-196a expression would promote cell survival and proliferation. Utilizing lung fibroblasts from AhR deficient (AhR(-/-)) and wild-type (AhR(+/+)) mice, we show that there is ligand-independent regulation of miRNA, including low miR-196a in AhR(-/-) cells. Validation by qRT-PCR revealed a significant decrease in basal expression of miR-196a in AhR(-/-) compared to AhR(+/+) cells. Exposure to AhR agonists benzo[a]pyrene (B[a]P) and FICZ as well as AhR antagonist CH-223191 decreased miR-196a expression in AhR(+/+) fibroblasts concomitant with decreased AhR protein levels. There was increased proliferation only in AhR(+/+) lung fibroblasts in response to serum, corresponding to a decrease in p27(KIP1) protein, a cyclin-dependent kinase inhibitor. Increasing the cellular levels of miR-196a had no effect on proliferation or expression of p27(KIP1) in AhR(-/-) fibroblasts but attenuated cigarette smoke-induced apoptosis. This study provides the first evidence that AhR expression is essential for the physiological regulation of cellular miRNA levels- including miR-196a. Future experiments designed to elucidate the functional relationship between the AhR and miR-196a may delineate additional novel ligand-independent roles for the AhR.
Cigarette smoke is associated with chronic and enhanced pulmonary inflammation characterized by increased cytokine production and leukocyte recruitment to the lung. Although the aryl hydrocarbon receptor (AhR) is well-known to mediate toxic effects of man-made environmental contaminants, the AhR has emerged as a suppressor of acute cigarette smoke-induced neutrophilia by a mechanism involving the NF-κB protein RelB. Yet individuals who smoke often smoke for many years and vary in their cigarette consumption. As there is currently no information on the AhR prevention of lung inflammation, including neutrophilia, due to varied and prolonged exposure regimes, we exposed control and AhR(-/-) mice to cigarette smoke for 2 weeks (sub-chronic exposure) utilizing low and high exposure protocols and evaluated pulmonary inflammation. Sub-chronic cigarette smoke exposure significantly increased pulmonary neutrophilia dose-dependently in AhR(-/-) mice. Surprisingly, there was no difference between smoke-exposed AhR(+/-) and AhR(-/-) mice in the expression of cytokines associated with neutrophil recruitment. Expression of pulmonary ICAM-1, an adhesion molecule involved in neutrophil migration and retention, was higher in pulmonary endothelial cells from AhR(-/-) mice. Although total lung RelB expression was increased by cigarette smoke, nuclear RelB was significantly lower in sub-chronically exposed AhR(-/-) mice. Inhibition of AhR activity by CH-223191 in endothelial cells potentiated ICAM-1 expression and prevented RelB nuclear translocation but had no effect on neutrophil adhesion. These data support that genetic absence of the AhR contributes to heightened pulmonary neutrophilia in response to on-going cigarette smoke exposure. Inter-individual variations in AhR expression may enhance the susceptibility to cigarette smoke-induced diseases.
- Jan 2014
Diseases due to cigarette smoke exposure, including chronic obstructive pulmonary disease (COPD) and lung cancer, are associated with chronic inflammation typified by the increased expression of cyclooxygenase-2 (COX-2) protein. RelB is an NF-κB family member that suppresses cigarette smoke induction of COX-2 through an unknown mechanism. The ability of RelB to regulate COX-2 expression may be via miR-146a, a miRNA that attenuates COX-2 in lung fibroblasts. In this study we tested whether RelB attenuation of cigarette smoke-induced COX-2 protein is due to miR-146a. Utilizing pulmonary fibroblasts deficient in RelB expression, together with siRNA knockdown of RelB, we show the essential role of RelB in diminishing smoke-induced COX-2 protein expression despite robust activation of the canonical NF-κB pathway and subsequent induction of Cox-2 mRNA. RelB did not regulate COX-2 protein expression at the level of mRNA stability. Basal levels of miR-146a were significantly lower in Relb-deficient cells and cigarette smoke increased miR-146a expression only in Relb-expressing cells. Inhibition of miR-146a had no effects on Relb expression or induction of Cox-2 mRNA by cigarette smoke but significantly increased COX-2 protein. These data highlight the potential of a RelB-miR-146a axis as a novel regulatory pathway that attenuates inflammation in response to respiratory toxicants.
Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder marked by relative resistance to steroids. Inflammation and apoptosis have been suggested to be important mechanisms for COPD. IL-17 superfamily has been associated with chronic inflammation and diminished responses to steroids. It is reasonable to consider that IL-17 may play a role in the pathogenesis of COPD. In this study, we examined IL-17 expression in mice exposed to cigarette smoke (CS), and investigated the contribution of IL-17 to CS-induced inflammation and alveolar cell apoptosis in IL-17(-/-) mice. After exposing wild-type and IL-17(-/-) mice to main-stream CS for 4 weeks, IL-17A, but not IL-17F, expression was increased in mice upon CS exposure. Neutrophil infiltration in the lung of IL-17(-/-) mice was significantly decreased. In IL-17(-/-) mice, there is reduced expression of IL-6, MIP-2 and MMP12 compared to wild-type mice after CS exposure. The number of apoptotic type II alveolar cells was significantly increased in CS-exposed wild-type mice but not in IL-17(-/-) mice. The effect of IL-17A on type II alveolar cell apoptosis was confirmed in vitro through either addition of IL-17A or transient knockdown of IL-17A by siRNA transfection in type II alveolar cells. These findings suggest that IL-17A plays an important role in the inflammatory response to CS exposure through increased multiple inflammatory mediators. Moreover, IL-17 may also contribute to type II alveolar cell apoptosis. This study opens a new option in targeting IL-17A to modulate inflammatory response to CS and may be the bases for new therapy for 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.
