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Role of Inducible Nitric Oxide Synthase Pathway in the Development of Non-eosinophilic Asthma
S130 Abstracts
511
SUNDAY
Role of Protein Tyrosine Phosphatase SHP-1 in Oxidative Stress and Development of Allergic Airway Inflammation Y. Cho, S. Oh, Z. Zhu; Johns Hopkins Asthma and Allergy Center, Baltimore, MD. RATIONALE: Oxidative stress has been implicated in allergic responses. SHP-1 can be inactivated, reversibly, by oxidants and has been reported as a negative regulator in a mouse asthma model. We investigated the effect and functioning mechanism of oxidative stress on the development of allergic airway inflammation in viable motheaten (mev) mice deficient of SHP-1. METHODS: Heterozygous mev mice were compared with wild type (WT) mice in these studies. Hydrogen peroxide and methyl viologen were used to generate oxidative stress for in vitro and in vivo experiments, respectively. We also evaluated whether heterozygous mev mice are more likely to break immune tolerance. Ovalbumin was given to mev and WT mice i.n. for 5 days, which usually will not elicit immunity in WT mice. RESULTS: In heterozygous mev mice, exposure to oxidative stress induced a different response in cells from bronchoalveolar lavage (BAL) and in the lung compared to that in WT mice. This includes a significant enhancement of intracellular ROS, transcription factor nrf2 nuclear translocation, and STAT-6 phosphorylation in vitro and increased CCL20, decreased IL-10 and TGF-b, and increased number of dendritic cells in BAL fluid in vivo. Furthermore, break of immune tolerance was evident as allergic airway inflammation was observed in mev mice after intranasal allergen exposures. CONCLUSIONS: These results suggest that SHP-1 plays a role in regulating oxidative stress. Thus increased intracellular oxidative stress and lack of SHP-1 in the presence of Th2-prone cellular activation may lead to the development of allergic airway inflammation.
512
Comparison of Central and Peripheral Airway Inflammation between Cough Variant Asthma and Mild Asthma by Examination of Hypertonic Saline Inhalation-Induced Sputum T. Shimoda1, Y. Obase2, R. Kishikawa1, S. Shoji1, S. Nishima1; 1Fukuoka National Hospital, Fukuoka, JAPAN, 2Kawasaki Medical School, Kurashiki, JAPAN. RATIONALE: Cough variant asthma (CVA) and bronchial asthma are both inflammatory disease. The intensity of central and peripheral airway inflammation was compared between CVA and mild asthma by examining induced sputum. METHODS: The subjects of the study were 28 patients with CVA, 27 patients with mild intermittent asthma (step 1) and 18 patients with mild persistent asthma (step 2) who had not previously been treated with inhaled corticosteroid. The study was conducted during an attack-free period. After inhalation of 3% hypertonic saline, the sputum collected during the initial 10 minutes was defined as a specimen from the central airway and the sputum obtained during the latter 10 minutes defined as a specimen from the peripheral airway, and collected specimens were examined. The expiratory nitric monoxide(eNO) was also measured. RESULTS: The percentages of eosinophils in the central and peripheral airways were 2.565.2 vs 2.266.2% (p 5 0.35) in CVA, 12.7620.4 vs 8.56615.7% (p 5 0.01) in step 1, and 21.2621.5 vs 16.2619.0% (p 5 0.01) in step 2. The eNO was 24.7617.1 ppb in CVA, 70.3664.7 ppb in step 1, and 99.0692.4 ppb in step 2 (CVA vs step1; p 5 0.01, CVA vs step2; p 5 0.01, step1 vs step2; p 5 0.58). The percentages of eosinophils in the central and peripheral airways tended to correlate with the eNO in combined data on all subjects (central: r 5 0.66, peripheral: r 5 0.67). CONCLUSIONS: Both central and peripheral airway inflammation was weaker in CVA than in mild asthma, suggesting that pathologically CVA is a precursor stage of bronchial asthma.
