ovalbumin and Metaplasia

People are often concurrently exposed to more than one air pollutant whether they are in outdoor or indoor environments. Therefore, inhalation studies that are designed to examine the toxicity of coexposures to two or more airborne toxicants may be more relevant for assessing human health risks than those studies that investigate the toxic effects of only one airborne toxicant at a time. Furthermore, airborne biogenic substances such as pollens, bacteria, fungi, and microbial toxins often coexist with common air pollutants in the ambient air, and when inhaled may also cause specific adverse effects on the respiratory tract. One such biogenic substance, bacterial endotoxin, is a potent stimulus of airway inflammation and is commonly found in domestic, agricultural, and industrial settings. Little is known about the interaction of exposures to biogenic substances and common air pollutants, such as ozone or airborne particulate matter. In the last few years, we have performed a series of in vivo studies using laboratory rodents that examined how airway surface epithelial cells are altered by coexposure to ozone and a biogenic substance, either bacterial endotoxin or a commonly used experimental aeroallergen (ovalbumin). Results from these studies indicate that the ozone-induced epithelial and inflammatory responses in laboratory rodents may be markedly enhanced by coexposure to an inhaled biogenic substance. Conversely, the adverse airway alterations caused by exposure to biogenic substances may be enhanced by coexposure to ozone. The results from these initial studies have also suggested some of the cellular and molecular mechanisms underlying the phenotypic epithelial alterations induced by these coexposures. Many more studies are needed to fully elucidate the potential risk to human health from coexposure to air pollutants and airborne biogenic substances.

Topics: Air Pollutants; Animals; Biological Factors; Disease Models, Animal; Drug Synergism; Endotoxins; Inflammation; Inhalation Exposure; Metaplasia; Neutrophils; Ovalbumin; Ozone; Rats; Rats, Inbred F344; Respiratory Mucosa; Respiratory Tract Diseases

Despite the substantial amount of efforts made to reduce morbidity and improve respiratory management, asthma control remained a major challenge for severe patients. Plant isoflavones, one of the most estrogenic compounds, are considered a potential alternative therapy for asthma. Iristectorigenin A, a naturally occurring isoflavone, is extracted from a variety of medical plants and its biological activity has not been reported previously.. In present study, we aim to reveal the potential therapeutic role of Iristectorigenin A against acute asthmatic mice.. We established ovalbumin (OVA) induced asthmatic murine model and orally administrated Iristectorigenin A at concentration of 5 and 10 mg/kg and dexamethasone as a positive control substance.. Asthmatic murine model was established with OVA sensitization and challenge. Lung function was assessed with FinePoint Ventilation system recording lung resistance (RI) and lung compliance (Cydn). White cells were sorted and counted in BALF. Histopathological assessment was conducted by H&E, PAS, and Masson's trichrome staining on paraffin embedded lung tissues. BALF content of IL-4, IL-5, IL-33, IL-13, INF-γ, IL-9 and serum IgE, IgG1 were measured using ELISA kit. Expression levels of mRNAs associated with inflammatory cytokines and goblet cell metaplasia were evaluated via quantitative RT-PCR. Protein expression levels of FOXA3, MUC5AC, SPDEF were estimated by immunohistochemistry on lung tissue, while NOTCH1 and NOTCH2 expressions were evaluated by western blotting analysis.. Iristectorigenin A resulted in improved airway hyperresponsiveness (AHR) mirrored by decreased RI and increased Cydn. With Iristectorigenin A, we also observed reduced number of BALF leukocytes, improved inflammatory cell infiltration in lung tissue, decreased content of BALF IL-4, IL-5, IL-33, but not IL-13, INF-γ, IL-9, and their mRNA levels, along with decreased levels of OVA-specific IgE, IgG1 in asthmatic mice. Additionally, Iristectorigenin A exhibited significant therapeutic potential on attenuating mucus production reflected by mitigated FOXA3 and MUC5AC immunostaining on the airway epithelium, as well as decreased mRNAs associated with goblet cell metaplasia. At last, a decrease in elevated expression level of NOTCH2, but not NOTCH1, in asthmatic mice lung tissue was observed by western blotting analysis.. Our study provides strong evidence that Iristectorigenin A can be potential therapeutic agent ameliorating airway inflammation and mucus hypersecretion in allergic asthma. This is a first research reported the potential of Iristectorigenin A as an alternative therapeutic agent.

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Immunoglobulin E; Immunoglobulin G; Inflammation; Interleukin-33; Interleukin-4; Interleukin-5; Interleukin-9; Lung; Metaplasia; Mice; Mice, Inbred BALB C; Mucus; Ovalbumin; Phenotype

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Topics: Animals; Anoctamin-1; Asthma; Chloride Channels; Goblet Cells; Metaplasia; Mice; Mucus; Ovalbumin; Respiratory Mucosa

As a chronic airway disease, asthma has two characteristics, tissue remodeling and airway inflammation. This research focused on miR-92a to explore how it works in asthma. We revealed that the expressions of miR-92a were decreased in both serum and lung tissues from ovalbumin-induced asthma mouse. Bioinformatics analysis, quantitative polymerase chain reaction (qPCR) and dual luciferase assay revealed that miR-92a targets MUC5AC, which was linked to mucus hypersecretion in the pulmonary tracts. By injecting miR-92a-mimics into the trachea, both the airway hyper-reactivity and airway inflammation can be alleviated in an asthma mouse model which is induced by ovalbumin. Moreover, the goblet cell phenotype of asthmatic mice is significantly reduced by the action of miR-92a. Furthermore, miR-92a blocked interleukin (IL)-13-induced MUC5AC luciferase activity in 16HBE. Together, upregulation of miR-92a expression in asthmatic mice plays a role in blocking goblet cell metaplasia by targeting MUC5AC, and thus in the treatment of chronic airway diseases, miR-92a can prevent epithelial remodeling, which is a reasonable method.

Topics: Animals; Asthma; Female; Gene Expression Regulation; Goblet Cells; Metaplasia; Mice; Mice, Inbred C57BL; MicroRNAs; Mucin 5AC; Ovalbumin

Notch1 has been linked to the pathogenesis of asthma due to its contribution on Th1/Th2 imbalance. γ-Secretase inhibitor (GSI) acts as an effective blocker of Notch1 signaling. Glucocorticoids (GCs) are the most effective anti-inflammatory drugs for asthma. The present study investigated the involvement of the Notch1 pathway in the anti-inflammatory effect of GCs and its association with Th1/Th2 balance.. The asthma model was established in BALB/c mice by sensitization with ovalbumin (OVA). Dexamethasone (DEX; 1 mg/kg) and/or GSI (0.03 mg/kg) was orally or intranasally administrated.. Compared to the OVA-sensitized mice, the administration of DEX and/or GSI significantly ameliorated the airway inflammation infiltration, goblet cell metaplasia, and airway hyper-responsiveness. The expression of IL-4 and IL-13, as well as the ratios of eosinophils and lymphocytes, were significantly decreased, whereas IFN-γ and IL-2 levels were significantly increased in bronchoalveolar lavage fluid after the administration of DEX and GSI. The expressions of the Notch1/NICD1 pathway were decreased after DEX and/or GSI administration in lung tissues, especially in CD4+ T cells. Also, a reduction of GATA3 and elevation of T-bet levels were correlated with the upregulation of Th1/Th2 ratios in lung tissues.. Through the inhibition of Notch1 signaling, both GSI and GCs could regulate Th1/Th2 balance involved in allergic airway inflammation in OVA-induced asthma.

Topics: Allergens; Animals; Asthma; Cells, Cultured; Cytokines; Dexamethasone; Disease Models, Animal; Glucocorticoids; Goblet Cells; Humans; Metaplasia; Mice; Mice, Inbred BALB C; Ovalbumin; Receptor, Notch1; Signal Transduction; T-Box Domain Proteins; Th1-Th2 Balance

Pidotimod is a synthetic dipeptide with biological and immuno‑modulatory properties. It has been widely used for treatment and prevention of recurrent respiratory infections. However, its impact on the regulation of allergic pulmonary inflammation is still not clear. In the current study, an ovalbumin (OVA)‑induced allergic asthma model was used to investigate the immune‑modulating effects of pidotimod on airway eosinophilia, mucus metaplasia and inflammatory factor expression compared with dexamethasone (positive control). The authors determined that treatment with pidotimod exacerbated pulmonary inflammation as demonstrated by significantly increased eosinophil infiltration, dramatically elevated immunoglobulin E production, and enhanced T helper 2 response. Moreover, treatment failed to attenuate mucus production in lung tissue, and did not reduce OVA‑induced high levels of FIZZ1 and Arg1 expression in asthmatic mice. In contrast, administration of dexamethasone was efficient in alleviating allergic airway inflammation in OVA‑induced asthmatic mice. These data indicated that pidotimod as an immunotherapeutic agent should be used cautiously and the effectiveness for controlling allergic asthma needs further evaluation and research.

Topics: Animals; Arginase; Asthma; Bronchoalveolar Lavage Fluid; Cell Differentiation; Disease Models, Animal; Down-Regulation; Eosinophils; Female; Hypersensitivity; Immunoglobulin E; Intercellular Signaling Peptides and Proteins; Lung; Metaplasia; Mice, Inbred C57BL; Mucus; Ovalbumin; Pyrrolidonecarboxylic Acid; Respiratory Tract Infections; Th2 Cells; Thiazolidines

Allergic asthma is a chronic inflammatory disease of the conducting airways characterized by the presence of allergen-specific IgE, Th2 cytokine production, eosinophilic airway inflammation, bronchial hyperreactivity, mucus overproduction, and structural changes in the airways. Investigators have tried to mimic these features of human allergic asthma in murine models. Whereas the surrogate allergen ovalbumin has been extremely valuable for unravelling underlying mechanisms of the disease, murine asthma models depend nowadays on naturally occurring allergens, such as house dust mite (HDM), cockroach, and Alternaria alternata. Here we describe a physiologically relevant model of acute allergic asthma based on sensitization and challenge with HDM extracts, and compare it with the ovalbumin/alum-induced asthma model. Moreover, we propose a detailed readout of the asthma phenotype, determining the degree of eosinophilia in bronchoalveolar lavage fluids by flow cytometry, visualizing goblet cell metaplasia, and measuring Th cytokine production by lung-draining mediastinal lymph node cells restimulated with HDM. © 2016 by John Wiley & Sons, Inc.

Topics: Acute Disease; Alum Compounds; Animals; Asthma; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Eosinophilia; Flow Cytometry; Goblet Cells; Humans; Metaplasia; Mice; Ovalbumin; Pyroglyphidae; Th2 Cells

The catalytical isoforms p110γ and p110δ of phosphatidylinositide 3-kinase γ (PI3Kγ) and PI3Kδ play an important role in the pathogenesis of asthma. Two key elements in allergic asthma are increased levels of eosinophils and IgE. Dual pharmacological inhibition of p110γ and p110δ reduces asthma-associated eosinophilic lung infiltration and ameliorates disease symptoms, whereas the absence of enzymatic activity in p110γKOδD910A mice increases IgE and basal eosinophil counts. This suggests that long-term inhibition of p110γ and p110δ might exacerbate asthma. Here, we analysed mice genetically deficient for both catalytical subunits (p110γ/δ-/-) and determined basal IgE and eosinophil levels and the immune response to ovalbumin-induced asthma. Serum concentrations of IgE, IL-5 and eosinophil numbers were significantly increased in p110γ/δ-/- mice compared to single knock-out and wildtype mice. However, p110γ/δ-/- mice were protected against OVA-induced infiltration of eosinophils, neutrophils, T and B cells into lung tissue and bronchoalveolar space. Moreover, p110γ/δ-/- mice, but not single knock-out mice, showed a reduced bronchial hyperresponsiveness. We conclude that increased levels of eosinophils and IgE in p110γ/δ-/- mice do not abolish the protective effect of p110γ/δ-deficiency against OVA-induced allergic airway inflammation.

Topics: Animals; B-Lymphocytes; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cell Count; Class I Phosphatidylinositol 3-Kinases; Eosinophilia; Eosinophils; Goblet Cells; Hypersensitivity; Immunoglobulin E; Interleukin-5; Lung; Metaplasia; Mice, Inbred C57BL; Neutrophils; Ovalbumin; Pneumonia; T-Lymphocytes

This study aimed to explore the effects of curcumin on experimental allergic rhinitis in rats.. Twenty-eight male Wistar albino rats were randomly divided into four groups: a control group; a group in which allergic rhinitis was induced and no treatment given; a group in which allergic rhinitis was induced followed by treatment with azelastine hydrochloride on days 21-28; and a group in which allergic rhinitis was induced followed by treatment with curcumin on days 21-28. Allergy symptoms and histopathological features of the nasal mucosa were examined.. The sneezing and nasal congestion scores were higher in the azelastine and curcumin treatment groups than in the control group. Histopathological examination showed focal goblet cell metaplasia on the epithelial surface in the azelastine group. In the curcumin group, there was a decrease in goblet cell metaplasia in the epithelium, decreased inflammatory cell infiltration and vascular proliferation in the lamina propria.. Curcumin is an effective treatment for experimentally induced allergic rhinitis in rats.