Structural cell migration plays a central role in the pathophysiology of several diseases, including asthma. Previously, we established that IL-17-induced (CXCL1, CXCL2, and CXCL3) production promoted airway smooth muscle cell (ASMC) migration, and consequently we sought to investigate the molecular mechanism of CXC-induced ASMC migration. Recombinant human CXCL1, CXCL2, and CXCL3 were used to assess migration of human primary ASMCs from normal and asthmatic subjects using a modified Boyden chamber. Neutralizing Abs or small interfering RNA (siRNA) knockdown and pharmacological inhibitors of PI3K, ERK1/2, and p38 MAPK pathways were used to investigate the receptors and the signaling pathways involved in CXC-induced ASMC migration, respectively. We established the ability of CXCL2 and CXCL3, but not CXCL1, to induce ASMC migration at the tested concentrations using normal ASMCs. We found CXCL2-induced ASMC migration to be dependent on p38 MAPK and CXCR2, whereas CXCL3-induced migration was dependent on p38 and ERK1/2 MAPK pathways via CXCR1 and CXCR2. While investigating the effect of CXCL2 and CXCL3 on asthmatic ASMC migration, we found that they induced greater migration of asthmatic ASMCs compared with normal ones. Interestingly, unlike normal ASMCs, CXCL2- and CXCL3-induced asthmatic ASMC migration was mainly mediated by the PI3K pathway through CXCR1. In conclusion, our results establish a new role of CXCR1 in ASMC migration and demonstrate the diverse mechanisms by which CXCL2 and CXCL3 mediate normal and asthmatic ASMC migration, suggesting that they may play a role in the pathogenesis of airway remodeling in asthma.
Telomeres are DNA-protein structures that protect chromosome ends from degradation.Previous studies suggest that analysis of telomere length is a predictor of disease progression in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. This study provides evidence of short telomeres in asthmatics relative to control subjects; telomere shortening may have a role in the process of immune aging in asthmatics.
Cigarette smoke is the principal cause of chronic obstructive pulmonary disease (COPD), a disorder characterised by airway inflammation. As epithelial cells are the first line of defense against foreign material, the response of normal epithelial cells to smoke has been extensively studied. However, little is known about how epithelial cells derived from COPD patients respond to ongoing smoke exposure. This study was aimed at comparing the intracellular response of normal human bronchial/tracheal epithelial cells (NHBE) and COPD-diseased human bronchial/ tracheal epithelial cells (DHBE) to cigarette smoke. NHBE and DHBE cells were treated with cigarette smoke condensate (CSC) for 24 hours. IL-8 production was measured by ELISA and western blot was used to measure TLR4 expression. Cells were pretreated with CLI-095, a TLR4 inhibitor, or the signaling pathway inhibitors PD184352, Helenalin, or PI-103, which inhibit the ERK1/2, NF-κB and PI3K pathways, respectively. NHBE cells increased IL-8 production in a dose-dependent manner in response to CSC while DHBE cells did not show any significant difference and had a much lower production of IL-8 in response to CSC compared to NHBE cells. There was no change in TLR4 expression with CSC exposure. CLI-095 and PD184352 attenuated IL-8 secretion, indicating that CSC-induced inflammation is both TLR4- and ERK1/2- dependent. These results demonstrate that NHBE and DHBE cells differentially respond to cigarette smoke. DHBE cells exhibit a dampened IL-8 release, indicating that COPD is associated with a reduced capacity of airway epithelial cells to respond to foreign material.
Increased airway smooth muscle (ASM) mass is a hallmark of airway remodeling in severe asthma. Th17-associated cytokines, particularly IL-17A, IL-17F, and IL-22, have been postulated to play a role in the pathogenesis of asthma. To investigate the in vitro effect of Th17 cytokines on the proliferation and survival of airway smooth muscle cells (ASMCs), human ASMCs from asthmatic and nonasthmatic subjects were incubated with IL-17A, IL-17F, or IL-22. The aforementioned cytokines demonstrated an ability to promote proliferation and survival of ASMCs from asthmatic and nonasthmatic subjects, which were mediated by selective activation of their corresponding receptors on ASMCs, including IL-17RA, IL-17RC, or IL-22R1, respectively. IL-17A and IL-17F-induced proliferation of ASMCs was dependent on ERK1/2 MAPK pathway, while IL-22-induced proliferation involved both ERK1/2 MAPK and NF-κB pathways. The involvement of signaling pathways was further confirmed by the inhibition of proliferation by knockdown of ERK1/2 MAPK or NF-κB p65 expression with pathway-specific siRNA. Together, our results show that Th17-associated cytokines promote proliferation and reduce the apoptotic rate of human ASMCs, raising the possibility that Th17 cytokines may contribute to increasing airway smooth muscle mass and airway remodeling in asthma.-Chang, Y., Al-Alwan, L., Risse, P.-A., Halayko, A. J., Martin, J. G.. Baglole, C. J., Eidelman, D. H., Hamid, Q. Th17-associated cytokines promote human airway smooth muscle cell proliferation.
