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A Murine Model Of Childhood Asthma
Abstracts S129
J ALLERGY CLIN IMMUNOL VOLUME 119, NUMBER 1
A Murine Model Of Childhood Asthma H. Mayuzumi1, Y. Ohki2, K. Tokuyama3, T. Mizuno2, H. Arakawa2, H. Mochizuki2, A. Morikawa2; 1Gunma Children‘s Medical Center, Shibukawa, JAPAN, 2Gunma University Graduate School of Medicine, Maebashi, JAPAN, 3Takasaki University of Health and Welfare, Takasaki, JAPAN. RATIONALE: Asthmatic children are more likely to outgrow their symptoms than adult patients. Thus, we wanted to know whether there were any age-related differences in the time course of the allergic airway inflammation. METHODS: BALB/C mice with different ages (young; 3 days after birth, and mature; 8 weeks of age) were sensitized with ovalbumin (OVA). Then, animals were challenged with aerosolized OVA during 1, 2, 4 or 8 consecutive weeks. Bronchial hyperresponsiveness (BHR), serum IgE levels, the degrees of inflammatory cell infiltration (ICI) and goblet cell metaplasia (GCM) in the airways, and the number of eosinophils and cytokine levels in bronchoalveolar lavage fluid (BALF) were examined. RESULTS: At one week, airway inflammation and BHR occurred similarly between young and mature mice. However, BHR disappeared at 4 week in young, whereas it persisted even at 8 weeks in mature mice. GCM, ICI and eosinophilia in BALF attenuated with time, with more remarkable reduction in young mice. BALF IL-4 level was high during first 2 weeks in both groups, while IL-2 level was significantly high at 2 weeks solely in young mice. CONCLUSIONS: Different time courses in airway inflammation and in BHR may relate to the different prognosis between childhood and adult asthma.
508
Nerve Growth Factor Induces Goblet Cell Hyperplasia And Muc5ac Production In A Human Epithelial Cell Line A. Micera1,2, B. Stampachiacchiere1, A. Lambiase2, S. Bonini2, S. Bonini3,4; 1IRCCS-G.B. Bietti Eye Foundation, Rome, ITALY, 2Dept. of Ophthalmology, University Campus Bio-Medico, Rome, ITALY, 3Internal Medicine, Second University of Naples, Naples, ITALY, 4CNR, Institute of Neurobiology and Molecular Medicine, Rome, ITALY. RATIONALE: Goblet cell hyperplasia and mucus hypersecretion are major pathophysiological features of inflammation and remodeling in asthma and other allergic and/or inflammatory disease. Airway epithelial cells produce several cytokines (IL9/IL13/IL4) and growth factors (EGF) that initiate and/or enhance inflammation and mucus hypersecretion. The aim of this study was to investigate whether Nerve Growth Factor (NGF), a pleiotropic factor taking part in both inflammation and tissue remodelling in allergic and inflammatory diseases, may influence mucus production in a human epithelial cell line. METHODS: A human bronchial epithelial cell line (EC) was expanded in dF12 medium supplemented with 5% inactivated Foetal Bovine Serum. Serum-starved sub-confluent and confluent EC were incubated with NGF (0-100ng/mL NGF) or EGF (20ng/mL) for 24-48-72 hrs. Periodic Acid Shiff staining was performed to quantify goblet cells. Total RNA and proteins were analysed for NGF, trkANGFR/p75NTR and muc1-4-5AC expression by real-time PCR, FACS/Western blot and confocal analysis. RESULTS: EC expressed both NGF and trkANGFR/p75NTR receptors and released NGF in their conditioned media. An intense PAS staining was induced 24hrs after NGF treatment. The addition of NGF or EGF induced a specific increase of muc5ACmRNA and protein in the medium. CONCLUSIONS: This is the first report showing that NGF, beside multiple activities on inflammatory and structural cells in allergic diseases and asthma, also exerts selective effects on goblet cell hyperplasia and mucus production in a human bronchial epithelial cell line. Funding: Supported in part by grants of the Italian Institute of Health and the Italian Ministry of Health, Anti-Doping Commission
509