Topics: Administration, Intranasal; Animals; Anti-Allergic Agents; Anti-Inflammatory Agents, Non-Steroidal; Chondrocytes; Cilia; Curcumin; Eosinophils; Goblet Cells; Hyperemia; Hypertrophy; Male; Mast Cells; Metaplasia; Nasal Mucosa; Ovalbumin; Phthalazines; Random Allocation; Rats; Rats, Wistar; Rhinitis, Allergic; Sneezing

Human studies demonstrated that allergen-specific immunotherapy (IT) represents an effective treatment for allergic diseases. IT involves repeated administration of the sensitizing allergen, indicating a crucial contribution of T cells to its medicinal benefit. However, the underlying mechanisms of IT, especially in a chronic disease, are far from being definitive. In the current study, we sought to elucidate the suppressive mechanisms of IT in a mouse model of chronic allergic asthma. OVA-sensitized mice were challenged with OVA or PBS for 4 wk. After development of chronic airway inflammation, mice received OVA-specific IT or placebo alternately to airway challenge for 3 wk. To analyze the T cell-mediated mechanisms underlying IT in vivo, we elaborated the role of T-bet-expressing Th1 cells, T cell-derived IL-10, and Ag-specific thymic as well as peripherally induced Foxp3(+) regulatory T (Treg) cells. IT ameliorated airway hyperresponsiveness and airway inflammation in a chronic asthma model. Of note, IT even resulted in a regression of structural changes in the airways following chronic inhaled allergen exposure. Concomitantly, IT induced Th1 cells, Foxp3(+), and IL-10-producing Treg cells. Detailed analyses revealed that thymic Treg cells crucially contribute to the effectiveness of IT by promoting IL-10 production in Foxp3-negative T cells. Together with the peripherally induced Ag-specific Foxp3(+) Treg cells, thymic Foxp3(+) Treg cells orchestrate the curative mechanisms of IT. Taken together, we demonstrate that IT is effective in a chronic allergic disease and dependent on IL-10 and thymic as well as peripherally induced Ag-specific Treg cells.

Topics: Airway Remodeling; Animals; Asthma; CD4-Positive T-Lymphocytes; Desensitization, Immunologic; Disease Models, Animal; Female; Goblet Cells; Immunoglobulin E; Immunomodulation; Immunophenotyping; Interleukin-10; Lung; Metaplasia; Mice; Mice, Knockout; Ovalbumin; Respiratory Hypersensitivity; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory

Surfactant protein D (SP-D) is a pulmonary collectin important in lung immunity. SP-D-deficient mice (Sftpd(-/-)) are reported to be susceptible to ovalbumin (OVA)- and fungal allergen-induced pulmonary inflammation, while treatment with exogenous SP-D has therapeutic effects in such disease models. β-Glucans are a diverse group of polysaccharides previously suggested to serve as fungal ligands for SP-D. We set out to investigate if SP-D could interact with 1,3-β-glucan and attenuate allergic pulmonary inflammation in the presence of 1,3-β-glucan. Allergic airway disease was induced in Sftpd(-/-) and Sftpd(+/+) mice by OVA sensitization and subsequent challenge with OVA, 1,3-β-glucan, or OVA/1,3-β-glucan together. Mice in the combined treatment group were further treated with a high dose of recombinant fragment of human SP-D (rfhSP-D). We demonstrated direct interaction between SP-D and 1,3-β-glucan. OVA-induced mucous cell metaplasia was increased in Sftpd(-/-) mice, supporting previously reported protective effects of endogenous SP-D in allergy. OVA-induced parenchymal CCL11 levels and eosinophilic infiltration in bronchoalveolar lavage were unaffected by 1,3-β-glucan, but were reversed with rfhSP-D treatment. 1,3-β-Glucan treatment did, however, induce pulmonary neutrophilic infiltration and increased TNF-α levels in bronchoalveolar lavage, independently of OVA-induced allergy. This infiltration was also reversed by treatment with rfhSP-D. 1,3-β-Glucan reduced OVA-induced mucous cell metaplasia, T helper 2 cytokines, and IFN-γ production. rfhSP-D treatment further reduced mucous metaplasia and T helper 2 cytokine secretion to background levels. In summary, rfhSP-D treatment resulted in attenuation of both allergic inflammation and 1,3-β-glucan-mediated neutrophilic inflammation. Our data suggest that treatment with high-dose SP-D protects from mold-induced exacerbations of allergic asthma.

Topics: Animals; beta-Glucans; Chemokine CCL11; Cytokines; Female; Humans; Hypersensitivity; Immunoglobulin E; Inflammation; Ligands; Metaplasia; Mice, Inbred C57BL; Microbiota; Ovalbumin; Protective Agents; Proteoglycans; Pulmonary Alveoli; Pulmonary Surfactant-Associated Protein D; Respiratory Hypersensitivity

Asthma is a chronic obstructive airway disease, characterized by inflammation and remodeling. Acetylcholine contributes to symptoms by inducing bronchoconstriction via the muscarinic M3 receptor. Recent evidence suggests that bronchoconstriction can regulate airway remodeling, and therefore implies a role for the muscarinic M3 receptor. The objective of this work was to study the contribution of the muscarinic M3 receptor to allergen-induced remodeling using muscarinic M3 receptor subtype-deficient (M3R(-/-)) mice. Wild-type (WT), M1R(-/-), and M2R(-/-) mice were used as controls. C57Bl/6 mice were sensitized and challenged with ovalbumin (twice weekly for 4 wk). Control animals were challenged with saline. Allergen exposure induced goblet cell metaplasia, airway smooth muscle thickening (1.7-fold), pulmonary vascular smooth muscle remodeling (1.5-fold), and deposition of collagen I (1.7-fold) and fibronectin (1.6-fold) in the airway wall of WT mice. These effects were absent or markedly lower in M3R(-/-) mice (30-100%), whereas M1R(-/-) and M2R(-/-) mice responded similarly to WT mice. In addition, airway smooth muscle and pulmonary vascular smooth muscle mass were 35-40% lower in saline-challenged M3R(-/-) mice compared with WT mice. Interestingly, allergen-induced airway inflammation, assessed as infiltrated eosinophils and T helper type 2 cytokine expression, was similar or even enhanced in M3R(-/-) mice. Our data indicate that acetylcholine contributes to allergen-induced remodeling and smooth muscle mass via the muscarinic M3 receptor, and not via M1 or M2 receptors. No stimulatory role for muscarinic M3 receptors in allergic inflammation was observed, suggesting that the role of acetylcholine in remodeling is independent of the allergic inflammatory response, and may involve bronchoconstriction.

Topics: Acetylcholine; Airway Remodeling; Allergens; Animals; Cytokines; Disease Models, Animal; Eosinophils; Extracellular Matrix; Female; Goblet Cells; Inflammation Mediators; Lung; Metaplasia; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth; Muscle, Smooth, Vascular; Ovalbumin; Pneumonia; Pulmonary Artery; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3

Gata5 is a transcription factor expressed in the lung, but its physiological role is unknown. To test whether and how Gata5 regulates airway constrictor responsiveness, we studied Gata5(-/-), Gata5(+/-), and wild-type mice on the C57BL/6J background. Cholinergic airway constrictor responsiveness was assessed invasively in mice without and with induction of allergic airway inflammation through ovalbumin sensitization and aerosol exposure. Gata5-deficient mice displayed native airway constrictor hyperresponsiveness (AHR) in the absence of allergen-induced inflammation. Gata5-deficient mice retained their relatively greater constrictor responsiveness even in ovalbumin-induced experimental asthma. Gata5 deficiency did not alter the distribution of cell types in bronchoalveolar lavage fluid, but bronchial epithelial mucus metaplasia was more prominent in Gata5(-/-) mice after allergen challenge. Gene expression profiles revealed that apolipoprotein E (apoE) was the fifth most down-regulated transcript in Gata5-deficient lungs, and quantitative RT-PCR and immunostaining confirmed reduced apoE expression in Gata5(-/-) mice. Quantitative RT-PCR also revealed increased IL-13 mRNA in the lungs of Gata5-deficient mice. These findings for the first time show that Gata5 regulates apoE and IL-13 expression in vivo and that its deletion causes AHR. Gata5-deficient mice exhibit an airway phenotype that closely resembles that previously reported for apoE(-/-) mice: both exhibit cholinergic AHR in native and experimental asthma states, and there is excessive goblet cell metaplasia after allergen sensitization and challenge. The Gata5-deficient phenotype also shares features that were previously reported for IL-13-treated mice. Together, these results indicate that Gata5 deficiency induces AHR, at least in part, by blunting apoE and increasing IL-13 expression.

Topics: Animals; Apolipoproteins E; Asthma; Bronchial Hyperreactivity; Bronchoconstriction; Disease Models, Animal; GATA5 Transcription Factor; Gene Expression Regulation; Genotype; Goblet Cells; Interleukin-13; Lung; Metaplasia; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocytes, Smooth Muscle; Ovalbumin; Phenotype; Pneumonia

Oral administration of bacterial extracts (eg, Broncho-Vaxom (BV)) has been proposed to attenuate asthma through modulating Treg cells. However, the underlying mechanism has not been fully characterized. This study sought to assess the effects of oral administration of BV on GSK-3β expression and Treg cells in ovalbumin (OVA)-induced asthmatic mice models.. Asthmatic mice models were established with OVA challenge and treated with oral administration of BV. Next, infiltration of inflammatory cells including eosinophil and neutrophils, mucous metaplasia, levels of Th1/Th2/Treg-typed cytokines and expression of GSK3β and Foxp3 were examined in asthmatic mice models by histological analysis, Bio-Plex and western blot, respectively. Moreover, the frequencies of Treg cells were evaluated in cultured splenocytes by flow cytometry in the presence of BV or GSK3β siRNA interference.. We found significant decrease of infiltrated inflammatory cells in bronchoalveolar lavage fluid (BALF) in asthmatic mice models after oral administration of BV. Oral administration of BV was shown to significantly suppress mucus metaplasia, Th2-typed cytokine levels and GSK3β expression while increasing Foxp3 production in asthmatic mice models. Moreover, BV significantly enhanced GSK3β-related expansion of Treg cells in cultured spleen cells in vitro.. Our findings provide evidence that oral administration of BV is capable of attenuating airway inflammation in asthmatic mice models, which may be associated with GSK3β-related expansion of Treg cells.

Topics: Administration, Oral; Animals; Asthma; Cell Extracts; Cytokines; Forkhead Transcription Factors; Gene Expression Regulation, Enzymologic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Male; Metaplasia; Mice; Mice, Inbred BALB C; Mucous Membrane; Ovalbumin; T-Lymphocytes, Regulatory

Epidemiologic studies suggest that increased concentrations of airborne spores of Aspergillus fumigatus closely relate to asthma aggravation. Chronic exposure to A. fumigatus aggravates airway inflammation, remodeling, and airway hyperresponsiveness in asthmatic rats. The effects of chronic exposure to A. fumigatus on epidermal growth factor receptor (EGFR) expression in the airway epithelial cells of asthmatic rats remain unclear. This study aimed to investigate the effects of chronic exposure to A. fumigatus on injury and shedding of airway epithelium, goblet cell metaplasia, and EGFR expression in the airway epithelial cells of asthmatic rats. A rat model of chronic asthma was established using ovalbumin (OVA) sensitization and challenge. Rats with chronic asthma were then exposed to long-term inhalation of spores of A. fumigatus, and the dynamic changes in injury and shedding of airway epithelium, goblet cell metaplasia, and EGFR expression were observed and analyzed. Chronic exposure to A. fumigatus could aggravate airway epithelial cell damage, upregulate the expression of EGFR and its ligands EGF and TGF-α, promote goblet cell metaplasia, and increase airway responsiveness in rats with asthma. Chronic exposure to A. fumigatus upregulates the expression of EGFR and its ligands in asthmatic rats. The EGFR pathway may play a role in asthma aggravation induced by exposure to A. fumigatus.