- Jun 2012
Airway smooth muscle cell (ASMC) migration is one of the proposed mechanisms underlying the increased airway smooth muscle mass seen in airway remodeling of patients with severe asthma. IL-17-related cytokines are a new subgroup of inflammatory mediators that have been suggested to play a role in regulating smooth muscle function. We hypothesized that IL-17-induced chemokine production from smooth muscle cells can contribute to migration of additional smooth muscle cells in the airways of asthmatic patients. We sought to investigate the effect of IL-17 on smooth muscle-derived chemokines and to examine the mechanisms involved in their production and contribution to the increase in airway smooth muscle migration. The effect of IL-17-induced supernatants on human ASMC migration was investigated. IL-17-induced growth-related oncogene (GRO) production and mRNA expression was assessed by using ELISA and RT-PCR, respectively. The direct effect of GROs on ASMC migration and the involvement of the CXCR2 receptor were also examined. IL-17-induced supernatants promoted ASMC migration. After IL-17 stimulation, GROs were the most abundant chemokines produced from ASMCs, and blocking their effect by using neutralizing antibodies significantly inhibited ASMC migration. In addition, a combination of recombinant human GRO-α, GRO-β, and GRO-γ was able to promote significant migration of ASMCs that was mediated through the CXCR2 receptor. These findings suggest that IL-17-induced GROs can be an important mediator of ASMC migration and therefore might contribute to the pathogenesis of airway remodeling in asthmatic patients.
- Feb 2012
Asthma affects all age groups and presents itself as a spectrum of severity and symptoms. Reactive oxidative species (ROS) play a pivotal role in asthma pathogenesis. Exhaled levels of mediators associated with ROS positively correlate with asthma severity.(1) Autophagy, the process of cellular waste disposal through lysosomal-dependent pathways, is induced by ROS to remove oxidized proteins or organelles to minimize tissue damage.(2) Although autophagy is augmented in the lungs of COPD patients compared to healthy control subjects (3), evidence for autophagy in asthma, particularly 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 histological approaches.
- Dec 2011
Cigarette smoke is the primary risk factor for chronic obstructive pulmonary disease (COPD). Alterations in the balance between apoptosis and proliferation are involved in the etiology of COPD. Fibroblasts and epithelial cells are sensitive to the oxidative properties of cigarette smoke, and whose loss may precipitate the development of COPD. Fibroblasts express the aryl hydrocarbon receptor (AhR), a transcription factor that attenuates pulmonary inflammation and may also regulate apoptosis. We hypothesized the AhR would prevent apoptosis caused by cigarette smoke. Using genetically deleted in vitro AhR expression models and an established method of cigarette smoke exposure, we report that AhR expression regulates fibroblasts proliferation and prevents morphological features of apoptosis, including membrane blebbing and chromatin condensation caused by cigarette smoke extract (CSE). Absence of AhR expression results in cleavage of PARP, lamin, and caspase-3. Mitochondrial dysfunction, including cytochrome c release, was associated with loss of AhR expression, indicating activation of the intrinsic apoptotic cascade. Heightened sensitivity of AhR-deficient fibroblasts was not the result of alterations in GSH, Nrf2, or HO-1 expression. Instead, AhR(-/-) cells had significantly less MnSOD and CuZn-SOD expression, enzymes that protects against oxidative stress. The ability of the AhR to suppress apoptosis was not restricted to fibroblasts, as siRNA-mediated knockdown of the AhR in lung epithelial cells also increased sensitivity to smoke-induced apoptosis. Collectively, these results suggest that cigarette smoke induced loss of lung structural support (i.e. fibroblasts, epithelial cells) caused by aberrations in AhR expression may explain why some smokers develop lung diseases such as COPD.
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. 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. 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. 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.
- Jul 2011
Acute lung inflammation can be caused by a variety of respirable agents, including cigarette smoke. Long-term cigarette smoke exposure can cause chronic obstructive pulmonary disease (COPD), a serious illness that affects >10 million Americans. Cigarette smoke is a known inducer of inflammation and is responsible for approximately 90% of all COPD cases. RelB, a member of the NF-κB family, attenuates cigarette smoke-induced inflammatory mediator production in mouse lung fibroblasts in vitro. We hypothesized that overexpression of RelB in the airways of mice would dampen acute smoke-induced pulmonary inflammation. Mice received a recombinant adenovirus encoding RelB by intranasal aspiration to induce transient RelB overexpression in the lungs and were subsequently exposed to mainstream cigarette smoke. Markers of inflammation were analyzed after smoke exposure. Neutrophil infiltration, normally increased by smoke exposure, was significantly and potently decreased after RelB overexpression. Cigarette smoke-induced proinflammatory cytokine and chemokine production, cyclooxygenase-2 expression, and prostaglandin E(2) production were also significantly decreased in the context of RelB overexpression. The expression of intercellular adhesion molecule 1, an NF-κB-dependent protein, was decreased, indicating a potential mechanism through which RelB can regulate inflammatory cell migration. Therefore, increased expression and/or activation of RelB could be a novel therapeutic strategy against acute lung inflammation caused by respirable agents and possibly against chronic injury, such as COPD.
Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myo-fibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treat-ment of fibrosis is limited by a lack of safe and effec-tive therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differen-tiation triggered by transforming growth factor-1 (TGF1). In this study, we demonstrate that TGF1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1=H-indole-3=-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGF1 sig-naling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR ago-nist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause se-vere toxic effects, ITE exhibits no toxicity. Interest-ingly, ITE effectively inhibits TGF1-driven myofi-broblast differentiation in AhR / fibroblasts: Its ability to inhibit TGF1 signaling is AhR indepen-dent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differen-tiation and may be useful clinically as an antiscar-ring agent.
Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-β1 (TGFβ1). In this study, we demonstrate that TGFβ1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFβ1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFβ1-driven myofibroblast differentiation in AhR(-/-) fibroblasts: Its ability to inhibit TGFβ1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent.
