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Inhibiting Pollen Nadph Oxidase-induced Signal By Intrapulmonary Administration Of Antioxidants Blocks Allergic Airway Inflammation
S128 Abstracts
503
SUNDAY
The Effect of Daily Apolipoprotein A-I Mimetic Therapy on T15Type Autoantibodies Against Oxidized Phosphatidylcholine in a Murine Model of Asthma K. S. Konduri1, S. D. Nandedkar1, D. Weilrauch1, D. A. Rickaby1, N. Duzgunes2, K. A. Pritchard1; 1Medical College of Wisconsin and Zablocki VA Medical Center, Milwaukee, WI, 2University of Pacific School of Dentistry, San Franscisco, CA. RATIONALE: Phospholipid derived proinflammatory lipids have been shown to regulate mononuclear and vascular cell dysfunction in vascular disease. T15-type antibodies bind proinflammatory lipids such as oxidized phosphatidylcholine (ox-PC) and therefore may be utilized as a marker of inflammation. The apolipoprotein (apo) A-I mimetic, D-4F has been shown to bind and remove pro-inflammatory lipids and therefore may play a pivotal role in decreasing pulmonary inflammation similar to what occurs in asthma. METHODS: C57/Black 6 mice were sensitized with ovalbumin, divided into two groups and received: (1) intranasal D-4F (20 mg) once a day for 4 weeks or (2) no treatment for 4 weeks. Normal mice were also maintained as additional controls. At the time of sacrifice samples were collected for eosinophil peroxidase activity (EPO) in the bronchioalveolar lavage fluid (BAL), lung histology, and serum IgE levels. Data were analyzed to compare sensitized D-4F treated group with the sensitized untreated and normal,untreated unsensitized groups using student t test. RESULTS: D-4F therapy resulted in decreases in ox-PC,EPO, serum IgE levels, and inflammation on lung histology. The therapy was tolerated without evidence of toxicity. CONCLUSIONS: D-4F therapy resulted in a significant decrease in lung inflammation,serum IgE levels and EPO in experimental asthma. This study also demonstrates efficacy of D-4F to decrease proinflammatory phospholipids in the lung in a murine model of asthma. This novel drug may provide a safe and effective alternative to currently available therapies in treating asthma. Funding: Children’s Research Foundation, NIH grants HL061417, HL071412
504
LILRB4 Suppresses the Development of Th2 Allergic Airway Inflammation J. J. Rao, N. A. Barrett, F. I. Hsu, Y. Kanaoka, K. F. Austen, H. R. Katz; Brigham and Women’s Hospital, Brookline, MA. RATIONALE: LILRB4 is an inhibitory receptor that suppresses effector mechanisms of pathologic inflammation driven by mast cell or neutrophil activation. However, it was not known whether LILRB4 inhibits the afferent phase of an immune response and ensuing inflammation. We therefore compared the responses of Lilrb41/1 and Lilrb4-/- mice in a model of Th2 allergic airway inflammation. METHODS: Lilrb41/1 and Lilrb4-/- mice were sensitized intranasally with 100 mg of LPS-depleted OVA and 100 ng of LPS in PBS (OVALPS) on days 0, 1, and 2. Negative control mice received PBS. On days 14, 15, 18, and 19, mice were challenged intranasally with 25 mg of LPS-depleted OVA. On day 21, BAL, lung, serum, and intrathoracic lymph node cells were obtained. The latter were cultured with 1 mg/ml of LPSdepleted OVA for 72 h. RESULTS: Both Lilrb41/1 and Lilrb4-/- OVA-LPS mice exhibited significant increases in pulmonary inflammation and Th2 cytokine production by OVA-restimulated lymph node cells, compared with PBS mice. However, Lilrb4-/- OVA-LPS mice demonstrated significantly greater increases in the number of BAL eosinophils and lymphocytes, as well as greater increases in perivascular and peribronchial inflammation, compared with Lilrb41/1 OVA-LPS mice. OVA-restimulated lymph node cells from Lilrb4-/- mice secreted significantly more IL-4, IL-5, and IL-13 than cells from Lilrb41/1 animals. In addition, serum levels of OVA-specific IgE and IgG1 were greater in Lilrb4-/- OVA-LPS mice than in Lilrb41/1 OVALPS mice. CONCLUSIONS: Our findings establish that LILRB4 counterregulates the development of Th2 adaptive immune and inflammatory responses in a model of allergic airway inflammation. Funding: NIH