Topics: Animals; Aspergillosis; Aspergillus fumigatus; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Goblet Cells; Male; Metaplasia; Ovalbumin; Pneumonia; Rats; Rats, Wistar

The peptide hormone relaxin plays a key role in the systemic hemodynamic and renovascular adaptive changes that occur during pregnancy, which is linked to its antiremodelling effects. Serelaxin (a recombinant form of human gene-2 relaxin) has been shown to inhibit lung fibrosis in various disease models and reverse airway remodelling and airway hyperresponsiveness (AHR) in allergic airways disease (AAD).. Although continuous systemic delivery of exogenous serelaxin alleviates allergic fibrosis and AHR, more direct routes for administration into the lung have not been investigated. Thus, intranasal administration of serelaxin was evaluated for its ability to reverse airway remodelling and AHR associated with AAD.. Female Balb/c mice were subjected to a 9-week model of chronic AAD. Subgroups of animals (n = 12/group) were then treated intranasally with serelaxin (0.8 mg/mL) or vehicle once daily for 14 days (from weeks 9-11). Saline-sensitized/challenged mice treated with intranasal saline served as additional controls. Differential bronchoalveolar lavage (BAL) cell counts, ovalbumin (OVA)-specific IgE levels, tissue inflammation, parameters of airway remodelling and AHR were then assessed.. Chronic AAD was associated with significant increases in differential BAL cell counts, OVA-specific IgE levels, inflammation, epithelial thickening, goblet cell metaplasia, TGF-β1 expression, epithelial Smad2 phosphorylation (pSmad2), subepithelial collagen thickness, total lung collagen concentration and AHR (all P < 0.05 vs. respective measurements from saline-treated mice). Daily intranasal delivery of serelaxin significantly diminished AAD-induced epithelial thickening, epithelial pSmad2, subepithelial and total lung collagen content (fibrosis) and AHR (all P < 0.05 vs. vehicle-treated AAD mice).. Intranasal delivery of serelaxin can effectively reduce airway remodelling and AHR, when administered once daily. Respirable preparations of serelaxin may have therapeutic potential for the prevention and/or reversal of established airway remodelling and AHR in asthma.

Topics: Administration, Intranasal; Airway Remodeling; Animals; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Female; Fibrosis; Goblet Cells; Immunoglobulin E; Lung; Metaplasia; Mice; Ovalbumin; Recombinant Proteins; Relaxin; Respiratory Hypersensitivity; Respiratory Mucosa

Asthma develops from injury to the airways/lungs, stemming from airway inflammation (AI) and airway remodeling (AWR), both contributing to airway hyperresponsiveness (AHR). Airway epithelial damage has been identified as a new etiology of asthma but is not targeted by current treatments. Furthermore, it is poorly studied in currently used animal models of AI and AWR. Therefore, this study aimed to incorporate epithelial damage/repair with the well-established ovalbumin (OVA)-induced model of chronic allergic airway disease (AAD), which presents with AI, AWR, and AHR, mimicking several features of human asthma. A 3-day naphthalene (NA)-induced model of epithelial damage/repair was superimposed onto the 9-week OVA-induced model of chronic AAD, before 6 weeks of OVA nebulization (NA+OVA group), during the second last OVA nebulization period (OVA/NA group) or 1 day after the 6-week OVA nebulization period (OVA+NA group), using 6-8-week-old female Balb/c mice (n=6-12/group). Mice subjected to the 9-week OVA model, 3-day NA model or respective vehicle treatments (saline and corn oil) were used as appropriate controls. OVA alone significantly increased epithelial thickness and apoptosis, goblet cell metaplasia, TGF-β1, subepithelial collagen (assessed by morphometric analyses of various histological stains), total lung collagen (hydroxyproline analysis), and AHR (invasive plethysmography) compared with that in saline-treated mice (all P<0.05 vs saline treatment). NA alone caused a significant increase in epithelial denudation and apoptosis, TGF-β1, subepithelial, and total lung collagen compared with respective measurements from corn oil-treated controls (all P<0.01 vs corn oil treatment). All three combined models underwent varying degrees of epithelial damage and AWR, with the OVA+NA model demonstrating the greatest increase in subepithelial/total lung collagen and AHR (all P<0.05 vs OVA alone or NA alone). These combined models of airway epithelial damage/AAD demonstrated that epithelial damage is a key contributor to AWR, fibrosis and related AHR, and augments the effects of AI on these parameters.

Topics: Animals; Apoptosis; Asthma; Bronchi; Bronchial Hyperreactivity; Collagen; Disease Models, Animal; Epithelium; Female; Fibrosis; Lung; Metaplasia; Mice; Mice, Inbred BALB C; Naphthalenes; Ovalbumin

Long-term and unresolved airway inflammation and airway remodeling, characteristic features of chronic asthma, if not treated could lead to permanent structural changes in the airways. Aldose reductase (AR), an aldo-sugar and lipid aldehyde metabolizing enzyme, mediates allergen-induced airway inflammation in mice, but its role in the airway remodeling is not known. In the present study, we have examined the role of AR on airway remodeling using ovalbumin (OVA)-induced chronic asthma mouse model and cultured human primary airway epithelial cells (SAECs) and mouse lung fibroblasts (mLFs).. Airway remodeling in chronic asthma model was established in mice sensitized and challenged twice a week with OVA for 6 weeks. AR inhibitor, fidarestat, was administered orally in drinking water after first challenge. Inflammatory cells infiltration in the lungs and goblet cell metaplasia, airway thickening, collagen deposition and airway hyper-responsiveness (AHR) in response to increasing doses of methacholine were assessed. The TGFβ1-induced epithelial-mesenchymal transition (EMT) in SAECs and changes in mLFs were examined to investigate AR-mediated molecular mechanism(s) of airway remodeling.. In the OVA-exposed mice for 6 wks inflammatory cells infiltration, levels of inflammatory cytokines and chemokines, goblet cell metaplasia, collagen deposition and AHR were significantly decreased by treatment with AR inhibitor, fidarestat. Further, inhibition of AR prevented TGFβ1-induced altered expression of E-cadherin, Vimentin, Occludin, and MMP-2 in SAECs, and alpha-smooth muscle actin and fibronectin in mLFs. Further, in SAECs, AR inhibition prevented TGFβ1- induced activation of PI3K/AKT/GSK3β pathway but not the phosphorylation of Smad2/3.. Our results demonstrate that allergen-induced airway remodeling is mediated by AR and its inhibition blocks the progression of remodeling via inhibiting TGFβ1-induced Smad-independent and PI3K/AKT/GSK3β-dependent pathway.

Topics: Airway Remodeling; Aldehyde Reductase; Animals; Asthma; Biomarkers; Chronic Disease; Disease Models, Animal; Enzyme Inhibitors; Epithelial Cells; Epithelial-Mesenchymal Transition; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Hypersensitivity; Imidazolidines; Inflammation; Lung; Metaplasia; Mice; Mucus; Ovalbumin; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Smad Proteins; Transforming Growth Factor beta1

The emergence of nanotechnology has produced a multitude of engineered nanomaterials such as carbon nanotubes (CNTs), and concerns have been raised about their effects on human health, especially for susceptible populations such as individuals with asthma. Multiwalled CNTs (MWCNTs) have been shown to exacerbate ovalbumin (OVA)-induced airway remodeling in mice. Moreover, cyclooxygenase-2 (COX-2) has been described as a protective factor in asthma. We postulated that COX-2-deficient (COX-2(-/-)) mice would be susceptible to MWCNT-induced exacerbations of allergen-induced airway remodeling, including airway inflammation, fibrosis, and mucus-cell metaplasia (i.e., the formation of goblet cells). Wild-type (WT) or COX-2(-/-) mice were sensitized to OVA to induce allergic airway inflammation before a single dose of MWCNTs (4 mg/kg) delivered to the lungs by oropharyngeal aspiration. MWCNTs significantly increased OVA-induced lung inflammation and mucus-cell metaplasia in COX-2(-/-) mice compared with WT mice. However, airway fibrosis after exposure to allergen and MWCNTs was no different between WT and COX-2(-/-) mice. Concentrations of certain prostanoids (prostaglandin D2 and thromboxane B2) were enhanced by OVA or MWCNTs in COX-2(-/-) mice. No differences in COX-1 mRNA concentrations were evident between WT and COX-2(-/-) mice treated with OVA and MWCNTs. Interestingly, MWCNTs significantly enhanced allergen-induced cytokines involved in Th2 (IL-13 and IL-5), Th1 (CXCL10), and Th17 (IL-17A) inflammatory responses in COX-2(-/-) mice, but not in WT mice. We conclude that exacerbations of allergen-induced airway inflammation and mucus-cell metaplasia by MWCNTs are enhanced by deficiencies in COX-2, and are associated with the activation of a mixed Th1/Th2/Th17 immune response.

Topics: Airway Remodeling; Allergens; Animals; Cyclooxygenase 1; Cyclooxygenase 2; Cytokines; Female; Inflammation; Male; Membrane Proteins; Metaplasia; Mice; Mice, Inbred C57BL; Mucus; Nanotubes, Carbon; Ovalbumin; T-Lymphocytes, Helper-Inducer

Exposure to irritants such as tobacco smoke (TS) causes acute airway inflammation. Chronic exposure may cause airway remodeling contributing to enhanced airway resistance. We hypothesize that combining airway sensitization and inhalation of irritants enhances the number of mucous producing cells beyond either agent alone. Guinea pigs were antigen sensitized or treated with its vehicle. These two groups were further divided into daily exposure to TS or air. After 3 months airway reactivity to ovalbumin (OA) was determined, airway and blood samples were examined and lung substance P quantified. Combining sensitization and TS exposure increased airway reactivity to OA, goblet cell and submucosal gland populations. Airway eosinophilia was greatest in the OA-sensitized group exposed to air rather than with its combination with TS exposure. Lung substance P levels were similarly elevated in both OA-sensitized groups. Airway irritant exposure in which airway sensitization exists enhances the potential of mucus production, airway resistance and mucus plugging of the airways through increasing the number of goblet cells and submucosal glands.

Topics: Administration, Inhalation; Airway Remodeling; Airway Resistance; Aluminum Hydroxide; Animals; Exocrine Glands; Goblet Cells; Guinea Pigs; Leukocytes; Metaplasia; Ovalbumin; Respiratory Hypersensitivity; Smoking; Time Factors

Mucous cell metaplasia is a hallmark of airway diseases, such as asthma and chronic obstructive pulmonary disease. The majority of human airway epithelium is pseudostratified, but the cell of origin of mucous cells has not been definitively established in this type of airway epithelium. There is evidence that ciliated, club cell (Clara), and basal cells can all give rise to mucus-producing cells in different contexts. Because pseudostratified airway epithelium contains distinct progenitor cells from simple columnar airway epithelium, the lineage relationships of progenitor cells to mucous cells may be different in these two epithelial types. We therefore performed lineage tracing of the ciliated cells of the murine basal cell-containing airway epithelium in conjunction with the ovalbumin (OVA)-induced murine model of allergic lung disease. We genetically labeled ciliated cells with enhanced Yellow Fluorescent Protein (eYFP) before the allergen challenge, and followed the fate of these cells to determine whether they gave rise to newly formed mucous cells. Although ciliated cells increased in number after the OVA challenge, the newly formed mucous cells were not labeled with the eYFP lineage tag. Even small numbers of labeled mucous cells could not be detected, implying that ciliated cells make virtually no contribution to the new goblet cell pool. This demonstrates that, after OVA challenge, new mucous cells do not originate from ciliated cells in a pseudostratified basal cell-containing airway epithelium.

Topics: Allergens; Animals; Asthma; Cell Proliferation; Cells, Cultured; Epithelial Cells; Goblet Cells; Hyperplasia; Male; Metaplasia; Mice; Mice, Inbred C57BL; Ovalbumin; Respiratory Mucosa; Stem Cells

Mucous cell metaplasia is a hallmark of asthma, and may be mediated by signal transducers and activators of transcription (STAT)-6 signaling. IL-17A is increased in the bronchoalveolar lavage fluid of patients with severe asthma, and IL-17A also increases mucus production in airway epithelial cells. Asthma therapeutics are being developed that inhibit STAT6 signaling, but the role of IL-17A in inducing mucus production in the absence of STAT6 remains unknown. We hypothesized that IL-17A induces mucous cell metaplasia independent of STAT6, and we tested this hypothesis in two murine models in which increased IL-17A protein expression is evident. In the first model, ovalbumin (OVA)-specific D011.10 Th17 cells were adoptively transferred into wild-type (WT) or STAT6 knockout (KO) mice, and the mice were challenged with OVA or PBS. WT-OVA and STAT6 KO-OVA mice demonstrated increased airway IL-17A and IL-13 protein expression and mucous cell metaplasia, compared with WT-PBS or STAT6 KO-PBS mice. In the second model, WT, STAT1 KO, STAT1/STAT6 double KO (DKO), or STAT1/STAT6/IL-17 receptor A (RA) triple KO (TKO) mice were challenged with respiratory syncytial virus (RSV) or mock viral preparation, and the mucous cells were assessed. STAT1 KO-RSV mice demonstrated increased airway mucous cell metaplasia compared with WT-RSV mice. STAT1 KO-RSV and STAT1/STAT6 DKO-RSV mice also demonstrated increased mucous cell metaplasia, compared with STAT1/STAT6/IL17RA TKO-RSV mice. We also treated primary murine tracheal epithelial cells (mTECs) from WT and STAT6 KO mice. STAT6 KO mTECs showed increased periodic acid-Schiff staining with IL-17A but not with IL-13. Thus, asthma therapies targeting STAT6 may increase IL-17A protein expression, without preventing IL-17A-induced mucus production.