J ALLERGY CLIN IMMUNOL JANUARY 2007
513
Role of Inducible Nitric Oxide Synthase Pathway in the Development of Non-eosinophilic Asthma H. Park1, S. Sohn1, C. Park1, H. Kwon1, D. Kim1, S. Kim1, Y. Chang2, S. Lee3, D. Choi4, S. Cho1, S. Chang5, K. Min1, Y. Kim1; 1Seoul National University Hospital, Seoul, REPUBLIC OF KOREA, 2Seoul National University Bundang Hospital, Seongnam, REPUBLIC OF KOREA, 3 Cheongju St. Mary’s Hospital, Cheongju, REPUBLIC OF KOREA, 4 Samsung Medical Center, Seoul, REPUBLIC OF KOREA, 5Sungae General Hospital, Gwangmyung, REPUBLIC OF KOREA. RATIONALE: Evidences are accumulated supporting that IFN-g-rich microenvironment plays an important role in the development of non-eosinophilic asthma (NEA). Meanwhile, expression and activity of inducible nitric oxide synthase (iNOS) triggered by proinflammatory cytokine, such as IFN-g, are increased in patients with asthma. This study was conducted to test the hypothesis that iNOS pathway plays an essential role in the development of NEA using murine model. METHODS: To produce the experimental NEA model, mice with C57BL/ 6 background were sensitized intranasally with ovalbumin (OVA) plus double-stranded RNA (dsRNA) and then challenged with OVA. This model was compared to the conventional eosinophilic-asthma (EA) model (IP sensitization). Then the effects of iNOS signal pathway on NEA model were assessed using mice deficient in the gene coding iNOS (iNOS-/-). RESULTS: Mice showed positive methacholine AHR, increased OVA-specific IgG2a, increased levels of IFN-g in BAL fluid along with non-eosinophilic airway inflammation characterized by lymphocyte and neutrophil. In addition, chronic airway challenge with OVA showed methacholine AHR comparable to the conventional EA model. Baseline airway obstruction, methacholine AHR, and lung inflammation in iNOS-/- mice significantly decreased. CONCLUSIONS: Intranasal sensitization with OVA plus dsRNA and subsequent challenge with OVA provides physiologic and suitable murine model of NEA. NEA phenotypes accompanied by increased IFN-g activity are dependent on the iNOS signaling pathway.
514
Increase of Amphiregulin Level in Sputum during Exacerbation of Acute Asthma Attacks in Children Y. Enomoto1,2, T. Takamasu2, M. Nakazawa3, K. Kurihara2, M. Minami3, H. Saito1, Y. Okayama1; 1RIKEN Yokohama Institute, Yokohama, JAPAN, 2Kanagawa Children’s Medical Center, Yokohama, JAPAN, 3Yokohama City University Graduate School of Medicine, Yokohama, JAPAN. RATIONALE: Since we have reported that FceRI-mediated amphiregulin (AREG) production by human mast cells increases mucin gene expression in epithelial cells, we examined changes in sputum level of AREG, tryptase and epidermal growth factor (EGF) during exacerbations of acute asthma attacks in children comparing with paired samples taken at remission. METHODS: Twelve asthmatic patients (median age 6.8 [0.9 to 15.2]), seven normal healthy volunteers (median age 9.2 [5.8 to 13.8]), and seven patients with common cold (median age 5.8 [0.2 to 12.5]) provided sputa. Concentrations of AREG, EGF and tryptase were measured by ELISA. Changes of MUC5AC mRNA in the NCI-H292 cells following incubation with sputum from patients were examined by real-time RT-PCR. RESULTS: AREG significantly increased during exacerbation (1069 pg/ ml [207 to 5108]) in comparison with that at remission (121 pg/ml [15 to 400]) (P < 0.001). AREG level during exacerbation of asthma were significantly higher than that from normal controls (p <0.001) and patients with common cold (p < 0.001). AREG level was significantly correlated to tryptase level (p <0.001) but not to EGF level. Changes in sputum level of AREG were significantly correlated to the changes of sputum volume and clinical symptom (p <0.05) but those of EGF were not. Sputum from asthmatics, which showed high-level of AREG, caused upregulation of MUC5AC mRNA in NCI-H292 cells. CONCLUSIONS: These results suggest that AREG in sputum that increases during exacerbation of acute asthma attacks may be responsible for overproduction of sputum, and that AREG in sputum may be produced by mast cells. Funding: RIKEN Yokohama institute, the National Institute of Biomedical Innovation