J ALLERGY CLIN IMMUNOL JANUARY 2007
505
Inhibiting Pollen Nadph Oxidase-induced Signal By Intrapulmonary Administration Of Antioxidants Blocks Allergic Airway Inflammation N. G. Dharajiya1, B. K. Choudhury1, A. Bacsi2, I. Boldogh1, R. Alama3, S. Sur1; 1University of Texas Medical Branch, Galveston, TX, 2Institute of Immunology, University of Debrecen, Debrecen, HUNGARY, 3National Jewish Medical & Research Center, Denver, CO. RATIONALE: We have shown that ragweed extract (RWE) contains NADPH oxidases that induce oxidative stress in the airways independent of adaptive immunity (signal 1) and augment antigen (signal 2)-induced allergic airway inflammation. Our objective was to test whether inhibiting signal 1 by administering antioxidants inhibits allergic airway inflammation in sensitized mice. METHODS: The ability of ascorbic acid (AA), N-acetyl cystenine (NAC), Tocopherol (Toc) and reduced glutathione (GSH) to scavenge pollen NADPH oxidase generated ROS was measured in cultured epithelial cells. These antioxidants were administered locally to inhibit signal 1 in the airways of RWE sensitized mice. Histology and special staining were performed to determine recruitment of inflammatory and mucin producing cells in the airways. Quantitative RT-PCR was performed to measure changes in Clca3 mRNA. RESULTS: Antioxidants inhibited ROS generation by pollen NADPH oxidases and intracellular ROS generation in cultured epithelial cells. AA in combination with NAC or Toc significantly decreased RWE induced ROS levels in cultured bronchial epithelial cells. Antioxidants increased antioxidant potential in the airways and decreased RWE challenge-induced 4-HNE adduct formation and upregulation of Clca3 in lungs, increase in airway mucin production, and recruitment of eosinophils and total inflammatory cells into the airways. However administration of AA1NAC 4 or 24 hrs after RWE challenge failed to inhibit allergic inflammation. CONCLUSIONS: Antioxidants scavenge ROS generated by RWE NADPH oxidases and inhibit intracellular ROS generation from bronchial epithelial cells cultured with RWE. Increasing antioxidant potential in the airways is sufficient to prevent pollen NADPH oxidases (signal 1)-mediated augmentation of allergic airway inflammation. Funding: NIH
506
IL-4 Bypasses Innate Immune Signals in Inhalational Priming A. Dittrich, K. Bottomly; Yale University School of Medicine, New Haven, CT. RATIONALE: Little is known about the mechanisms that facilitate sensitization towards new allergens in mono-sensitized individuals. We have shown that IL-4 is involved in facilitating priming of antigen-specific T cells and therefore asked if IL-4 can bypass signals via the innate immune system, a prerequisite for inhalational priming towards inocuous proteins. METHODS: Mice were primed with antigen 1 IL-4 or LPS and challenged with antigen alone. Flow cytometry was used to analyze phenotypic changes in lung and mediastinal lymph node DCs. DCs were isolated from mediastinal lymph nodes and co-cultured with transgenic T cells. In vivo T cell proliferation was assessed after transfer of CFSE-labelled transgenic T cells. RESULTS: Our studies show that co-administration of IL-4 with antigen during priming obviates the need for signals via LPS or the common adaptor protein MyD88 in the induction of a pulmonary Th2 response. Co-administration of IL-4 with antigen induces distinct phenotypical changes of lung and lymph node DCs and cocultures indicate that IL-4 induces sufficient migration and maturation of DCs to prime T cells. This is underlined by results showing that in vivo T cell proliferation in animals treated with antigen and IL-4 or LPS is equivocal. CONCLUSIONS: Our results indicate that priming via IL-4 is governed by mechanisms different from the mechanisms governing priming via innate immune signals, such as LPS. They suggest that IL-4 plays a crucial role in facilitating priming towards secondary allergens in mono-sensitized individuals and may thus provide further insight into the mechanisms underlying phenomenon. Funding: Deutsche Forschungsgemeinschaft