Topics: Adoptive Transfer; Animals; Bronchoalveolar Lavage Fluid; Female; Interleukin-13; Interleukin-17; Lung; Metaplasia; Mice; Mice, Inbred BALB C; Mice, Knockout; Mucus; Neutrophils; Ovalbumin; Peptide Fragments; Receptors, Interleukin-17; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; STAT1 Transcription Factor; STAT6 Transcription Factor; Th17 Cells; Transcriptional Activation

Aqueous extract of Kalanchoe pinnata (Kp) have been found effective in models to reduce acute anaphylactic reactions. In the present study, we investigate the effect of Kp and the flavonoid quercetin (QE) and quercitrin (QI) on mast cell activation in vitro and in a model of allergic airway disease in vivo. Treatment with Kp and QE in vitro inhibited degranulation and cytokine production of bone marrow-derived mast cells following IgE/FcɛRI crosslinking, whereas treatment with QI had no effect. Similarly, in vivo treatment with Kp and QE decreased development of airway hyperresponsiveness, airway inflammation, goblet cell metaplasia and production of IL-5, IL-13 and TNF. In contrast, treatment with QI had no effect on these parameters. These findings demonstrate that treatment with Kp or QE is effective in treatment of allergic airway disease, providing new insights to the immunomodulatory functions of this plant.

Topics: Animals; Basophil Degranulation Test; Bronchial Hyperreactivity; Goblet Cells; Interleukin-13; Interleukin-5; Kalanchoe; Mast Cells; Metaplasia; Mice; Mice, Inbred BALB C; Ovalbumin; Phytotherapy; Plant Extracts; Quercetin; Tumor Necrosis Factor-alpha

There is very limited knowledge about the effects of alcohol on airway hyperresponsiveness and inflammation in asthma. Historical accounts of alcohol administration to patients with breathing problems suggest that alcohol may have bronchodilating properties. We hypothesized that alcohol exposure will alter airway hyperresponsiveness (AHR) and pulmonary inflammation in a mouse model of allergic asthma. To test this hypothesis, BALB/c mice were fed either 18% alcohol or water and then sensitized and challenged with ovalbumin (OVA). AHR was assessed by means of ventilation or barometric plethysmography and reported as either total lung resistance or enhanced pause, respectively. Airway inflammation was assessed by total and differential cell counts in bronchoalveolar lavage fluid (BALF), cytokine levels in BALF, lung histology, and serum immunoglobulin E (IgE) levels. Alcohol feeding significantly blocked methacholine-induced increases in AHR compared with water-fed controls. Alcohol feeding significantly reduced total cell numbers (64%) as well as the number of eosinophils (84%) recruited to the lungs of these mice. Modest changes in lung pathology were also observed. Alcohol exposure led to a reduction of IgE in the serum of the EtOH OVA mice. These data demonstrate that alcohol exposure blunts AHR and dampens allergic airway inflammation indices in allergic mice and suggest that there may be an important role for alcohol in the modulation of asthma. These data provide an in vivo basis for previous clinical observations in humans substantiating the bronchodilator properties of alcohol and for the first time demonstrates an alcohol-induced reduction of allergic inflammatory cells in a mouse model of allergic asthma.

Topics: Animals; Anti-Inflammatory Agents; Asthma; Bronchoalveolar Lavage Fluid; Bronchoconstriction; Bronchoconstrictor Agents; Calcitonin Gene-Related Peptide; Cell Count; Cell Line; Cytokines; Eosinophils; Ethanol; Goblet Cells; Immunoglobulin E; Inflammation; Lung; Male; Metaplasia; Methacholine Chloride; Mice; Mice, Inbred BALB C; Ovalbumin; PPAR gamma; Th2 Cells

The glucocorticoid receptor (GR) is a transcription factor able to support either target gene activation via direct binding to DNA or gene repression via interfering with the activity of various proinflammatory transcription factors. An improved therapeutic profile for combating chronic inflammatory diseases has been reported through selectively modulating the GR by only triggering its transrepression function. We have studied in this paper the activity of Compound A (CpdA), a dissociated GR modulator favoring GR monomer formation, in a predominantly Th2-driven asthma model. CpdA acted similarly to the glucocorticoid dexamethasone (DEX) in counteracting OVA-induced airway hyperresponsiveness, recruitment of eosinophils, dendritic cells, neutrophils, B and T cells, and macrophages in bronchoalveolar lavage fluid, lung Th2, Tc2, Th17, Tc17, and mast cell infiltration, collagen deposition, and goblet cell metaplasia. Both CpdA and DEX inhibited Th2 cytokine production in bronchoalveolar lavage as well as nuclear translocation of NF-κB and its subsequent recruitment onto the IκBα promoter in the lung. By contrast, DEX but not CpdA induces expression of the GR-dependent model gene MAPK phosphatase 1 in the lung, confirming the dissociative action of CpdA. Mechanistically, we demonstrate that CpdA inhibited IL-4-induced STAT6 translocation and that GR is essential for CpdA to mediate chemokine repression. In conclusion, we clearly show in this study the anti-inflammatory effect of CpdA in a Th2-driven asthma model in the absence of transactivation, suggesting a potential therapeutic benefit of this strategy.

Topics: Acetates; Animals; Anti-Asthmatic Agents; Anti-Inflammatory Agents, Non-Steroidal; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cytokines; Dexamethasone; Disease Models, Animal; Drug Evaluation, Preclinical; Dual Specificity Phosphatase 1; Enzyme Induction; Gene Expression Regulation; Goblet Cells; Inflammation; Leukocytes; Lung; Mast Cells; Metaplasia; Mice; Mice, Inbred BALB C; Ovalbumin; Quaternary Ammonium Compounds; Receptors, Glucocorticoid; STAT6 Transcription Factor; Transcriptional Activation; Tyramine

Airway remodeling in bronchial asthma results from chronic, persistent airway inflammation. The effects of the reversal of airway remodeling by drug interventions remain to be elucidated. We investigated the effects of ONO-1301, a novel prostacyclin agonist with thromboxane inhibitory activity, on the prevention and reversibility of airway remodeling in an experimental chronic asthma model. Mice sensitized and challenged to ovalbumin (OVA) three times a week for 5 consecutive weeks were administered ONO-1301 or vehicle twice a day from the fourth week of OVA challenges. Twenty-four hours after the final OVA challenge, airway hyperresponsiveness (AHR) was assessed, and bronchoalveolar lavage was performed. Lung specimens were excised for staining to detect goblet-cell metaplasia, airway smooth muscle, and submucosal fibrosis. Mice administered ONO-1301 showed limited increases in AHR compared with mice administered the vehicle. The histological findings of airway remodeling were improved in ONO-1301-treated mice compared with vehicle-treated mice. Presumably, these therapeutic effects of ONO-1301 are attributable to the up-regulation of production of hepatocyte growth factor (HGF) in lung tissue, because the neutralization of HGF by antibodies prevented the effects of ONO-1301 on AHR and airway remodeling. Mice administered ONO-1301 showed similar levels of AHR and airway remodeling as mice administered montelukast, a cysteinyl-leukotriene-1 receptor antagonist, and lower levels were observed in mice administered dexamethasone. These data suggest that ONO-1301 exerts the effect of reversing airway remodeling, at least in part through an elevation of HGF in the lungs, and may be effective as an anti-remodeling drug in the treatment of asthma.

Topics: Acetates; Airway Remodeling; Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cyclopropanes; Dexamethasone; Epoprostenol; Female; Goblet Cells; Hepatocyte Growth Factor; Inflammation; Lung; Metaplasia; Mice; Mice, Inbred BALB C; Muscle, Smooth; Ovalbumin; Pulmonary Fibrosis; Pyridines; Quinolines; Receptors, Leukotriene; Sulfides; Thromboxanes; Up-Regulation

Asthma is an atopic disorder with increasing frequency and severity in developed nations. Interleukin-13 (IL-13) is one of the most critical mediators of asthma pathology. In the present study, we developed a vaccine comprised of a keyhole limpet hemocyanin-mIL-13 heterocomplex immunogen to persistently neutralize excessive endogenous IL-13. Our results showed that the IL-13 peptide kinoid vaccine could induce sustained and high titer of IL-13-specific IgG when using aluminum hydroxide as an adjuvant, and could suppress the accumulation of eosinophils as well as IL-13 levels in bronchoalveolar lavage fluid (BALF). In addition, total IgE and ovalbumin (OVA)-specific IgE in serum were significantly inhibited. This study also showed that vaccination could prevent airway inflammation and epithelial cell proliferation with goblet cell hyperplasia in a mouse model of acute asthma. In summary, our findings suggest that the IL-13 peptide kinoid can serve as an innovative and effective vaccine against asthma.

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Eosinophils; Female; Goblet Cells; Immunoglobulin E; Immunoglobulin G; Inflammation; Interleukin-13; Metaplasia; Mice; Mice, Inbred BALB C; Ovalbumin; Peptides; Vaccines, Subunit

Protein-S-glutathionylation (PSSG) is an oxidative modification of reactive cysteines that has emerged as an important player in pathophysiological processes. Under physiological conditions, the thiol transferase, glutaredoxin-1 (Glrx1) catalyses deglutathionylation. Although we previously demonstrated that Glrx1 expression is increased in mice with allergic inflammation, the impact of Glrx1/PSSG in the development of allergic airways disease remains unknown. In the present study we examined the impact of genetic ablation of Glrx1 in the pathogenesis of allergic inflammation and airway hyperresponsiveness (AHR) in mice. Glrx1(-/-) or WT mice were subjected to the antigen, ovalbumin (OVA), and parameters of allergic airways disease were evaluated 48 h after three challenges, and 48 h or 7 days after six challenges with aerosolized antigen. Although no clear increases in PSSG were observed in WT mice in response to OVA, marked increases were detected in lung tissue of mice lacking Glrx1 48 h following six antigen challenges. Inflammation and expression of proinflammatory mediators were decreased in Glrx1(-/-) mice, dependent on the time of analysis. WT and Glrx1(-/-) mice demonstrated comparable increases in AHR 48 h after three or six challenges with OVA. However, 7 days postcessation of six challenges, parameters of AHR in Glrx1(-/-) mice were resolved to control levels, accompanied by marked decreases in mucus metaplasia and expression of Muc5AC and GOB5. These results demonstrate that the Glrx1/S-glutathionylation redox status in mice is a critical regulator of AHR, suggesting that avenues to increase S-glutathionylation of specific target proteins may be beneficial to attenuate AHR.

Topics: Animals; Bronchial Hyperreactivity; Glutaredoxins; Glutathione; Lung; Lung Diseases; Metaplasia; Mice; Mucus; Ovalbumin; Pneumonia; Proteins

Airway mucous cell metaplasia and chronic inflammation are pathophysiological features that influence morbidity and mortality associated with asthma and other chronic pulmonary disorders. Elucidation of the molecular mechanisms regulating mucous metaplasia and hypersecretion provides the scientific basis for diagnostic and therapeutic opportunities to improve the care of chronic pulmonary diseases.. To determine the role of the airway epithelial–specific transcription factor NK2 homeobox 1 (NKX2-1, also known as thyroid transcription factor-1 [TTF-1]) in mucous cell metaplasia and lung inflammation.. Expression of NKX2-1 in airway epithelial cells from patients with asthma was analyzed. NKX2-1 +/-gene targeted or transgenic mice expressing NKX2-1 in conducting airway epithelial cells were sensitized to the aeroallergen ovalbumin. In vitro studies were used to identify mechanisms by which NKX2-1 regulates mucous cell metaplasia and inflammation.. NKX2-1 was suppressed in airway epithelial cells from patients with asthma. Reduced expression of NKX2-1 in heterozygous NKX2-1 +/- gene targeted mice increased mucous metaplasia in the small airways after pulmonary sensitization to ovalbumin. Conversely, mucous cell metaplasia induced by aeroallergen was inhibited by expression of NKX2-1 in the respiratory epithelium in vivo. Genome-wide mRNA analysis of lung tissue from ovalbumin-treated mice demonstrated that NKX2-1 inhibited mRNAs associated with mucous metaplasia and Th2-regulated inflammation,including Spdef, Ccl17, and Il13. In vitro, NKX2-1 inhibited SPDEF, a critical regulator of airway mucous cell metaplasia,and the Th2 chemokine CCL26.. The present data demonstrate a novel function for NKX2-1 in a gene network regulating mucous cell metaplasia and allergic inflammation in the respiratory epithelium.