Role of Insulin-like Growth Factor (IGF)-I in Human and Experimental Asthma H. Yoshihara1, T. Adachi1, H. Nagase1, N. Yamashita2, K. Ohta1; 1Teikyo University School Medicine, Tokyo, JAPAN, 2Musashino University, Tokyo, JAPAN. RATIONALE: One characteristic feature of bronchial asthma is airway inflammation elicited by inflammatory cells such as T cells and eosinophils. The chronic inflammation leads to epithelial remodeling which is an important mechanism of irreversible airway obstruction. Insulin-like growth factor (IGF)-I acts on fibroblasts as a progression factor to push cells toward proliferation and activation to synthesize collagen. Although PDGF is involved in remodeling of asthma, the role of IGF-I is unclear so far. METHODS: A/J mice were immunized and challenged with ovalbumin (OVA) in the presence or absence of anti-IGF-I antibody. After BALF was obtained, the lungs were fixed with 20% formaldehyde for hematoxylin-eosin and elastica van Gieson stainings. To investigate a role of IGF-I in human asthma, we measured IGF-I concentration in exhaled breath condensates (EBCs) using ECoScreen (EAGER, Germany). RESULTS: Following OVA sensitization and challenge in mice, we observed increased number of eosinophils in BALF and enhanced tissue eosinophilia. Mice treated with OVA had both thickening of the airway wall and deposition of pink-stained elastic fibers. The aforementioned phenomenon induced by OVA was markedly attenuated by anti-IGF-I treatment. The level of IGF-I in EBCs was significantly higher than those from normal subjects. CONCLUSIONS: Our results suggest that IGF-I has a critical role in inflammation and remodeling of asthma. The pharmacological targeting of IGF-I can be a new strategy to treat asthma, especially that with irreversible airway remodeling. Funding: Teikyo University School of Medicine
510
Effect of Inflammatory Cytokines on the Expression of Tie-2 Receptors in Structural Cells of the Lung T. O. Makinde1, D. K. Agrawal2; 1Creighton University, Department of Biomedical Sciences, Omaha, NE, 2Creighton University, Departments of Biomedical Sciences, Internal Medicine, Medical Microbiology and Immunology., Omaha, NE. RATIONALE: Angiopoietin-1 (Ang-1) is a primary angiogenic growth factor that induces both pro- and anti-inflammatory effects including eosinophil chemotaxis and inhibition of VEGF-induced matrix metalloproteases expression via tie-2 receptors (tie-2Rs). Here, we examined the effect of pro-inflammatory cytokines on the expression of tie2-Rs in various structural cells of the lungs. METHODS: BEAS-2b airway epithelial cells, Human lung fibroblasts, and human bronchial smooth muscle cells (HBSMCs) were stimulated for 24 hours with TNF-a (100 ng/ml), IL-1b (10 ng/ml), TGFb1 (10 ng/ml), or IL-13 (10 ng/ml) alone or in combination. Total protein and mRNA transcripts of Tie-2Rs were analyzed. RESULTS: The structural cells differentially expressed tie-2R mRNA with BEAS-2b cells being the lowest. TNF-a, IL-13, and TGF-b1 increased the mRNA expression of tie-2Rs in HBSMCs. HBSMCs expressed the tie-2R proteins only if cultured with 1o% FBS. BEAS-2b cells lost tie2R expression with increasing passages. TNF-a, IL-1 b, and IL-13 increased tie-2R expression in BEAS-2b and HBSMCs. TGF-b1 minimally increased tie-2R expression in HBSMCs with no effect in BEAS-2b. The up-regulatory actions of TNF-a and IL-1b were inhibited by IL-13 and TGF-b1 in BEAS 2b but not in HBSMCs. CONCLUSIONS: Structural cells in airways express tie-2Rs, which are regulated by pro-inflammatory cytokines. Co-stimulation of different cytokines may have an inhibitory or an additive effect on tie-2R expression, suggesting that tie-2R expression is dependent on the micro-environment and cellular condition. Potential determining factors include generation of the cell, length of culturing, absence or presence of serum. Such information could be useful to elucidate the role of tie-2Rs in airway remodeling in asthma. Funding: NIH Grant #RO1HL070885