- Dec 2010
Heme-oxygenase-1 (HO-1) is induced in response to oxidative stress and is believed to be a cytoprotective and anti-inflammatory enzyme. It is unknown whether normal or malignant human B-lineage cells express HO-1. 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is an interesting electrophilic lipid mediator able to increase oxidative stress in B cells. Here, we tested normal and malignant human B-lineage cells for their ability to express HO-1 in response to 15d-PGJ(2), as well as the signaling pathways required for HO-1 expression. 15d-PGJ(2) potently induced HO-1 protein expression in normal and malignant B cells. Malignant B cells exhibited a greater induction of HO-1 protein compared to normal B lymphocytes. Using siRNA directed against the transcription factor Nrf2 and B cells isolated from Nrf2-deficient mice, we show that HO-1 induction by 15d-PGJ(2) is dependent on Nrf2. These results show that, compared to normal B lymphocytes, malignant B cells have a greater capacity to increase their HO-1 protein levels in response to 15d-PGJ(2). We speculate that the ability to highly express HO-1 by malignant B cells could confer a survival advantage.
- May 2010
- American Thoracic Society 2010 International Conference, May 14-19, 2010 • New Orleans
- Mar 2010
Thyroid eye disease (TED) is a debilitating disorder characterized by the accumulation of adipocytes and hyaluronan (HA). Production of HA by fibroblasts leads to remarkable increases in tissue volume and to the anterior displacement of the eyes. Prostaglandin D(2) (PGD(2)), mainly produced by mast cells, promotes orbital fibroblast adipogenesis. The mechanism by which PGD(2) influences orbital fibroblasts and their synthesis of HA is poorly understood. We report here that mast cell-derived PGD(2) is a key factor that promotes HA biosynthesis by orbital fibroblasts. Primary orbital fibroblasts from TED patients were isolated and used to test the effects of PGD(2), prostaglandin J(2), as well as prostaglandin D receptor (DP) agonists and antagonists on HA synthesis. The expression of HA synthase (HAS), hyaluronidase, DP1, and DP2 mRNA levels was assessed by PCR. Small interfering RNAs against HAS1 or HAS2 were used to assess the importance of HAS isoforms on HA production. Treatment of human orbital fibroblasts with PGD(2) and PGJ(2) increased HA synthesis and HAS mRNA. HAS2 was the dominant isoform responsible for HA production by PGD(2). The effect of PGD(2) on HA production was mimicked by the selective DP1 agonist BW245C. The DP1 antagonist MK-0524 completely blocked PGD(2)-induced HA synthesis. Human mast cells (HMC-1) produced PGD(2). Co-culture of HMC-1 cells with orbital fibroblasts induced HA production and inhibition of mast cell-derived PGD(2) prevented HA synthesis. Mast cell-derived PGD(2) increased HA production via activation of DP1. Selectively targeting the production of PGD(2) and/or activation of DP1 may prevent pathological changes associated with TED.
Protective humoral immune responses critically depend on the optimal differentiation of B cells into Ab-secreting cells. Because of the important role of Abs in fighting infections and in successful vaccination, it is imperative to identify mediators that control B cell differentiation. Activation of B cells through TLR9 by CpG-DNA induces plasma cell differentiation and Ab production. Herein, we examined the role of the peroxisome proliferator-activated receptor (PPAR)gamma/RXRalpha pathway on human B cell differentiation. We demonstrated that activated B cells up-regulate their expression of PPARgamma. We also show that nanomolar levels of natural (15-deoxy-Delta(12,14)-prostaglandin J(2)) or synthetic (rosiglitazone) PPARgamma ligands enhanced B cell proliferation and significantly stimulated plasma cell differentiation and Ab production. Moreover, the addition of GW9662, a specific PPARgamma antagonist, abolished these effects. Retinoid X receptor (RXR) is the binding partner for PPARgamma and is required to produce an active transcriptional complex. The simultaneous addition of nanomolar concentrations of the RXRalpha ligand (9-cis-retinoic acid) and PPARgamma ligands to CpG-activated B cells resulted in additive effects on B cell proliferation, plasma cell differentiation, and Ab production. Furthermore, PPARgamma ligands alone or combined with 9-cis-retinoic acid enhanced CpG-induced expression of Cox-2 and the plasma cell transcription factor BLIMP-1. Induction of these important regulators of B cell differentiation provides a possible mechanism for the B cell-enhancing effects of PPARgamma ligands. These new findings indicate that low doses of PPARgamma/RXRalpha ligands could be used as a new type of adjuvant to stimulate Ab production.
Oxidative stress plays an important role in the pathogenesis of pulmonary fibrosis. Heme oxygenase-1 (HO-1) is a key antioxidant enzyme, and overexpression of HO-1 significantly decreases lung inflammation and fibrosis in animal models. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that the PPARgamma ligands 15d-PGJ2 and 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), which have potent antifibrotic effects in vitro, also strongly induce HO-1 expression in primary human lung fibroblasts. Pharmacological and genetic approaches are used to demonstrate that induction of HO-1 is PPARgamma independent. Upregulation of HO-1 coincides with decreased intracellular glutathione (GSH) levels and can be inhibited by N-acetyl cysteine (NAC), a thiol antioxidant and GSH precursor. Upregulation of HO-1 is not inhibited by Trolox, a non-thiol antioxidant, and does not involve the transcription factors AP-1 or Nrf2. CDDO and 15d-PGJ2 contain an alpha/beta unsaturated ketone that acts as an electrophilic center that can form covalent bonds with free reduced thiols. Rosiglitazone, a PPARgamma ligand that lacks an electrophilic center, does not induce HO-1. These data suggest that in human lung fibroblasts, 15d-PGJ2 and CDDO induce HO-1 via a GSH-dependent mechanism involving the formation of covalent bonds between 15d-PGJ2 or CDDO and GSH. Inhibiting HO-1 upregulation with NAC has only a small effect on the antifibrotic properties of 15d-PGJ2 and CDDO in vitro. These results suggest that CDDO and similar electrophilic PPARgamma ligands may have great clinical potential as antifibrotic agents, not only through direct effects on fibroblast differentiation and function, but indirectly by bolstering antioxidant defenses.