Topics: Adolescent; Adult; Allergens; Animals; Asthma; Chemokine CCL17; Down-Regulation; Female; Gene Regulatory Networks; Hepatocyte Nuclear Factor 3-beta; Heterozygote; Humans; Immunization; In Vitro Techniques; Lung; Male; Metaplasia; Mice; Mice, Transgenic; Middle Aged; Nuclear Proteins; Ovalbumin; Pneumonia; Proto-Oncogene Proteins c-ets; Respiratory Mucosa; RNA, Messenger; Th2 Cells; Thyroid Nuclear Factor 1; Transcription Factors; Young Adult

Activin A is a member of the TGF-beta superfamily and plays a role in allergic inflammation and asthma pathogenesis. Recent evidence suggests that activin A regulates proinflammatory cytokine production and is regulated by inflammatory mediators. In a murine model of acute allergic airway inflammation, we observed previously that increased activin A concentrations in bronchoalveolar lavage (BAL) fluid coincide with Th2 cytokine production in lung-draining lymph nodes and pronounced mucus metaplasia in bronchial epithelium. We therefore hypothesized that IL-13, the key cytokine for mucus production, regulates activin A secretion into BAL fluid in experimental asthma. IL-13 increased BAL fluid activin A concentrations in naive mice and dose dependently induced activin A secretion from cultured human airway epithelium. A key role for IL-13 in the secretion of activin A into the BAL fluid during allergic airway inflammation was confirmed in IL-13-deficient mice. Eosinophils were not involved in this response because there was no difference in BAL fluid activin A concentrations between wild-type and eosinophil-deficient mice. Our data highlight an important role for IL-13 in the regulation of activin A intraepithelially and in BAL fluid in naive mice and during allergic airway inflammation. Given the immunomodulatory and fibrogenic effects of activin A, our findings suggest an important role for IL-13 regulation of activin A in asthma pathogenesis.

Topics: Activin Receptors; Activins; Animals; Asthma; Bronchoalveolar Lavage Fluid; Cells, Cultured; Disease Models, Animal; Eosinophils; Epithelial Cells; Female; Humans; Inhibin-beta Subunits; Interleukin-13; Interleukin-5; Metaplasia; Mice; Mice, Inbred BALB C; Mice, Knockout; Ovalbumin; Pneumonia; Recombinant Proteins; Respiratory Mucosa; Signal Transduction; Time Factors; Transforming Growth Factor beta

NF-kappaB activation in the airway epithelium has been established as a critical pathway in ovalbumin (Ova)-induced airway inflammation in BALB/c mice (Poynter ME, Cloots R, van Woerkom T, Butnor KJ, Vacek P, Taatjes DJ, Irvin CG, Janssen-Heininger YM. J Immunol 173: 7003-7009, 2004). BALB/c mice are susceptible to the development of allergic airway disease, whereas other strains of mice, such as C57BL/6, are considered more resistant. The goal of the present study was to determine the proximal signals required for NF-kappaB activation in the airway epithelium in allergic airway disease and to unravel whether these signals are strain-dependent. Our previous studies, conducted in the BALB/c mouse background, demonstrated that transgenic mice expressing a dominant-negative version of IkappaBalpha in the airway epithelium (CC10-IkappaBalpha(SR)) were protected from Ova-induced inflammation. In contrast to these earlier observations, we demonstrate here that CC10-IkappaBalpha(SR) transgenic mice on the C57BL/6 background were not protected from Ova-induced allergic airway inflammation. Consistent with this finding, Ova-induced nuclear localization of the RelA subunit of NF-kappaB was not observed in C57BL/6 mice, in contrast to the marked nuclear presence of RelA in BALB/c mice. Evaluation of cytokine profiles in bronchoalveolar lavage demonstrated elevated expression of TNF-alpha in BALB/c mice compared with C57BL/6 mice after an acute challenge with Ova. Finally, neutralization of TNF-alpha by a blocking antibody prevented nuclear localization of RelA in BALB/c mice after Ova challenge. These data suggest that the mechanism of response of the airway epithelium of immunized C57BL/6 mice to antigen challenge is fundamentally different from that of immunized BALB/c mice and highlight the potential importance of TNF-alpha in regulating epithelial NF-kappaB activation in allergic airway disease.

Topics: Animals; Antigens; Enzyme Activation; Epithelial Cells; Epithelium; Female; I-kappa B Kinase; I-kappa B Proteins; Metaplasia; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mucus; Neutralization Tests; NF-kappa B; NF-KappaB Inhibitor alpha; Ovalbumin; Pneumonia; Respiratory Hypersensitivity; Species Specificity; Trachea; Transgenes; Tumor Necrosis Factor-alpha; Uteroglobin

Natural killer T (NK T) cells have been shown to play an essential role in the development of allergen-induced airway hyperresponsiveness (AHR) and/or airway inflammation in mouse models of acute asthma. Recently, NK T cells have been reported to be required for the development of AHR in a virus induced chronic asthma model. We investigated whether NK T cells were required for the development of allergen-induced AHR, airway inflammation and airway remodelling in a mouse model of chronic asthma. CD1d-/- mice that lack NK T cells were used for the experiments. In the chronic model, AHR, eosinophilic inflammation, remodelling characteristics including mucus metaplasia, subepithelial fibrosis and increased mass of the airway smooth muscle, T helper type 2 (Th2) immune response and immunoglobulin (Ig)E production were equally increased in both CD1d-/- mice and wild-type mice. However, in the acute model, AHR, eosinophilic inflammation, Th2 immune response and IgE production were significantly decreased in the CD1d-/- mice compared to wild-type. CD1d-dependent NK T cells may not be required for the development of allergen-induced AHR, eosinophilic airway inflammation and airway remodelling in chronic asthma model, although they play a role in the development of AHR and eosinophilic inflammation in acute asthma model.

Topics: Acute Disease; Airway Remodeling; Airway Resistance; Allergens; Animals; Antigens, CD1d; Asthma; Bronchial Hyperreactivity; Bronchitis; Chronic Disease; Disease Models, Animal; Female; Fibrosis; Immunoglobulin E; Male; Metaplasia; Mice; Mice, Inbred BALB C; Mice, Knockout; Muscle, Smooth; Natural Killer T-Cells; Ovalbumin; Pulmonary Eosinophilia; Th2 Cells

Inflammation plays a key role in lung injury and in the pathogenesis of asthma. Two murine models of allergic airway inflammation-sensitization and challenge to ovalbumin (OVA) and intratracheal exposure to interleukin-13 (IL13)-were used to evaluate the expression of poly(ADP-ribose) polymerase-1 (PARP-1) in allergic airway inflammation. Inflammation is prominent in OVA-induced allergic asthma, but this inflammation is greatly reduced by a PARP-1 inhibitor and almost eliminated when PARP-1 knockout mice are subjected to the OVA model. The present study temporally evaluated PARP-1 protein expression, localization, and activity, as well as inflammation and goblet cell metaplasia (GCM), in murine lungs following a single OVA challenge or IL13 exposure. Following OVA challenge PARP-1 protein expression and activity were greatly increased, being maximal at 3 to 5 days following OVA exposure and beginning to decrease by day 8. These changes correlated with the timing and degree of inflammation and GCM. In contrast, PARP-1 protein or activity did not change following single IL13 exposure, though GCM was manifested without inflammation. This study demonstrates that both PARP-1 protein and activity are increased by allergen-activated inflammatory mediators, excluding IL13, and that PARP-1 increase does not appear necessary for GCM, one of the characteristic markers of allergic airway inflammation in murine models.

Topics: Allergens; Animals; Asthma; Disease Models, Animal; Goblet Cells; Interleukin-13; Lung; Male; Metaplasia; Mice; Mice, Inbred BALB C; Mice, Knockout; Ovalbumin; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases

Collagen deposition is observed in a diverse set of pulmonary diseases, and the unraveling of the molecular signaling pathways that facilitate collagen deposition represents an ongoing area of investigation. The stress-activated protein kinase, c-Jun N-terminal kinase 1 (JNK1), is activated by a large variety of cellular stresses and environmental insults. Recent work from our laboratory demonstrated the critical role of JNK1 in epithelial to mesenchymal transition. The goal of the present study was to examine the involvement of JNK1 in subepithelial collagen deposition in mice subjected to models of allergic airways disease and interstitial pulmonary fibrosis. Activation of JNK was slightly enhanced in lungs from mice subjected to sensitization and challenge with ovalbumin (Ova), and predominant localization of phospho-JNK was observed in the bronchial epithelium. While mice lacking JNK1 (JNK1-/- mice) displayed enhanced lung inflammation and cytokine production compared with wild-type (WT) mice, JNK1-/- mice accumulated less subepithelial collagen deposition in response to antigen, and showed decreased expression of profibrotic genes compared with WT animals. Furthermore, transforming growth factor (TGF)-beta1 content in the bronchoalveolar lavage was diminished in JNK1-/- mice compared with WT animals subjected to antigen. Finally, we demonstrated that mice lacking JNK1 were protected against TGF-beta1 and bleomycin-induced pro-fibrotic gene expression and pulmonary fibrosis. Collectively, these findings demonstrate an important requirement for JNK1 in promoting collagen deposition in multiple models of fibrosis.

Topics: Animals; Antigens; Bleomycin; Collagen; Enzyme Activation; Epithelium; Gene Expression Regulation; Immunization; Lung; Metaplasia; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 8; Mucus; Ovalbumin; Phosphorylation; Pulmonary Fibrosis; Transforming Growth Factor beta1

Asthma is a dynamic disorder of airway inflammation and airway remodeling with an imbalance in T helper type 1 (Th(1))/Th(2) immune response. Increased Th(2) cytokines such as IL-4 and IL-13 induce arginase either directly or indirectly through transforming growth factor-beta(1) (TGF-beta(1)) and lead to subepithelial fibrosis, which is a crucial component of airway remodeling. Synthetic antimalarials have been reported to have immunomodulatory properties. Mepacrine is known for its reduction of airway inflammation in short-term allergen challenge model by reducing Th(2) cytokines and cysteinyl leukotrienes, which has an important role in the development of airway remodeling features. Therefore, we hypothesized that mepacrine may reduce airway remodeling. For this, extended subacute ovalbumin mice model of asthma was developed; these mice showed an increased expression of profibrotic mediators, subepithelial fibrosis, and goblet cell metaplasia along with airway inflammation, increased Th(2) cytokines, allergen-specific IgE, IgG(1), increased cytosolic PLA(2) (cPLA(2)), and airway hyperresponsiveness. Presence of intraepithelial eosinophils and significant TGF-beta(1) expression in subepithelial mesenchymal regions by repeated allergen exposures indicate that asthmatic mice of this study have developed human mimicking as well as late stages of asthma. However, mepacrine treatment decreased Th(2) cytokines and subepithelial fibrosis and alleviated asthma features. These reductions by mepacrine were associated with a decrease in levels and expression of TGF-beta(1) and the reduction in activity, expression of arginase in lung cytosol, and immunolocalization in inflammatory cells present in perivascular and peribronchial regions. These results suggest that mepacrine might reduce the development of subepithelial fibrosis by reducing the arginase and TGF-beta(1). These effects of mepacrine likely underlie its antiairway remodeling action in asthma.

Topics: Animals; Arginase; Asthma; Bronchial Hyperreactivity; Disease Models, Animal; Epithelial Cells; Fibrosis; Goblet Cells; Hydroxyeicosatetraenoic Acids; Inflammation; Lung; Metaplasia; Mice; Mice, Inbred BALB C; Ovalbumin; Phospholipases A2, Cytosolic; Quinacrine; Transforming Growth Factor beta1

One of the major aspects of airway remodeling in asthma is the development of mucous cell metaplasia (MCM). The role of cytokines in the generation and resolution of MCM has been studied in mice and in isolated airway epithelial cells in culture. However, studies using organ cultures that keep the tubular structure of the airways intact and allow studies in the absence of inflammatory cells have not been reported. We established an organ culture system that replicates the allergen-induced MCM in mice and analyzed the role of Bax in the IFN-gamma-induced resolution of MCM. IL-9 or IL-13 induced MCM independently, but a combined IL-9/IL-13 treatment enhanced MCM synergistically. Addition of IFN-gamma at 0.1 ng/ml concentration further increased MCM to levels observed in allergen-exposed mice in vivo. However, MCM was reduced when explants were treated with 50 ng/ml IFN-gamma after MCM was established. While IL-9/IL-13 induced MCM in bronchioles microdissected from bax(+/+) and bax(-/-) mice to a similar extent, IFN-gamma treatment reduced MCM only in bronchioles from bax(+/+) but not in bax(-/-) bronchioles. Restoration of Bax expression in bax(-/-) bronchioles using an adenoviral expression system reduced IL-9/IL-13-induced MCM while MCM was similar in noninfected or adenoviral green fluorescent protein-infected bax(-/-) bronchioles. Furthermore, expressing Bax using an adenoviral expression system reduced allergen-induced MCM in mice. These studies show that allergen-induced MCM is a response to a combination of various cytokines at defined concentrations and that IFN-gamma requires Bax for the resolution of MCM.