511
SUNDAY
Role of Protein Tyrosine Phosphatase SHP-1 in Oxidative Stress and Development of Allergic Airway Inflammation Y. Cho, S. Oh, Z. Zhu; Johns Hopkins Asthma and Allergy Center, Baltimore, MD. RATIONALE: Oxidative stress has been implicated in allergic responses. SHP-1 can be inactivated, reversibly, by oxidants and has been reported as a negative regulator in a mouse asthma model. We investigated the effect and functioning mechanism of oxidative stress on the development of allergic airway inflammation in viable motheaten (mev) mice deficient of SHP-1. METHODS: Heterozygous mev mice were compared with wild type (WT) mice in these studies. Hydrogen peroxide and methyl viologen were used to generate oxidative stress for in vitro and in vivo experiments, respectively. We also evaluated whether heterozygous mev mice are more likely to break immune tolerance. Ovalbumin was given to mev and WT mice i.n. for 5 days, which usually will not elicit immunity in WT mice. RESULTS: In heterozygous mev mice, exposure to oxidative stress induced a different response in cells from bronchoalveolar lavage (BAL) and in the lung compared to that in WT mice. This includes a significant enhancement of intracellular ROS, transcription factor nrf2 nuclear translocation, and STAT-6 phosphorylation in vitro and increased CCL20, decreased IL-10 and TGF-b, and increased number of dendritic cells in BAL fluid in vivo. Furthermore, break of immune tolerance was evident as allergic airway inflammation was observed in mev mice after intranasal allergen exposures. CONCLUSIONS: These results suggest that SHP-1 plays a role in regulating oxidative stress. Thus increased intracellular oxidative stress and lack of SHP-1 in the presence of Th2-prone cellular activation may lead to the development of allergic airway inflammation.
512
Comparison of Central and Peripheral Airway Inflammation between Cough Variant Asthma and Mild Asthma by Examination of Hypertonic Saline Inhalation-Induced Sputum T. Shimoda1, Y. Obase2, R. Kishikawa1, S. Shoji1, S. Nishima1; 1Fukuoka National Hospital, Fukuoka, JAPAN, 2Kawasaki Medical School, Kurashiki, JAPAN. RATIONALE: Cough variant asthma (CVA) and bronchial asthma are both inflammatory disease. The intensity of central and peripheral airway inflammation was compared between CVA and mild asthma by examining induced sputum. METHODS: The subjects of the study were 28 patients with CVA, 27 patients with mild intermittent asthma (step 1) and 18 patients with mild persistent asthma (step 2) who had not previously been treated with inhaled corticosteroid. The study was conducted during an attack-free period. After inhalation of 3% hypertonic saline, the sputum collected during the initial 10 minutes was defined as a specimen from the central airway and the sputum obtained during the latter 10 minutes defined as a specimen from the peripheral airway, and collected specimens were examined. The expiratory nitric monoxide(eNO) was also measured. RESULTS: The percentages of eosinophils in the central and peripheral airways were 2.565.2 vs 2.266.2% (p 5 0.35) in CVA, 12.7620.4 vs 8.56615.7% (p 5 0.01) in step 1, and 21.2621.5 vs 16.2619.0% (p 5 0.01) in step 2. The eNO was 24.7617.1 ppb in CVA, 70.3664.7 ppb in step 1, and 99.0692.4 ppb in step 2 (CVA vs step1; p 5 0.01, CVA vs step2; p 5 0.01, step1 vs step2; p 5 0.58). The percentages of eosinophils in the central and peripheral airways tended to correlate with the eNO in combined data on all subjects (central: r 5 0.66, peripheral: r 5 0.67). CONCLUSIONS: Both central and peripheral airway inflammation was weaker in CVA than in mild asthma, suggesting that pathologically CVA is a precursor stage of bronchial asthma.