- Sep 2009
Cigarette smoke is the most important environmental risk factor for developing age-related macular degeneration (AMD). Damage to the retinal pigment epithelium (RPE) caused by cigarette smoke may underlie the etiology of AMD. This study investigated the molecular and cellular effects of cigarette smoke exposure on human RPE cells. ARPE-19 or primary human RPE cells were exposed to cigarette smoke extract (CSE) or hydroquinone (HQ), a component of cigarette smoke. The effect of this exposure on key aspects of RPE vitality including viability, cell size, mitochondrial membrane potential (DeltaPsi(m)), superoxide production, 4-hydroxy-2-nonenal (4-HNE), vascular endothelial growth factor (VEGF), and heme oxygenase-1 (HO-1) expression was determined. Exposure of RPE cells to CSE or HQ caused oxidative damage and apoptosis, characterized by a reduction in cell size and nuclear condensation. Evidence of oxidative damage also included increased lipid peroxidation (4-HNE) and mitochondrial superoxide production, as well as a decrease in intracellular glutathione (GSH). Exogenous administration of antioxidants (GSH and N-acetyl-cysteine) prevented oxidative damage to the RPE cells caused by CSE. Cigarette smoke also induced expression of VEGF, HO-1, and the transcription factor nuclear factor erythroid-derived 2, like 2 (NRF2). However, NRF2 was only modestly involved in CSE-induced HO-1 expression, as shown by the NRF2 small interfering RNA studies. These new findings demonstrate that cigarette smoke is a potent inducer of oxidative damage and cell death in human RPE cells. These data support the hypothesis that cigarette smoke contributes to AMD pathogenesis by causing oxidative damage and cell death to RPE cells.
Peroxisome proliferator-activated receptor gamma (PPARgamma) is a multifunctional transcription factor that regulates adipogenesis, immunity and inflammation. Our laboratory previously demonstrated that PPARgamma ligands induce apoptosis in malignant B cells. While malignant B lineage cells such as B cell lymphoma express PPARgamma, its physiological function remains unknown. Herein, we demonstrate that silencing PPARgamma expression by RNAi in human Burkitt's type B lymphoma cells increased basal and mitogen-induced proliferation and survival, which was accompanied by enhanced NF-kappaB activity and increased expression of Bcl-2. These cells also had increased survival upon exposure to PPARgamma ligands and exhibited a less differentiated phenotype. In contrast, PPARgamma overexpression in B lymphoma cells inhibited cell growth and decreased their proliferative response to mitogenic stimuli. These cells were also more sensitive to PPARgamma-ligand induced growth arrest and displayed a more differentiated phenotype. Collectively, these findings support a regulatory role for PPARgamma in the proliferation, survival and differentiation of malignant B cells. These findings further suggest the potential of PPARgamma as a therapeutic target for B cell malignancy.
- Nov 2008
Platelet production is an intricate process that is poorly understood. Recently, we demonstrated that the natural peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), augments platelet numbers by increasing platelet release from megakaryocytes through the induction of reactive oxygen species (ROS). 15d-PGJ(2) can exert effects independent of PPARgamma, such as increasing oxidative stress. Heme oxygenase-1 (HO-1) is a potent antioxidant and may influence platelet production. To further investigate the influence of 15d-PGJ(2) on megakaryocytes and to understand whether HO-1 plays a role in platelet production. Meg-01 cells (a primary megakaryoblastic cell line) and primary human megakaryocytes derived from cord blood were used to examine the effects of 15d-PGJ(2) on HO-1 expression in megakaryocytes and their daughter platelets. The role of HO-1 activity in thrombopoiesis was studied using established in vitro models of platelet production. 15d-PGJ(2) potently induced HO-1 protein expression in Meg-01 cells and primary human megakaryocytes. The platelets produced from these megakaryocytes also expressed elevated levels of HO-1. 15d-PGJ(2)-induced HO-1 was independent of PPARgamma, but could be replicated using other electrophilic prostaglandins, suggesting that the electrophilic properties of 15d-PGJ(2) were important for HO-1 induction. Interestingly, inhibiting HO-1 activity enhanced ROS generation and augmented 15d-PGJ(2)-induced platelet production, which could be attenuated by antioxidants. These new data reveal that HO-1 negatively regulates thrombopoiesis by inhibiting ROS.
Fibroblasts are key structural cells that can be damaged by cigarette smoke. Cigarette smoke contains many components capable of eliciting oxidative stress, which may induce heme oxygenase (HO)-1, a cytoprotective enzyme. There are no data on HO-1 expression in primary human lung fibroblasts after cigarette smoke extract (CSE) exposure. We hypothesized that human lung fibroblasts exposed to cigarette smoke would increase HO-1 though changes in intracellular glutathione (GSH). Primary human lung fibroblasts were exposed to CSE, and changes in HO-1 expression and GSH levels were assessed. CSE induced a time- and dose-dependent increase in expression of HO-1, but not HO-2 or biliverdin reductase, in two different primary human lung fibroblast strains, a novel finding. This induction of HO-1 paralleled a decrease in intracellular GSH, and a sustained reduction in GSH resulted in a dramatic increase in HO-1. Treatment with the antioxidants N-acetyl-l-cysteine or GSH reduced the expression of HO-1 induced by CSE. We also examined the signal transduction mechanism responsible for HO-1 induction. Nuclear factor erythroid-derived 2, like 2 (Nrf2) was not involved in HO-1 induction by CSE. Activator protein-1 (AP-1) is a redox-sensitive transcription factor shown in other systems to regulate HO-1 expression. CSE exposure resulted in nuclear accumulation of c-Fos and c-Jun, two key AP-1 components. Reduction of c-Fos and c-Jun nuclear translocation by SP-600125 attenuated the CSE-induced expression of HO-1. These data support the concept that changes in the cellular redox status brought on by cigarette smoke induce HO-1 in fibroblasts. This increase in HO-1 may help protect against cigarette smoke-induced inflammation and/or cell death.