Topics: Adenoviridae; Alcian Blue; Allergens; Animals; bcl-2-Associated X Protein; Bronchi; Coloring Agents; Dose-Response Relationship, Drug; Drug Interactions; Eosine Yellowish-(YS); Fluorescent Dyes; Goblet Cells; Green Fluorescent Proteins; Hemagglutinins; Hematoxylin; Immunohistochemistry; Interferon-gamma; Interleukin-13; Interleukin-9; Metaplasia; Mice; Mice, Inbred C57BL; Models, Biological; Organ Culture Techniques; Ovalbumin; Periodic Acid-Schiff Reaction; Time Factors

IL-13 is a critical cytokine at sites of Th2 inflammation. In these locations it mediates its effects via a receptor complex, which contains IL-4Ralpha and IL-13Ralpha1. A third, high-affinity IL-13 receptor, IL-13Ralpha2, also exists. Although it was initially felt to be a decoy receptor, this has not been formally demonstrated and the role(s) of this receptor has recently become controversial. To define the role(s) of IL-13Ralpha2 in IL-13-induced pulmonary inflammation and remodeling, we compared the effects of lung-targeted transgenic IL-13 in mice with wild-type and null IL-13Ralpha2 loci. We also investigated the effect of IL-13Ralpha2 deficiency on the OVA-induced inflammatory response. In this study, we show that in the absence of IL-13Ralpha2, IL-13-induced pulmonary inflammation, mucus metaplasia, subepithelial fibrosis, and airway remodeling are significantly augmented. These changes were accompanied by increased expression and production of chemokines, proteases, mucin genes, and TGF-beta1. Similarly, an enhanced inflammatory response was observed in an OVA-induced phenotype. In contrast, disruption of IL-13Ralpha2 had no effect on the tissue effects of lung-targeted transgenic IL-4. Thus, IL-13Ralpha2 is a selective and powerful inhibitor of IL-13-induced inflammatory, remodeling, and physiologic responses in the murine lung.

Topics: Animals; Fibrosis; Interleukin-13; Interleukin-13 Receptor alpha2 Subunit; Interleukin-4; Lung; Metaplasia; Mice; Mice, Mutant Strains; Mucus; Ovalbumin; Pneumonia

The Th2 cytokines IL-4 and IL-13 mediate allergic pulmonary inflammation and airways hyperreactivity (AHR) in asthma models through signaling dependent upon the IL-4 receptor-alpha chain (IL-4Ralpha). IL-13 has been further implicated in the overproduction of mucus by the airway epithelium and in lung remodeling that commonly accompanies chronic inflammation. IL-4Ralpha-deficient mice are resistant to allergen-induced asthma, highlighting the therapeutic promise of selective molecular inhibitors of IL-4Ralpha. We designed a chemically modified IL-4Ralpha antisense oligonucleotide (IL-4Ralpha ASO) that specifically inhibits IL-4Ralpha protein expression in lung eosinophils, macrophages, dendritic cells, and airway epithelium after inhalation in allergen-challenged mice. Inhalation of IL-4Ralpha ASO attenuated allergen-induced AHR, suppressed airway eosinophilia and neutrophilia, and inhibited production of airway Th2 cytokines and chemokines in previously allergen-primed and -challenged mice. Histologic analysis of lungs from these animals demonstrated reduced goblet cell metaplasia and mucus staining that correlated with inhibition of Muc5AC gene expression in lung tissue. Therapeutic administration of inhaled IL-4Ralpha ASO in chronically allergen-challenged mice produced a spectrum of anti-inflammatory activity similar to that of systemically administered Dexamethasone with the added benefit of reduced airway neutrophilia. These data support the potential utility of a dual IL-4 and IL-13 oligonucleotide inhibitor in allergy/asthma, and suggest that local inhibition of IL-4Ralpha in the lung is sufficient to suppress allergen-induced pulmonary inflammation and AHR.

Topics: Administration, Inhalation; Aerosols; Animals; Anti-Inflammatory Agents; Asthma; Bronchial Hyperreactivity; Bronchial Provocation Tests; Chemokines; Dendritic Cells; Disease Models, Animal; Eosinophils; Gene Expression Regulation; Goblet Cells; Inflammation; Lung; Macrophages, Alveolar; Male; Metaplasia; Mice; Mucins; Oligonucleotides, Antisense; Ovalbumin; Receptors, Cell Surface; Th2 Cells; Treatment Outcome

IL-18 is known to induce IFN-gamma production, which is enhanced when combined with IL-2. In the present study, we investigated whether the combination of exogenous IL-2 and IL-18 alters airway hyperresponsiveness (AHR) and airway inflammation. Sensitized mice exposed to ovalbumin (OVA) challenge developed AHR, inflammatory cells in the bronchoalveolar lavage (BAL) fluid, and increases in levels of Th2 cytokines and goblet cell numbers. The combination of IL-2 and IL-18, but neither alone, prevented these changes while increasing levels of IL-12 and IFN-gamma. The combination of IL-2 and IL-18 was ineffective in IFN-gamma-deficient and signal transducer and activator of transcription (STAT)4-deficient mice. Flow cytometry analysis showed significant increases in numbers of IFN-gamma-positive natural killer (NK) cells in the lung after treatment with the combination therapy, and transfer of lung NK cells isolated from sensitized and challenged mice treated with the combination significantly suppressed AHR and BAL eosinophilia. These data demonstrate that the combination of IL-2 and IL-18 prevents AHR and airway inflammation, likely through IL-12-mediated induction of IFN-gamma production in NK cells.

Topics: Adoptive Transfer; Animals; Cell Count; Drug Synergism; Female; Goblet Cells; Humans; Inflammation; Injections, Intraperitoneal; Interferon-gamma; Interleukin-18; Interleukin-2; Killer Cells, Natural; Lung; Male; Metaplasia; Mice; Mice, Inbred C57BL; Ovalbumin; Respiratory Hypersensitivity; STAT4 Transcription Factor

Epidemiological studies suggest that infections with helminths protect from the development of asthma. Supporting this view is our published finding that infection with Nippostrongylus brasiliensis decreased ovalbumin-induced Th2 responses in the lung of mice.. To evaluate if N. brasiliensis excretory-secretory products also prevent the development of asthma.. Mice were immunized with ovalbumin/alum intraperitoneally in the absence or presence of helminthic products and then challenged intranasally with ovalbumin. Six days later, we analyzed if the mice developed Th2 responses in the lung.. The application of the helminthic products together with ovalbumin/alum during the sensitization period totally inhibited the development of eosinophilia and goblet cell metaplasia in the airways and also strongly reduced the development of airway hyperreactivity. Allergen-specific IgG1 and IgE serum levels were also strongly reduced. These findings correlated with decreased levels of IL-4 and IL-5 in the airways in product-treated animals. The suppressive effects on the development of allergic responses were independent of the presence of Toll-like receptors 2 and 4, IFN-gamma, and most important, IL-10. Interestingly, suppression was still observed when the helminthic products were heated or treated with proteinase K. Paradoxically, we found that strong helminth product-specific Th2 responses were induced in parallel with the inhibition of ovalbumin-specific responses.. Our results suggest that helminths suppress the development of asthma by secreting substances that modulate allergic responses without affecting the generation of helminth-specific Th2 immunity. The identification of these products may lead to the design of novel therapeutic intervention strategies for the treatment of asthma.

Topics: Animals; Bronchial Provocation Tests; Endopeptidase K; Female; Goblet Cells; Helminthiasis, Animal; Immunoglobulin E; Immunoglobulin G; Metaplasia; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Nippostrongylus; Ovalbumin; Pulmonary Eosinophilia; Respiratory Hypersensitivity; Th2 Cells

Allergen sensitization and allergic airway disease are likely to come about through the inhalation of Ag with immunostimulatory molecules. However, environmental pollutants, including nitrogen dioxide (NO2), may promote adaptive immune responses to innocuous Ags that are not by themselves immunostimulatory. We tested in C57BL/6 mice whether exposure to NO2, followed by inhalation of the innocuous protein Ag, OVA, would result in allergen sensitization and the subsequent development of allergic airway disease. Following challenge with aerosolized OVA alone, mice previously exposed via inhalation to NO2 and OVA developed eosinophilic inflammation and mucus cell metaplasia in the lungs, as well as OVA-specific IgE and IgG1, and Th2-type cytokine responses. One hour of exposure to 10 parts per million NO2 increased bronchoalveolar lavage fluid levels of total protein, lactate dehydrogenase activity, and heat shock protein 70; promoted the activation of NF-kappaB by airway epithelial cells; and stimulated the subsequent allergic response to Ag challenge. Furthermore, features of allergic airway disease were not induced in allergen-challenged TLR2-/- and MyD88-/- mice exposed to NO2 and aerosolized OVA during sensitization. These findings offer a mechanism whereby allergen sensitization and asthma may result under conditions of high ambient or endogenous NO2 levels.

Topics: Administration, Inhalation; Aerosols; Allergens; Animals; Bronchial Hyperreactivity; Eosinophilia; Immunologic Factors; Lung; Metaplasia; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucus; Myeloid Differentiation Factor 88; Nitrogen Dioxide; Ovalbumin; Respiratory Hypersensitivity; Toll-Like Receptor 2

The effect of ageing on several pathologic features of allergic asthma (pulmonary inflammation, eosinophilia, mucus hypersecretion), and their relationship with airway hyperresponsiveness (AHR) is not well characterized.. To evaluate lung inflammation, mucus metaplasia and AHR in relationship with age in murine models of allergic asthma comparing young and older mice.. Young (6 weeks) and older (6, 12, 18 months) BALB/c mice were sensitized and challenged with ovalbumin (OVA). AHR and bronchoalveolar fluid (BALF), total inflammatory cell count and differential were measured. To evaluate mucus metaplasia, quantitative PCR for the major airway mucin-associated gene, MUC-5AC, from lung tissue was measured, and lung tissue sections stained with periodic acid-Schiff (PAS) for goblet-cell enumeration. Lung tissue cytokine gene expression was determined by quantitative PCR, and systemic cytokine protein levels by ELISA from spleen-cell cultures. Antigen-specific serum IgE was determined by ELISA.. AHR developed in both aged and young OVA-sensitized/challenged mice (OVA mice), and was more significantly increased in young OVA mice than in aged OVA mice. However, BALF eosinophil numbers were significantly higher, and lung histology showed greater inflammation in aged OVA mice than in young OVA mice. MUC-5AC expression and numbers of PAS+ staining bronchial epithelial cells were significantly increased in the aged OVA mice. All aged OVA mice had increased IL-5 and IFN-gamma mRNA expression in the lung and IL-5 and IFN-gamma protein levels from spleen cell cultures compared with young OVA mice. OVA-IgE was elevated to a greater extent in aged OVA mice.. Although pulmonary inflammation and mucus metaplasia after antigen sensitization/challenge occurred to a greater degree in older mice, the increase in AHR was significantly less compared with younger OVA mice. Antigen treatment produced a unique cytokine profile in older mice (elevated IFN-gamma and IL-5) compared with young mice (elevated IL-4 and IL-13). Thus, the airway response to inflammation is lessened in ageing animals, and may represent age-associated events leading to different phenotypes in response to antigen provocation.