503
SUNDAY
The Effect of Daily Apolipoprotein A-I Mimetic Therapy on T15Type Autoantibodies Against Oxidized Phosphatidylcholine in a Murine Model of Asthma K. S. Konduri1, S. D. Nandedkar1, D. Weilrauch1, D. A. Rickaby1, N. Duzgunes2, K. A. Pritchard1; 1Medical College of Wisconsin and Zablocki VA Medical Center, Milwaukee, WI, 2University of Pacific School of Dentistry, San Franscisco, CA. RATIONALE: Phospholipid derived proinflammatory lipids have been shown to regulate mononuclear and vascular cell dysfunction in vascular disease. T15-type antibodies bind proinflammatory lipids such as oxidized phosphatidylcholine (ox-PC) and therefore may be utilized as a marker of inflammation. The apolipoprotein (apo) A-I mimetic, D-4F has been shown to bind and remove pro-inflammatory lipids and therefore may play a pivotal role in decreasing pulmonary inflammation similar to what occurs in asthma. METHODS: C57/Black 6 mice were sensitized with ovalbumin, divided into two groups and received: (1) intranasal D-4F (20 mg) once a day for 4 weeks or (2) no treatment for 4 weeks. Normal mice were also maintained as additional controls. At the time of sacrifice samples were collected for eosinophil peroxidase activity (EPO) in the bronchioalveolar lavage fluid (BAL), lung histology, and serum IgE levels. Data were analyzed to compare sensitized D-4F treated group with the sensitized untreated and normal,untreated unsensitized groups using student t test. RESULTS: D-4F therapy resulted in decreases in ox-PC,EPO, serum IgE levels, and inflammation on lung histology. The therapy was tolerated without evidence of toxicity. CONCLUSIONS: D-4F therapy resulted in a significant decrease in lung inflammation,serum IgE levels and EPO in experimental asthma. This study also demonstrates efficacy of D-4F to decrease proinflammatory phospholipids in the lung in a murine model of asthma. This novel drug may provide a safe and effective alternative to currently available therapies in treating asthma. Funding: Children’s Research Foundation, NIH grants HL061417, HL071412
504
LILRB4 Suppresses the Development of Th2 Allergic Airway Inflammation J. J. Rao, N. A. Barrett, F. I. Hsu, Y. Kanaoka, K. F. Austen, H. R. Katz; Brigham and Women’s Hospital, Brookline, MA. RATIONALE: LILRB4 is an inhibitory receptor that suppresses effector mechanisms of pathologic inflammation driven by mast cell or neutrophil activation. However, it was not known whether LILRB4 inhibits the afferent phase of an immune response and ensuing inflammation. We therefore compared the responses of Lilrb41/1 and Lilrb4-/- mice in a model of Th2 allergic airway inflammation. METHODS: Lilrb41/1 and Lilrb4-/- mice were sensitized intranasally with 100 mg of LPS-depleted OVA and 100 ng of LPS in PBS (OVALPS) on days 0, 1, and 2. Negative control mice received PBS. On days 14, 15, 18, and 19, mice were challenged intranasally with 25 mg of LPS-depleted OVA. On day 21, BAL, lung, serum, and intrathoracic lymph node cells were obtained. The latter were cultured with 1 mg/ml of LPSdepleted OVA for 72 h. RESULTS: Both Lilrb41/1 and Lilrb4-/- OVA-LPS mice exhibited significant increases in pulmonary inflammation and Th2 cytokine production by OVA-restimulated lymph node cells, compared with PBS mice. However, Lilrb4-/- OVA-LPS mice demonstrated significantly greater increases in the number of BAL eosinophils and lymphocytes, as well as greater increases in perivascular and peribronchial inflammation, compared with Lilrb41/1 OVA-LPS mice. OVA-restimulated lymph node cells from Lilrb4-/- mice secreted significantly more IL-4, IL-5, and IL-13 than cells from Lilrb41/1 animals. In addition, serum levels of OVA-specific IgE and IgG1 were greater in Lilrb4-/- OVA-LPS mice than in Lilrb41/1 OVALPS mice. CONCLUSIONS: Our findings establish that LILRB4 counterregulates the development of Th2 adaptive immune and inflammatory responses in a model of allergic airway inflammation. Funding: NIH