Diseases such as chronic obstructive pulmonary disease and lung cancer caused by cigarette smoke affect millions of people worldwide. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that influences responses to certain environmental pollutants such as tobacco smoke. However, the physiological function(s) of the AhR is unknown. Herein we propose that the physiologic role of the AhR is to limit inflammation. We show that lung fibroblasts from AhR–/– mice produce a heightened inflammatory response to cigarette smoke, typified by increased levels of cyclooxygenase-2 (COX-2) and prostaglandins (PGs), when compared with wild type (AhR+/+) fibroblasts. This response was dependent on AhR expression as transient transfection of an AhR expression plasmid into AhR–/– fibroblasts significantly attenuated the smoke-induced COX-2 and PG production, confirming the anti-inflammatory role of the AhR. The AhR can interact with NF-κB. However, the heightened inflammatory response observed in AhR–/– fibroblasts was not the result of NF-κB (p50/p65) activation. Instead it was coupled with a loss of the NF-κB family member RelB in AhR–/– fibroblasts. Taken together, these studies provide compelling evidence that AhR expression limits proinflammatory COX-2 and PG production by maintaining RelB expression. The association between RelB and AhR may represent a new therapeutic and more selective target with which to combat inflammation-associated diseases.
- Jul 2007
Acute sympathetic denervation of the small intestine up-regulates alpha1-adrenoceptors on villus enterocytes and activation of these alpha1-adrenoceptors inhibits chloride secretion. We tested whether alpha1-adrenoceptor-mediated inhibition of chloride secretion was the result of reduced ClC-2 chloride channel expression. Phorbol myristate acetate (PMA) (a protein kinase C (PKC) activator) had no effect on ClC-2 levels. In contrast, alpha1-adrenoceptor activation significantly decreased ClC-2 protein levels in both the villus (1.58+/-0.19 to 0.75+/-0.19 arbitrary units) and crypt (1.69+/-0.15 to 0.37+/-0.23 arbitrary units) epithelial cells from the acutely denervated jejunum but not innervated controls. These data suggest that inhibition of chloride secretion following alpha1-adrenoceptor activation in the acutely denervated small intestine may be through ClC-2 down-regulation.
- Apr 2007
The transcription factor aryl hydrocarbon receptor (AhR) plays an important role in the response to environmental pollutants. However, its role in normal physiology is unclear. To investigate the role of AhR in acute lung inflammation, control and AhR knockout (KO) mice were exposed to inhaled cigarette smoke or bacterial endotoxin. Smoke-induced lung inflammation was twofold to threefold more severe in AhR KO mice than controls. Intriguingly, levels of tumor necrosis factor-alpha and interleukin-6 in the bronchoalveolar lavage of air-exposed KO mice were equal to the levels seen in smoke-exposed controls, suggesting that AhR-deficient mice are inflammation prone. AhR KO mice challenged with inhaled endotoxin, which does not contain AhR ligands, also developed greater lung neutrophilia than controls, and bronchoalveolar lavage cells from AhR KO mice produced elevated levels of tumor necrosis factor-alpha and interleukin-6 when treated with endotoxin in vitro. Nuclear factor-kappaB DNA-binding activity was elevated in smoke-exposed AhR KO mice compared with controls and was associated with a rapid loss of RelB only in the KO mice. We propose that AhR is a previously unrecognized regulator of inflammation that interacts with nuclear factor-kappaB so that in the absence of AhR RelB is prematurely degraded, resulting in heightened inflammatory responses to multiple proinflam-matory stimuli.
- Feb 2007
- Tissue Repair, Contraction and the Myofibroblast
Fibroblasts are a heterogeneous population of structural cells whose primary function is the production of extracellular matrix for normal tissue maintenance and repair. However, fibroblasts provide much more than structural support as they synthesize and respond to many different cytokines and lipid mediators and are intimately involved in the processes of inflammation. It is now appreciated that fibroblasts exhibit phenotypic heterogeneity, differing not only between organ systems, but also within a given anatomical site. Subtypes of fibroblasts can be identified by the expression of markers such as Thy-1, a cell surface glycoprotein of unknown function. Initial characterization of fibroblasts as Thy-1+ or Thy-1− can be performed by immunofluorescence or flow cytometry. They can be sorted according to their expression of Thy-1 by fluorescence-activated cell sorting (FACS), cloning and/or magnetic beading, yielding greater than 99% purity. Fibroblasts that are separated into Thy-1+ and Thy-1− subsets exhibit differences in their morphological, immunological and proliferative responses and ability to differentiate into a-smooth muscle actin-expressing myofibroblasts and adipocyte-like lipofibroblasts, key cells for wound healing and fibrotic disorders. The identification of Thy-1 as a surface marker by which to separate fibroblast subtypes has yielded vital insight into diseases such as scarring and wound healing and highlights the concept of fibroblast heterogeneity. Future research into fibroblast subsets may lead to the tissue-specific treatment of disease such as idiopathic pulmonary fibrosis and Graves’ ophthalmopathy.