Topics: Aging; Animals; Asthma; B-Lymphocytes; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Enzyme-Linked Immunosorbent Assay; Eosinophilia; Female; Gene Expression; Interferon-gamma; Interleukin-13; Interleukin-4; Interleukin-5; Metaplasia; Mice; Mice, Inbred BALB C; Mucin 5AC; Mucins; Mucus; Ovalbumin; Pneumonia; Polymerase Chain Reaction; T-Lymphocytes

We evaluated the role of Syk, using an inhibitor, on allergen-induced airway hyperresponsiveness (AHR) and airway inflammation in a system shown to be B cell- and mast cell-independent. Sensitization of BALB/c mice with ovalbumin (OVA) and alum after three consecutive OVA challenges resulted in AHR to inhaled methacholine and airway inflammation. The Syk inhibitor R406 (30 mg/kg, administered orally, twice daily) prevented the development of AHR, increases in eosinophils and lymphocytes and IL-13 levels in bronchoalveolar lavage (BAL) fluid, and goblet cell metaplasia when administered after sensitization and before challenge with OVA. Levels of IL-4, IL-5, and IFN-gamma in BAL fluid and allergen-specific antibody levels in serum were not affected by treatment. Because many of these responses may be influenced by dendritic cell function, we investigated the effect of R406 on bone marrow-derived dendritic cell (BMDC) function. Co-culture of BMDC with immune complexes of OVA and IgG anti-OVA together with OVA-sensitized spleen mononuclear cells resulted in increases in IL-13 production. IL-13 production was inhibited if the BMDCs were pretreated with the Syk inhibitor. Intratracheal transfer of immune complex-pulsed BMDCs (but not nonpulsed BMDCs) to naive mice before airway allergen challenge induced the development of AHR and increases in BAL eosinophils and lymphocytes. All of these responses were inhibited if the transferred BMDCs were pretreated with R406. These results demonstrate that Syk inhibition prevents allergen-induced AHR and airway inflammation after systemic sensitization and challenge, at least in part through alteration of DC function.

Topics: Allergens; Animals; B-Lymphocytes; Bone Marrow Cells; Bronchial Hyperreactivity; Cells, Cultured; Coculture Techniques; Cytokines; Dendritic Cells; Enzyme Activation; Female; Goblet Cells; Inflammation; Interleukin-13; Intracellular Signaling Peptides and Proteins; Mast Cells; Metaplasia; Methacholine Chloride; Mice; Mice, Inbred BALB C; Ovalbumin; Oxazines; Protein-Tyrosine Kinases; Pyridines; Respiratory System; Syk Kinase

Mucin hypersecretion is a prominent feature of obstructive airway diseases such as asthma. Clara cells conditionally produce mucin in response to inflammatory signals in a process termed mucous metaplasia. This can be followed by mucin secretion stimulated by various signaling molecules. The cellular and molecular mechanisms that regulate mucin production and secretion are not well understood. Adenosine is a signaling nucleoside that has been implicated in airway diseases in which mucus obstruction is prominent. Furthermore, the A(3) adenosine receptor (A(3)AR) is upregulated in mucin-producing goblet cells of the airway, thereby implicating it in processes involved in mucous cell biology. Here we use genetic approaches to investigate the contribution of A(3)AR signaling to mucus production and secretion in a mouse model of allergen-induced pulmonary disease. We found that the degree of mucin production in response to allergen is similar in wild-type and A(3)AR-deficient mice, and that overexpression of this receptor in Clara cells neither induces mucin production itself, nor enhances mucin production in response to allergen challenge. Collectively, these experiments demonstrate that the A(3)AR is neither necessary nor sufficient for mucous cell metaplasia. In contrast to the lack of effect on mucin production, agonist-induced mucin secretion was increased in goblet cells overexpressing the A(3)AR, and was absent in A(3)AR-deficient mice. Thus, the A(3)AR contributes to mucin secretion in allergen-induced metaplasia. Signaling through this receptor may contribute to mucus airway obstruction seen in pulmonary disorders in which adenosine levels are elevated.

Topics: Animals; Bronchial Provocation Tests; Cell Shape; Metaplasia; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mucins; Ovalbumin; Receptor, Adenosine A3; Respiratory Mucosa; Signal Transduction

Immunological tolerance during prolonged exposure to allergen is accompanied by a shift in the lymphocyte content and a reduction of goblet cell metaplasia (GCM). Bim initiates negative selection of autoreactive T and B cells and shut down of T cell immune responses in vivo. The present study investigated whether Bim plays a role in the resolution of GCM during prolonged exposure to allergen. Loss of Bim increased T lymphocyte numbers in the bronchoalveolar lavage at 4 and 15 days of allergen exposure. The numbers of pulmonary CD4(+)8(-), CD4(-)8(+), and gammadelta T cells were significantly higher in naive and allergen-challenged bim(-/-) mice compared with wild-type (WT) littermates. When activated, pulmonary bim(-/-) T cells produced increased levels of IFNgamma compared with bim(+/+) T cells. No differences were noted in the total numbers of epithelial cells per millimeter of basal lamina between bim(+/+) and bim(-/-) mice, and the rate of resolution over 15 days of exposure was similar in both groups of mice. However, GCM was significantly enhanced and expression of IL-13Ralpha2 was reduced in bim(-/-) mice compared with WT mice at 4 days. Furthermore, treatment of bronchiolar explant cultures with increasing IFNgamma levels reduced immunostaining for IL-13Ralpha2. Collectively, these studies suggest that, during prolonged exposure to allergen, Bim plays no role in the resolution of GCM, but increased IFNgamma levels in bim(-/-) mice may be responsible for reduced expression of IL-13Ralpha2 and enhanced GCM despite similar levels of IL-13 in bim(+/+) and bim(-/-) mice.

Topics: Allergens; Animals; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; CD8-Positive T-Lymphocytes; Goblet Cells; Interferon-gamma; Interleukin-13 Receptor alpha2 Subunit; Lung; Male; Membrane Proteins; Metaplasia; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Ovalbumin; Proto-Oncogene Proteins; Receptors, Antigen, T-Cell, gamma-delta; Th1 Cells; Th2 Cells

Airway cyclooxygenase-2 (COX-2) is induced by cytokine-mediated inflammation such as occurs in asthma. However, the role of COX-2 in the pathophysiology of asthma is not fully understood.. Allergic inflammation, airway responsiveness to methacholine and mucous cell metaplasia after ovalbumin sensitization in the airways of COX-2 deficient (-/-) mice, COX-2 (+/+) mice and C57BL/6J mice treated with a selective COX-2 inhibitor, nimesulide were assessed. Histology, cell analysis, measurements of arachidonic acid metabolites and Th2 cytokine levels in bronchoalveolar lavage fluid (BALF), and measurement of serum IgE were performed.. Eosinophil infiltration into the airway wall, and the number of eosinophils in BALF were greater in sensitized COX-2 (-/-) mice than in sensitized COX-2 (+/+) mice. The levels of cysteinyl leukotrienes (LTC4/D4/E4), prostaglandin E2 (PGE2) and interleukin (IL)-13 as well as airway responsiveness did not differ in COX-2 (-/-) mice and COX-2 (+/+) mice. However, sensitized COX-2 (-/-) mice had higher LTC4/D4/E4 and lower PGE2 concentrations compared with non-sensitized COX-2 (-/-) mice. The number of PAS/alcian blue-positive airway epithelial cells and serum IgE were elevated in COX-2 (-/-) mice. Nimesulide-treated mice showed augmented eosinophilic inflammation, LTC4/D4/E4 concentrations and mucous cell metaplasia.. These data indicate that COX-2 deficiency augments allergic inflammation and mucous cell metaplasia.

Topics: Animals; Arachidonic Acid; Bronchi; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Bronchoconstrictor Agents; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Cysteine; Dinoprostone; Female; Immunoglobulin E; Inflammation; Interleukin-13; Leukotrienes; Lung; Male; Metaplasia; Methacholine Chloride; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Sulfonamides

The pathophysiology of infantile asthma may differ from that in older children or in adults, partly because of the different immune response depending upon maturation. In adult mice, the sensitizing dose of antigen is known to be critical to the polarized development of helper T cell subsets and allergic airway inflammation. We wanted to know the characteristics of allergic airway inflammation of infantile asthma by developing a murine model.. BALB/C mice at different stages of maturation (juvenile: 3 days after birth; adult: 8 weeks of age) were sensitized with 10 or 1,000 microg ovalbumin (OVA) or vehicle. The animals were then challenged with aerosolized OVA or saline once a day during 6 consecutive days. After the final challenge, bronchial hyperresponsiveness (BHR), bronchoalveolar lavage fluid (BALF), histological changes in the airways and immunological status were examined.. In both juvenile and adult animals, sensitization with 10 microg OVA induced the T helper 2 response (elevated IL-4 and decreased IFN-gamma levels). BHR, airway eosinophilia, the inflammatory cell infiltration, goblet cell metaplasia (GCM), and IgE antibody production were more prominent in animals given this dose than 1,000 microg OVA. Among these responses, GCM as well as BALF IL-4, and BHR were comparable between juvenile and adult animals, whereas other parameters were lower in juvenile animals, especially in those given 1,000 microg OVA.. GCM and, consequently, airway mucus hypersecretion may be an important component of allergic airway inflammation in infantile asthma.

Topics: Age Factors; Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Eosinophilia; Goblet Cells; Immunoglobulin E; Inflammation; Lung; Metaplasia; Mice; Mice, Inbred BALB C; Mucus; Ovalbumin; Th2 Cells

Mucus overproduction from goblet cells, a characteristic feature of the allergic asthmatic inflammation induced by ovalbumin (OVA) in mice, was examined morphologically. In OVA-untreated (normal) mice, there were no goblet cells in intrapulmonary bronchus and bronchiole. However, goblet cells with or without hyperplasia in the mucosa of inflamed bronchus-bronchiole were recognized in the allergic asthmatic mice. The non-ciliated epithelium containing electron lucent granules (mucus) showed many similarities to Clara cells, which have characteristic secretory granules and many mitochondria, except for the less-developed smooth endoplasmic reticulum seen in normal mice. Ciliated Clara cells with or without mucus were rarely recognized. In addition, mucus was found in neither ciliated nor basal epithelium. The present study suggests that goblet-cell metaplasia in the bronchus and bronchiole of inflamed mucosa may be derived, at least in part, from Clara cells.

Topics: Animals; Asthma; Bronchi; Disease Models, Animal; Endoplasmic Reticulum, Smooth; Female; Goblet Cells; Metaplasia; Mice; Mice, Inbred DBA; Microscopy, Electron; Mitochondria; Mucous Membrane; Ovalbumin; Secretory Vesicles

In mice, intratracheal challenges with antigen (ovalbumin) or recombinant murine interleukin-13 (IL-13) induce lung inflammation, bronchial hyperreactivity (BHR), and mucus accumulation as independent events (Singer M, Lefort J, and Vargaftig BB. Am J Respir Cell Mol Biol 26: 74-84, 2002), largely mediated by leukotrienes (LT). We previously showed that LTC(4) was released 15 min after ovalbumin, and we show that it induces the expression of monocyte chemoattractant proteins 1 and 5 and KC in the lungs, as well as IL-13 mRNA. Instilled intratracheally, these chemokines induced BHR and mucus accumulation, which were inhibited by the 5-lipoxygenase inhibitor zileuton and by the cysteinyl-LT receptor antagonist MK-571, suggesting mediation by cysteinyl-LT. Because these chemokines also induced release of LT into the bronchoalveolar lavage fluid and IL-13 into the lungs, we hypothesize that LT- and chemokine-based loops for positive-feedback regulations cooperate to maintain and amplify BHR and lung mucus accumulation after allergic challenge and in situations where IL-13, LT, or chemokines are generated.

Topics: Animals; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Chemokines; Hypersensitivity; Interleukin-13; Leukotriene C4; Leukotrienes; Lung; Male; Metaplasia; Mice; Mice, Inbred Strains; Mucins; Mucus; Ovalbumin; Recombinant Proteins; Respiratory Mucosa; RNA, Messenger

Most infections with respiratory viruses induce Th1 responses characterized by the generation of Th1 and CD8(+) T cells secreting IFN-gamma, which in turn have been shown to inhibit the development of Th2 cells. Therefore, it could be expected that respiratory viral infections mediate protection against asthma. However, the opposite seems to be true, because viral infections are often associated with the exacerbation of asthma. For this reason, we investigated what effect an influenza A (flu) virus infection has on the development of asthma. We found that flu infection 1, 3, 6, or 9 wk before allergen airway challenge resulted in a strong suppression of allergen-induced airway eosinophilia. This effect was associated with strongly reduced numbers of Th2 cells in the airways and was not observed in IFN-gamma- or IL-12 p35-deficient mice. Mice infected with flu virus and immunized with OVA showed decreased IL-5 and increased IFN-gamma, eotaxin/CC chemokine ligand (CCL)11, RANTES/CCL5, and monocyte chemoattractant protein-1/CCL2 levels in the bronchoalveolar lavage fluid, and increased airway hyperreactivity compared with OVA-immunized mice. These results suggest that the flu virus infection reduced airway eosinophilia by inducing Th1 responses, which lead to the inefficient recruitment of Th2 cells into the airways. However, OVA-specific IgE and IgG1 serum levels, blood eosinophilia, and goblet cell metaplasia in the lung were not reduced by the flu infection. Flu virus infection also directly induced AHR and goblet cell metaplasia. Taken together, our results show that flu virus infections can induce, exacerbate, and suppress features of asthmatic disease in mice.