J ALLERGY CLIN IMMUNOL JANUARY 2007
505
Inhibiting Pollen Nadph Oxidase-induced Signal By Intrapulmonary Administration Of Antioxidants Blocks Allergic Airway Inflammation N. G. Dharajiya1, B. K. Choudhury1, A. Bacsi2, I. Boldogh1, R. Alama3, S. Sur1; 1University of Texas Medical Branch, Galveston, TX, 2Institute of Immunology, University of Debrecen, Debrecen, HUNGARY, 3National Jewish Medical & Research Center, Denver, CO. RATIONALE: We have shown that ragweed extract (RWE) contains NADPH oxidases that induce oxidative stress in the airways independent of adaptive immunity (signal 1) and augment antigen (signal 2)-induced allergic airway inflammation. Our objective was to test whether inhibiting signal 1 by administering antioxidants inhibits allergic airway inflammation in sensitized mice. METHODS: The ability of ascorbic acid (AA), N-acetyl cystenine (NAC), Tocopherol (Toc) and reduced glutathione (GSH) to scavenge pollen NADPH oxidase generated ROS was measured in cultured epithelial cells. These antioxidants were administered locally to inhibit signal 1 in the airways of RWE sensitized mice. Histology and special staining were performed to determine recruitment of inflammatory and mucin producing cells in the airways. Quantitative RT-PCR was performed to measure changes in Clca3 mRNA. RESULTS: Antioxidants inhibited ROS generation by pollen NADPH oxidases and intracellular ROS generation in cultured epithelial cells. AA in combination with NAC or Toc significantly decreased RWE induced ROS levels in cultured bronchial epithelial cells. Antioxidants increased antioxidant potential in the airways and decreased RWE challenge-induced 4-HNE adduct formation and upregulation of Clca3 in lungs, increase in airway mucin production, and recruitment of eosinophils and total inflammatory cells into the airways. However administration of AA1NAC 4 or 24 hrs after RWE challenge failed to inhibit allergic inflammation. CONCLUSIONS: Antioxidants scavenge ROS generated by RWE NADPH oxidases and inhibit intracellular ROS generation from bronchial epithelial cells cultured with RWE. Increasing antioxidant potential in the airways is sufficient to prevent pollen NADPH oxidases (signal 1)-mediated augmentation of allergic airway inflammation. Funding: NIH
506
IL-4 Bypasses Innate Immune Signals in Inhalational Priming A. Dittrich, K. Bottomly; Yale University School of Medicine, New Haven, CT. RATIONALE: Little is known about the mechanisms that facilitate sensitization towards new allergens in mono-sensitized individuals. We have shown that IL-4 is involved in facilitating priming of antigen-specific T cells and therefore asked if IL-4 can bypass signals via the innate immune system, a prerequisite for inhalational priming towards inocuous proteins. METHODS: Mice were primed with antigen 1 IL-4 or LPS and challenged with antigen alone. Flow cytometry was used to analyze phenotypic changes in lung and mediastinal lymph node DCs. DCs were isolated from mediastinal lymph nodes and co-cultured with transgenic T cells. In vivo T cell proliferation was assessed after transfer of CFSE-labelled transgenic T cells. RESULTS: Our studies show that co-administration of IL-4 with antigen during priming obviates the need for signals via LPS or the common adaptor protein MyD88 in the induction of a pulmonary Th2 response. Co-administration of IL-4 with antigen induces distinct phenotypical changes of lung and lymph node DCs and cocultures indicate that IL-4 induces sufficient migration and maturation of DCs to prime T cells. This is underlined by results showing that in vivo T cell proliferation in animals treated with antigen and IL-4 or LPS is equivocal. CONCLUSIONS: Our results indicate that priming via IL-4 is governed by mechanisms different from the mechanisms governing priming via innate immune signals, such as LPS. They suggest that IL-4 plays a crucial role in facilitating priming towards secondary allergens in mono-sensitized individuals and may thus provide further insight into the mechanisms underlying phenomenon. Funding: Deutsche Forschungsgemeinschaft