SUNDAY
507
J ALLERGY CLIN IMMUNOL VOLUME 119, NUMBER 1
A Murine Model Of Childhood Asthma H. Mayuzumi1, Y. Ohki2, K. Tokuyama3, T. Mizuno2, H. Arakawa2, H. Mochizuki2, A. Morikawa2; 1Gunma Children‘s Medical Center, Shibukawa, JAPAN, 2Gunma University Graduate School of Medicine, Maebashi, JAPAN, 3Takasaki University of Health and Welfare, Takasaki, JAPAN. RATIONALE: Asthmatic children are more likely to outgrow their symptoms than adult patients. Thus, we wanted to know whether there were any age-related differences in the time course of the allergic airway inflammation. METHODS: BALB/C mice with different ages (young; 3 days after birth, and mature; 8 weeks of age) were sensitized with ovalbumin (OVA). Then, animals were challenged with aerosolized OVA during 1, 2, 4 or 8 consecutive weeks. Bronchial hyperresponsiveness (BHR), serum IgE levels, the degrees of inflammatory cell infiltration (ICI) and goblet cell metaplasia (GCM) in the airways, and the number of eosinophils and cytokine levels in bronchoalveolar lavage fluid (BALF) were examined. RESULTS: At one week, airway inflammation and BHR occurred similarly between young and mature mice. However, BHR disappeared at 4 week in young, whereas it persisted even at 8 weeks in mature mice. GCM, ICI and eosinophilia in BALF attenuated with time, with more remarkable reduction in young mice. BALF IL-4 level was high during first 2 weeks in both groups, while IL-2 level was significantly high at 2 weeks solely in young mice. CONCLUSIONS: Different time courses in airway inflammation and in BHR may relate to the different prognosis between childhood and adult asthma.
508
Nerve Growth Factor Induces Goblet Cell Hyperplasia And Muc5ac Production In A Human Epithelial Cell Line A. Micera1,2, B. Stampachiacchiere1, A. Lambiase2, S. Bonini2, S. Bonini3,4; 1IRCCS-G.B. Bietti Eye Foundation, Rome, ITALY, 2Dept. of Ophthalmology, University Campus Bio-Medico, Rome, ITALY, 3Internal Medicine, Second University of Naples, Naples, ITALY, 4CNR, Institute of Neurobiology and Molecular Medicine, Rome, ITALY. RATIONALE: Goblet cell hyperplasia and mucus hypersecretion are major pathophysiological features of inflammation and remodeling in asthma and other allergic and/or inflammatory disease. Airway epithelial cells produce several cytokines (IL9/IL13/IL4) and growth factors (EGF) that initiate and/or enhance inflammation and mucus hypersecretion. The aim of this study was to investigate whether Nerve Growth Factor (NGF), a pleiotropic factor taking part in both inflammation and tissue remodelling in allergic and inflammatory diseases, may influence mucus production in a human epithelial cell line. METHODS: A human bronchial epithelial cell line (EC) was expanded in dF12 medium supplemented with 5% inactivated Foetal Bovine Serum. Serum-starved sub-confluent and confluent EC were incubated with NGF (0-100ng/mL NGF) or EGF (20ng/mL) for 24-48-72 hrs. Periodic Acid Shiff staining was performed to quantify goblet cells. Total RNA and proteins were analysed for NGF, trkANGFR/p75NTR and muc1-4-5AC expression by real-time PCR, FACS/Western blot and confocal analysis. RESULTS: EC expressed both NGF and trkANGFR/p75NTR receptors and released NGF in their conditioned media. An intense PAS staining was induced 24hrs after NGF treatment. The addition of NGF or EGF induced a specific increase of muc5ACmRNA and protein in the medium. CONCLUSIONS: This is the first report showing that NGF, beside multiple activities on inflammatory and structural cells in allergic diseases and asthma, also exerts selective effects on goblet cell hyperplasia and mucus production in a human bronchial epithelial cell line. Funding: Supported in part by grants of the Italian Institute of Health and the Italian Ministry of Health, Anti-Doping Commission
509