- Aug 2006
Cigarette smoke is the principal cause of emphysema. Recent attention has focused on the loss of alveolar fibroblasts in the development of emphysema. Fibroblasts may become damaged by oxidative stress and undergo apoptosis as a result of cigarette smoke exposure. Not all smokers develop lung diseases associated with tobacco smoke, a fact that may reflect individual variation among human fibroblast strains. We hypothesize that fibroblasts from different human beings vary in their ability to undergo apoptosis after cigarette smoke exposure. This could account for emphysematous changes that occur in the lungs of some but not all smokers. Primary human lung fibroblast strains were exposed to cigarette smoke extract (CSE) and assessed for viability, morphological changes, and mitochondrial transmembrane potential as indicators of apoptosis. We also examined the generation of intracellular reactive oxygen species (ROS), 4-hydroxy-2-nonenal, and changes in glutathione (GSH) and glutathione disulfide (GSSG) levels. Each human lung fibroblast strain exhibited a differential sensitivity to CSE as judged by changes in mitochondrial membrane potential, viability, ROS generation, and glutathione production. Interestingly, the thiol antioxidants N-acetyl-L-cysteine and GSH eliminated CSE-induced changes in fibroblast morphology such as membrane blebbing, nuclear condensation, and cell size and prevented alterations in mitochondrial membrane potential and the generation of ROS. These findings support the concept that oxidative stress and apoptosis are responsible for fibroblast death associated with exposure to tobacco smoke. Variations in the sensitivity of fibroblasts to cigarette smoke may account for the fact that only some smokers develop emphysema.
The tumor microenvironment comprises many cell types including infiltrating immune cells such as lymphocytes, endothelial cells and a complex stroma consisting mainly of fibroblasts. Fibroblasts are heterogeneous and consist of Thy-1+ and Thy-1- subsets that define different biosynthetic and differentiation potential. They produce mediators linked to carcinogenesis and metastasis, including Cox-2 and PGE2, both of which are also increased in most cancers. This review will highlight the emerging role of the complex fibroblastic stroma in establishing a microenvironment supporting malignant transformation, tumor growth and attenuation of host anti-tumor immune responses.
Loss of sympathetic input due to intestinal denervation results in hypersensitivity and increased intestinal secretion. It is unknown whether denervation-induced alterations in intestinal epithelial physiology are the result of changes in adrenoceptors on enterocytes (ENTs). The purpose of this study was to examine adrenoceptor distribution and pharmacology on small intestinal ENTs following acute intestinal denervation. Lewis rats underwent small bowel transplantation (SBT) or sham operation and proximal small intestinal segments were harvested 1, 2 and 4 weeks postoperatively. Intestinal electrolyte movement was assessed using short-circuit current (Isc) measurements of stripped epithelial sheets following stimulation with phenylephrine (PE), an alpha(1)-adrenoceptor agonist. The presence of adrenoceptor subtypes on separated villus and crypt ENTs was assessed using flow cytometry. Alpha(1)-adrenoceptors were found on approximately 27% of jejunal villus ENTs, but not crypt ENTs, following acute extrinsic denervation. ENTs from the Lewis rat have few beta-adrenoceptors. Alpha(1)-adrenoceptor stimulation of acutely denervated intestinal epithelial sheets decreased Isc by -13.45%. This effect was mediated by a reduction in chloride (Cl(-)) secretion; the absence of Cl(-) reversed the Isc to +13.79%. In conclusion, loss of sympathetic innervation to the gastrointestinal epithelium causes acute upregulation of alpha(1)-adrenoceptors on villus ENTs, leading to inhibition of Cl(-) secretion at the villus tip. The increase in adrenoceptors may reflect a compensatory mechanism to combat the increased secretory state of the bowel due to the loss of the sympathetic innervation and tonic control over intestinal secretion.
- Oct 2005
Cigarette smoking can lead to chronic lung inflammation and lung cancer. Chronic inflammation, associated with expression of cyclooxygenase-2 (COX-2) and prostaglandins, predisposes to malignancy. We recently demonstrated that human lung fibroblasts are activated by cigarette smoke to express COX-2 and prostaglandin E(2) (PGE(2)). Little is known about the mechanism whereby smoke activates human lung fibroblasts to produce proinflammatory mediators. Herein, we report the central role of the aryl hydrocarbon receptor (AHR) in cigarette smoke extract (CSE)-induced COX-2, microsomal PGE(2) synthase (mPGES), and PGE(2) production in human lung fibroblasts. Western blot analysis revealed that primary strains of human lung fibroblasts express AHR and aryl hydrocarbon nuclear translocator protein, supporting the possibility that smoke activates lung fibroblasts through this pathway. Experiments were subsequently performed to determine whether the AHR was activated by CSE. Immunocytochemistry and EMSA analysis revealed that CSE induced nuclear translocation of the AHR in human lung fibroblasts. CSE decreased protein levels of the AHR, consistent with AHR ligand-induced proteosome-mediated degradation. CSE also induced mPGES-1 and COX-2 protein and increased PGE(2) production. Treatment of human fibroblasts with AHR antagonists in the presence of CSE inhibited AHR nuclear translocation as well as COX-2, mPGES-1, and PGE(2) production. These data indicate that the AHR pathway plays an important role in cigarette smoke-mediated COX-2 and PG production in human lung fibroblasts and may contribute to tobacco-associated inflammation and lung disease.
- May 2005
Neural and paracrine agents, such as dopamine, epinephrine, and histamine, affect intestinal epithelial function, but it is unclear if these agents act on receptors directly at the enterocyte level. The cellular localization and villus-crypt distribution of adrenergic, dopamine, and histamine receptors within the intestinal epithelium is obscure and needs to be identified. Single cell populations of villus or crypt epithelial cells were isolated from the jejunum of adult guinea pigs. Enterocytes were separated from intraepithelial lymphocytes by flow cytometry and specific binding was determined using fluorescent probes. Alpha1-adrenergic receptors were located on villus and crypt intraepithelial lymphocytes and enterocytes. Beta-adrenergic receptors were found on villus and crypt enterocytes. Dopamine receptors were found on all cell types examined, whereas histamine receptors were not detected (<10% for each cell population). These studies demonstrated that (1) receptors for epinephrine and dopamine exist on epithelial cells of the guinea pig jejunum, (2) beta-adrenergic receptors are found primarily on villus and crypt enterocytes and (3) intraepithelial lymphocytes contain alpha1-adrenergic, but have few beta-adrenergic, receptors. The presence of neural receptors suggests that these agents are acting, at least in part, at the enterocyte or intraepithelial lymphocyte levels to modulate intestinal and immune function.