J ALLERGY CLIN IMMUNOL JANUARY 2007
513
Role of Inducible Nitric Oxide Synthase Pathway in the Development of Non-eosinophilic Asthma H. Park1, S. Sohn1, C. Park1, H. Kwon1, D. Kim1, S. Kim1, Y. Chang2, S. Lee3, D. Choi4, S. Cho1, S. Chang5, K. Min1, Y. Kim1; 1Seoul National University Hospital, Seoul, REPUBLIC OF KOREA, 2Seoul National University Bundang Hospital, Seongnam, REPUBLIC OF KOREA, 3 Cheongju St. Mary’s Hospital, Cheongju, REPUBLIC OF KOREA, 4 Samsung Medical Center, Seoul, REPUBLIC OF KOREA, 5Sungae General Hospital, Gwangmyung, REPUBLIC OF KOREA. RATIONALE: Evidences are accumulated supporting that IFN-g-rich microenvironment plays an important role in the development of non-eosinophilic asthma (NEA). Meanwhile, expression and activity of inducible nitric oxide synthase (iNOS) triggered by proinflammatory cytokine, such as IFN-g, are increased in patients with asthma. This study was conducted to test the hypothesis that iNOS pathway plays an essential role in the development of NEA using murine model. METHODS: To produce the experimental NEA model, mice with C57BL/ 6 background were sensitized intranasally with ovalbumin (OVA) plus double-stranded RNA (dsRNA) and then challenged with OVA. This model was compared to the conventional eosinophilic-asthma (EA) model (IP sensitization). Then the effects of iNOS signal pathway on NEA model were assessed using mice deficient in the gene coding iNOS (iNOS-/-). RESULTS: Mice showed positive methacholine AHR, increased OVA-specific IgG2a, increased levels of IFN-g in BAL fluid along with non-eosinophilic airway inflammation characterized by lymphocyte and neutrophil. In addition, chronic airway challenge with OVA showed methacholine AHR comparable to the conventional EA model. Baseline airway obstruction, methacholine AHR, and lung inflammation in iNOS-/- mice significantly decreased. CONCLUSIONS: Intranasal sensitization with OVA plus dsRNA and subsequent challenge with OVA provides physiologic and suitable murine model of NEA. NEA phenotypes accompanied by increased IFN-g activity are dependent on the iNOS signaling pathway.
514
Increase of Amphiregulin Level in Sputum during Exacerbation of Acute Asthma Attacks in Children Y. Enomoto1,2, T. Takamasu2, M. Nakazawa3, K. Kurihara2, M. Minami3, H. Saito1, Y. Okayama1; 1RIKEN Yokohama Institute, Yokohama, JAPAN, 2Kanagawa Children’s Medical Center, Yokohama, JAPAN, 3Yokohama City University Graduate School of Medicine, Yokohama, JAPAN. RATIONALE: Since we have reported that FceRI-mediated amphiregulin (AREG) production by human mast cells increases mucin gene expression in epithelial cells, we examined changes in sputum level of AREG, tryptase and epidermal growth factor (EGF) during exacerbations of acute asthma attacks in children comparing with paired samples taken at remission. METHODS: Twelve asthmatic patients (median age 6.8 [0.9 to 15.2]), seven normal healthy volunteers (median age 9.2 [5.8 to 13.8]), and seven patients with common cold (median age 5.8 [0.2 to 12.5]) provided sputa. Concentrations of AREG, EGF and tryptase were measured by ELISA. Changes of MUC5AC mRNA in the NCI-H292 cells following incubation with sputum from patients were examined by real-time RT-PCR. RESULTS: AREG significantly increased during exacerbation (1069 pg/ ml [207 to 5108]) in comparison with that at remission (121 pg/ml [15 to 400]) (P < 0.001). AREG level during exacerbation of asthma were significantly higher than that from normal controls (p <0.001) and patients with common cold (p < 0.001). AREG level was significantly correlated to tryptase level (p <0.001) but not to EGF level. Changes in sputum level of AREG were significantly correlated to the changes of sputum volume and clinical symptom (p <0.05) but those of EGF were not. Sputum from asthmatics, which showed high-level of AREG, caused upregulation of MUC5AC mRNA in NCI-H292 cells. CONCLUSIONS: These results suggest that AREG in sputum that increases during exacerbation of acute asthma attacks may be responsible for overproduction of sputum, and that AREG in sputum may be produced by mast cells. Funding: RIKEN Yokohama institute, the National Institute of Biomedical Innovation