Topics: Allergens; Animals; Bronchial Hyperreactivity; Cell Migration Inhibition; Cell Movement; Cells, Cultured; Chemokine CCL11; Chemokine CCL2; Chemokine CCL5; Chemokines, CC; Down-Regulation; Epitopes, T-Lymphocyte; Goblet Cells; Influenza A virus; Interferon-gamma; Interleukin-5; Lung; Lymphocyte Count; Lymphopenia; Metaplasia; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Nippostrongylus; Orthomyxoviridae Infections; Ovalbumin; Peptide Fragments; Pulmonary Eosinophilia; Strongylida Infections; Th2 Cells; Up-Regulation

Increased mucus production in asthma is an important cause of airflow obstruction during severe exacerbations. To better understand the changes in airway epithelium that lead to increased mucus production, ovalbumin-sensitized and -challenged mice were used. The phenotype of the epithelium was dramatically altered, resulting in increased numbers of mucous cells, predominantly in the proximal airways. However, the total numbers of epithelial cells per unit area of basement membrane did not change. A 75% decrease in Clara cells and a 25% decrease in ciliated cells were completely compensated for by an increase in mucous cells. Consequently, by day 22, 70% of the total epithelial cell population in the proximal airways was mucous cells. Electron microscopy illustrated that Clara cells were undergoing metaplasia to mucous cells. Conversely, epithelial proliferation, detected with 5-chloro-2-deoxyuridine immunohistochemistry, was most marked in the distal airways. Using ethidium homodimer cell labeling to evaluate necrosis and terminal dUTP nick-end labeling immunohistochemistry to evaluate apoptosis, this proliferation was accompanied by negligible cell death. In conclusion, epithelial cell death did not appear to be the stimulus driving epithelial proliferation and the increase in mucous cell numbers was primarily a result of Clara cell metaplasia.

Topics: Animals; Asthma; Bronchi; Cell Division; Disease Models, Animal; Epithelial Cells; Male; Metaplasia; Mice; Mice, Inbred BALB C; Microscopy, Electron; Ovalbumin; Respiratory Mucosa

Allergic airway responses cause proliferation of epithelial cells and mucus cell metaplasia (MCM), and the resolution of MCM involves reduction of cell numbers. The role of inflammation and apoptosis on this process was investigated in P-selectin +/+ and -/- mice sensitized and challenged with OVA by analyzing the expression and the role of regulators of apoptosis in metaplastic mucus cells. No differences were observed in MCM at 5 days of allergen exposure between +/+ and -/- mice, despite reduced IL-13 levels in -/- mice. Although IL-4 levels were similar in both -/- and +/+ mice, IL-13 and IL-5 levels had decreased and IFN-gamma levels were increased earlier in -/- compared with +/+ mice. MCM levels were decreased 4-fold at 7 days of allergen exposure in -/- mice and at 15 days in +/+ mice. The percentage of Bax-expressing mucus cells increased significantly at 7 days in -/- mice and at 10 days in +/+ mice. The Bax-positive mucus cells exhibited caspase-specific cleavage of cytokeratin 18. IFN-gamma caused Bax expression in IL-13-induced MCM in microdissected airway cultures. MCM remained significantly elevated in Bax -/- mice following 15 days of allergen exposure compared with +/+ mice, while the number of eosinophils was reduced in both Bax +/+ and -/- mice at 15 days. Together, these data demonstrate that reduced IL-13 levels were sufficient to elicit maximum MCM, that IFN-gamma induces Bax in metaplastic mucus cells, and that Bax plays a critical role in the resolution of MCM, but not in the resolution of eosinophils.

Topics: Allergens; Animals; bcl-2-Associated X Protein; Bronchi; Cell Count; Culture Techniques; Dissection; Eosinophilia; Inflammation; Interferon-gamma; Lung; Male; Metaplasia; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucus; Ovalbumin; P-Selectin; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Respiratory Hypersensitivity

Inflammatory responses induced by allergen exposure cause mucous cell metaplasia (MCM) by differentiation of existing and proliferating epithelial cells into mucus-storing cells. Airway epithelia have various mechanisms that resolve these changes to form normal airway epithelia. In this report, we first investigated the state of mucous cell metaplasia and the mechanisms by which MCM is reduced despite continued exposures to allergen. After 5 days of allergen exposure, extensive MCM had developed but was reduced when allergen challenge was continued for 15 days. During this exposure period, IL-13 levels decreased and IFN-gamma levels increased in the bronchoalveolar lavage fluid. In contrast, IL-13 levels decreased but IFN-gamma was not detected at any time point during the resolution of MCM following cessation of allergen exposure. Instillation of IFN-gamma but not anti-Fas caused accelerated resolution of MCM and MCM was not resolved in Stat1-deficient mice exposed to allergen for 15 days, confirming that IFN-gamma is crucial for reducing MCM during prolonged exposures to allergen. IFN-gamma but not anti-Fas induced apoptotic cell death in proliferating normal human bronchial epithelial cells and in human bronchial epithelial cells from subjects with asthma. The apoptotic effect of IFN-gamma was caspase dependent and was inhibited by IL-13, indicating that the Th2 milieu in asthmatics may maintain MCM by preventing cell death in metaplastic mucous cells. These studies could be useful in the understanding of deficiencies leading to chronicity in airway changes and designing novel therapies to reverse MCM and airway obstruction in asthmatics.

Topics: Adult; Allergens; Animals; Apoptosis; Asthma; Bronchi; Cells, Cultured; DNA-Binding Proteins; fas Receptor; Humans; Hypersensitivity; Interferon-gamma; Interleukin-13; Kinetics; Male; Metaplasia; Mice; Mice, Inbred C57BL; Ovalbumin; Respiratory Mucosa; STAT1 Transcription Factor; Trans-Activators

IL-4 and IL-13 are key regulators in atopic disorders and both signal through the receptor chain IL-4Ralpha. IL-4 and IL-13 are also the only cytokines known to induce class switching to IgE. We sought to compare allergen-specific IgE responses and allergic reactivity of wild-type (wt) mice with IL-4-/- and IL-4Ralpha-/- mice, which lack both IL-4 and IL-13 functions.. BALB/c wt, IL-4-/- and IL-4Ralpha-/- mice were immunized with ovalbumin intranasally or intraperitoneally and specific antibody titers were measured by ELISA. Bronchoalveolar lavage fluids and lung tissue were analyzed cytologically and histologically. Allergic reactivity was determined by active cutaneous anaphylaxis and anaphylactic shock.. wt mice immunized intranasally or intraperitoneally showed high titers of specific IgE 3 and 6 weeks after primary sensitization, resulting in cutaneous anaphylaxis and anaphylactic shock upon challenge. Intranasal sensitization resulted in airway eosinophilia and goblet cell metaplasia. In contrast, IL-4-/- and IL-4Ralpha-/- mice showed no specific IgE after 3 weeks, but produced high titers after 6 weeks. At this time cutaneous anaphylaxis and anaphylactic shock could be induced as in wt mice, but lung pathology was absent.. We conclude that upon long-term allergen exposure, alternative switch mechanisms independent of IL-4 and IL-4Ralpha may induce IgE but not asthma-like lung pathology. This may be relevant for the development of allergic disease, since long-term allergen exposure is a frequent condition during allergic sensitization.

Topics: Allergens; Anaphylaxis; Animals; Eosinophilia; Female; Immunoglobulin E; Immunoglobulin G; Interleukin-13; Interleukin-4; Metaplasia; Mice; Mice, Inbred BALB C; Mice, Knockout; Ovalbumin; Receptors, Interleukin-4

Airway inflammation, hyperreactivity, increased number of goblet cells, and mucus overproduction characterize asthma. Respiratory challenge with ovalbumin (OVA) of sensitized mice has been shown by several laboratories to cause pulmonary pathology similar to that observed in human allergic asthma. Recently, interleukin (IL)-13 has been shown to be a central mediator in this process. Because the airways of healthy mice have few, if any, mucus-producing cells, an increase in the number of these cells likely reflects induction of mucin-gene expression. The purpose of this study was to identify mucin genes induced as a result of airway goblet-cell metaplasia (GCM) in mice sensitized and challenged with OVA or in mice treated with IL-13 alone. BALB/c mice were sensitized by intraperitoneal injection (Days 0, 4, 7, 11, and 14) and intranasal instillation (Day 14) of 100 microg of OVA in saline, and then challenged by intranasal instillation (Days 25, 26, and 27) of the same. IL-13-treated mice received 5 microg of IL-13 by intranasal instillation on three consecutive days. Control mice were given saline alone. All mice were studied 24 h after the last challenge. Histologic analysis of the lungs revealed both a striking peribronchial and perivascular lymphocytic and eosinophilic inflammation and airway GCM in OVA-treated mice, and also airway GCM without inflammation in IL-13-treated mice. Northern blot analysis of lung RNA demonstrated (1) expression of Muc-5/5ac messenger RNA (mRNA) in OVA-treated and IL-13-treated mice, but not in control mice; (2) expression of Muc-1 mRNA at comparable levels in all mice regardless of treatment; and (3) no expression of Muc-2 or Muc-3 mRNA in control or treated mice. Western blot analysis demonstrated the expression of Muc-5/5ac protein (both apomucin and glycosylated mucin) in lung lysates of OVA-treated (but not control) mice, and also the expression of Muc-5/5ac mucins in the bronchoalveolar lavage fluid of OVA-treated and IL-13-treated mice. These findings demonstrate that airway GCM is associated with the induction of pulmonary expression of Muc-5/5ac mRNA and mucin in murine models of allergic asthma.

Topics: Alveolitis, Extrinsic Allergic; Animals; Antibodies; Biomarkers; Bronchoalveolar Lavage Fluid; Gene Expression; Goblet Cells; Interleukin-13; Lung; Male; Metaplasia; Mice; Mice, Inbred BALB C; Mucin 5AC; Mucin-5B; Mucins; Ovalbumin; RNA, Messenger

IL-4 and IL-13 play a putative role in mucus hypersecretion in asthma. Suplatast tosilate prevents the synthesis of T(H2) cytokines.. Because suplatast tosilate inhibits T(H2) cytokines but does not inhibits IFN-gamma production, we examined the effect of suplatast on IL-4- or IL-13- and ovalbumin (OVA)-induced mucin synthesis in NCI-H292 cells in vitro and in bronchi of pathogen-free BALB/c mice in vivo.. In vitro, NCI-H292 cells were preincubated with suplatast tosilate (0.1-100 microgram/mL) 1 hour before adding human recombinant IL-4 (10 ng/mL). In vivo, mouse recombinant IL-4 or IL-13 (250 ng per/mouse) was instilled intranasally in mice pretreated with suplatast tosilate (50 mg.kg(-1).d(-1)). Mucous glycoconjugates were stained with Alcian blue (AB)/periodic acid-Schiff (PAS) stain. To evaluate effects of suplatast tosilate on goblet-cell metaplasia in OVA-sensitized mice, animals were pretreated with suplatast tosilate (1-50 mg.kg(-1).d(-1)) intragastrically. IL-4 and IL-13 were measured, and allergic inflammatory cells were analyzed in bronchoalveolar lavage fluid of OVA-sensitized mice.. Pretreatment with suplastast did not prevent IL-4- or IL-13-induced increase in mucous glycoconjugate production in NCI-H292 cells or in mice. OVA sensitization increased AB/PAS-stained area of the epithelium (48.1% +/- 2.4%, P <.01 compared with control mice). Suplatast tosilate inhibited OVA-induced goblet-cell metaplasia in airway epithelium in a dose-dependent fashion; 50 mg.kg(-1).d(-1) decreased the AB/PAS area to 22.7% +/- 2.7% (P <.05 compared with OVA sensitization alone). Pretreatment with suplatast tosilate also prevented OVA-induced increase in IL-4 and IL-13 levels and decreased the number of lymphocytes and eosinophils in bronchoalveolar lavage fluid (P <.05 compared with values of mice given OVA alone).. These results indicate that suplatast tosilate prevents allergen-induced goblet-cell metaplasia and the recruitment of eosinophils and lymphocytes into the airways. These results suggest that this effect is due to the prevention of the production of T(H2) cytokines in airways.

Topics: Animals; Anti-Allergic Agents; Arylsulfonates; Bronchi; Cell Line; Goblet Cells; Humans; Immunization; Interleukin-4; Male; Metaplasia; Mice; Mice, Inbred BALB C; Mucins; Ovalbumin; Sulfonium Compounds