- Feb 2005
Primary fibroblasts represent a heterogeneous population of cells that can be separated into subsets on the basis of cell surface markers such as Thy-1. Deriving fibroblasts initially involves obtaining tissue explants from tissues such as the lung, heart, cornea, skin, and orbit. The tissue is mechanically dissociated and cells are allowed to proliferate from the fragments. Following establishment of a primary culture of fibroblasts, it is necessary to characterize the new strain of cells to ensure their purity and fibroblastic phenotype using immunofluorescence and immunohistochemistry to detect the presence or absence of cell-specific surface markers. Characterizing the cells as expressing or lacking Thy-1 can also be performed by immunofluorescence in concert with microscopy or by flow cytometry using an anti-human Thy-1 antibody. In addition, fibroblasts may be sorted according to their expression of Thy-1 by fluorescence-activated cell sorting and/or magnetic beading; use of these techniques can yield greater than 99% purity. Once separated, the pure Thy-1 expressing or lacking fibroblast subsets can be propagated. These subsets can then be used for experimentation to determine functional differences between fibroblasts derived from normal and pathological tissue such as scarred lung.
Cigarette smoking can lead to many human pathologies including cardiovascular and respiratory disease. Recent studies have defined a role for fibroblasts in the development of colon cancer. Moreover, fibroblasts are now thought of as key "sentinel" cells that initiate inflammation by releasing proinflammatory mediators including prostaglandins (PGs). Pathological overexpression of cyclooxygenase-2 (COX-2) and excess eicosanoid production are found in the early stages of carcinogenesis. By promoting chronic inflammation, COX-2 and eicosanoid production may actually cause a predisposition to malignancy. Furthermore, the associated inflammation induced by production of these mediators is central to the pathogenesis of chronic obstructive pulmonary disease. Little is known of the responses of normal lung fibroblasts to cigarette smoke, despite their abundance. We report herein that normal human lung fibroblasts, when exposed to cigarette smoke extract, induce COX-2 with concurrent synthesis of prostaglandin E2 (PGE2). The mechanisms by which cigarette-derived toxicants lead to increased COX-2 levels and PGE2 synthesis include increases in steady-state COX-2 mRNA levels (approximately four- to fivefold), phosphorylation of ERK1/2, and nuclear translocation of the p50 and p65 subunits of the transcription factor NF-kappaB, which are important elements in COX-2 expression. Furthermore, there was a dramatic 25-fold increase in microsomal prostaglandin E synthase, the key enzyme involved in the production of PGE2. We propose that normal human lung fibroblasts, when exposed to cigarette smoke constituents, elicit COX-2 expression with consequent prostaglandin synthesis, thus creating a proinflammatory environment. This chronic inflammatory state may act as one of the first steps towards epithelial transformation.
The distribution of digestive enzymes was studied using histoenzymological methods in yellowtail and winter flounder larvae reared on three different diets: live food, weaned at day 15, or starved. Alkaline phosphatase, dipeptidyl peptidase IV, aminopeptidase M and esterase were present at 3 days post-hatch and became differentially distributed coinciding with morphological development. For larvae fed a live diet, activity of these enzymes was present in the intestine of both species and rectum of yellowtail flounder. Alkaline phosphatase was also present in the post-oesophageal swelling (stomach anlage) of winter flounder. Activity of all enzymes was absent in starved winter flounder larvae and a decrease in aminopeptidase M and esterase activity occurred in starved yellowtail flounder larvae. Acid phosphatase was not identified in either species. The eVect of weaning on enzymatic activity was not evaluated fully as the larvae did not survive long enough after the introduction of the artificial diet to complete experimentation.
- Aug 1997
The histological development and mucous histochemistry of the alimentary tract in larval yellowtail flounder were studied using light microscopy. Samples were taken when the larvae were first offered food at 3 days post-hatch, then at 7, 10, 29, 36, and 46 days post-hatch, at which time they were metamorphosing. Regional partitioning of the digestive tract into the buccal cavity, pharynx, oesophagus, post-oesophageal swelling (PES), intestine, and rectum was complete by day 10. Goblet cells were present only in the buccal cavity, pharynx and intestine by day 7, but increased in number and distribution as development continued. By day 29, the posterior zone of the oesophagus had a marked increase in goblet cell density and mucosal folding. At the transition from oesophagus to PES/stomach stratified epithelium with goblet cells changed abruptly to a columnar epithelium with no goblet cells. Multicellular glands in the PES of 36-day larvae allowed it to be defined as a stomach. The distinct brush border of columnar epithelium and the presence of goblet cells characterize the intestine and rectum. All goblet cells throughout the digestive tract were strongly positive for acid mucins as was the luminal layer of the stratified epithelia lining the buccal cavity, pharynx and oesophagus. The PES/stomach epithelium stained weakly for neutral mucins. No mucin staining was associated with the gastric glandular epithelium. The brush borders of the intestine and rectum were strongly positive for combinations of neutral and acid mucins.
"NQ-87018." Thesis (Ph.D.)--University of Calgary, Dept. of Gastrointestinal Sciences, 2002. Includes bibliographical references. Microfiche.