Role of Insulin-like Growth Factor (IGF)-I in Human and Experimental Asthma H. Yoshihara1, T. Adachi1, H. Nagase1, N. Yamashita2, K. Ohta1; 1Teikyo University School Medicine, Tokyo, JAPAN, 2Musashino University, Tokyo, JAPAN. RATIONALE: One characteristic feature of bronchial asthma is airway inflammation elicited by inflammatory cells such as T cells and eosinophils. The chronic inflammation leads to epithelial remodeling which is an important mechanism of irreversible airway obstruction. Insulin-like growth factor (IGF)-I acts on fibroblasts as a progression factor to push cells toward proliferation and activation to synthesize collagen. Although PDGF is involved in remodeling of asthma, the role of IGF-I is unclear so far. METHODS: A/J mice were immunized and challenged with ovalbumin (OVA) in the presence or absence of anti-IGF-I antibody. After BALF was obtained, the lungs were fixed with 20% formaldehyde for hematoxylin-eosin and elastica van Gieson stainings. To investigate a role of IGF-I in human asthma, we measured IGF-I concentration in exhaled breath condensates (EBCs) using ECoScreen (EAGER, Germany). RESULTS: Following OVA sensitization and challenge in mice, we observed increased number of eosinophils in BALF and enhanced tissue eosinophilia. Mice treated with OVA had both thickening of the airway wall and deposition of pink-stained elastic fibers. The aforementioned phenomenon induced by OVA was markedly attenuated by anti-IGF-I treatment. The level of IGF-I in EBCs was significantly higher than those from normal subjects. CONCLUSIONS: Our results suggest that IGF-I has a critical role in inflammation and remodeling of asthma. The pharmacological targeting of IGF-I can be a new strategy to treat asthma, especially that with irreversible airway remodeling. Funding: Teikyo University School of Medicine
510
Effect of Inflammatory Cytokines on the Expression of Tie-2 Receptors in Structural Cells of the Lung T. O. Makinde1, D. K. Agrawal2; 1Creighton University, Department of Biomedical Sciences, Omaha, NE, 2Creighton University, Departments of Biomedical Sciences, Internal Medicine, Medical Microbiology and Immunology., Omaha, NE. RATIONALE: Angiopoietin-1 (Ang-1) is a primary angiogenic growth factor that induces both pro- and anti-inflammatory effects including eosinophil chemotaxis and inhibition of VEGF-induced matrix metalloproteases expression via tie-2 receptors (tie-2Rs). Here, we examined the effect of pro-inflammatory cytokines on the expression of tie2-Rs in various structural cells of the lungs. METHODS: BEAS-2b airway epithelial cells, Human lung fibroblasts, and human bronchial smooth muscle cells (HBSMCs) were stimulated for 24 hours with TNF-a (100 ng/ml), IL-1b (10 ng/ml), TGFb1 (10 ng/ml), or IL-13 (10 ng/ml) alone or in combination. Total protein and mRNA transcripts of Tie-2Rs were analyzed. RESULTS: The structural cells differentially expressed tie-2R mRNA with BEAS-2b cells being the lowest. TNF-a, IL-13, and TGF-b1 increased the mRNA expression of tie-2Rs in HBSMCs. HBSMCs expressed the tie-2R proteins only if cultured with 1o% FBS. BEAS-2b cells lost tie2R expression with increasing passages. TNF-a, IL-1 b, and IL-13 increased tie-2R expression in BEAS-2b and HBSMCs. TGF-b1 minimally increased tie-2R expression in HBSMCs with no effect in BEAS-2b. The up-regulatory actions of TNF-a and IL-1b were inhibited by IL-13 and TGF-b1 in BEAS 2b but not in HBSMCs. CONCLUSIONS: Structural cells in airways express tie-2Rs, which are regulated by pro-inflammatory cytokines. Co-stimulation of different cytokines may have an inhibitory or an additive effect on tie-2R expression, suggesting that tie-2R expression is dependent on the micro-environment and cellular condition. Potential determining factors include generation of the cell, length of culturing, absence or presence of serum. Such information could be useful to elucidate the role of tie-2Rs in airway remodeling in asthma. Funding: NIH Grant #RO1HL070885
SUNDAY
507