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Eosinophil Granular Proteins Specifically Major Basic Protein Damage Bronchial Epithelial Cells Infected with Respiratory Syncytial Virus
AB116 Abstracts
441
SUNDAY
Induced-Tolerogenic Dendritic Cells That Promote Tolerance And De Novo Differentiation Of Regulatory T Cells R. A. Maldonado1,2, L. C. Perdomo2, F. Vascotto2, L. Francisco2, A. T. Bauquet3, V. K. Kuchroo3, A. H. Sharpe2, U. H. von Andrian2; 1Selecta Biosciences Inc, Watertown, MA, 2Harvard Medical School, Boston, MA, 3Center for Neurologic Diseases and Brigham and Womens Hospital, Boston, MA. RATIONALE: We hypothesized that incubation of Dendritic Cells (DC) with tolerogenic factors may induce tolerogenic function artificially in order to use these cells as research and therapeutic tools. METHODS: several combinations of compounds were tested for their effect on the tolerogenic function of DC. The capacity of these cells to induce deletion of antigen-specific cells and induce the differentiation of na€ıve CD4+ T cells (Tn) into Treg was surveyed in vitro as well as their capacity to ameliorate autoimmune conditions in vivo. RESULTS: mouse splenic and bone marrow-derived DC and human monocyte-derived DC treated in vitro with rapamycin and TGFb induce Treg differentiation. The resulting adaptive Treg (aTreg) are phenotypically and functionally equivalent to natural-occurring Treg (nTreg) in vitro. These induced-tolerogenic DC (itDC) cotreated with toll-like receptor (TLR) agonists remain tolerogenic, required direct itDC-Tn contact, TCR stimulation, but no cell division or IL-6, IL-10, TGFb, IDO1, FasL or EBI3 to stimulate Treg differentiation. Further, a marked reduction in the numbers of Tn in the first two days could be observed previous to Treg differentiation. Using the Experimental Autoimmune Encephalomyelitis model (EAE) we demonstrate that infusions of itDC blocked the progress of the disease while significantly diminishing the number of effector T cells and increasing Treg. CONCLUSIONS: itDC can shape the repertoire of antigen specific T cells through the elimination of antigen specific cells and/or the induction aTreg that suppress autoimmune responses in vivo.
442
Modulation of Eosinophil Responsiveness to TSLP-Mediated Degranulation E. B. Cook, J. L. Stahl, E. A. Schwantes, N. P. Barney, S. A. Mathur; University Wisconsin, Madison, WI. RATIONALE: Eosinophils and TSLP are increased during allergic inflammation in the epithelium of the respiratory tract, skin, and on the ocular surface. Our previous studies demonstrated that eosinophils respond to high concentrations of TSLP with degranulation. The purpose of these studies was to examine whether priming with TNFa and IL-3 (previously shown to upregulate eosinophil TSLP receptor expression) or co-incubation with primary human conjunctival epithelial cells could affect the responsiveness of eosinophils to TSLP. METHODS: Eosinophils were stimulated with various concentrations of TSLP and evaluated for release of eosinophil derived neurotoxin by ELISA. For priming experiments, degranulation was evaluated in the presence and absence of pre-treatment with TNFa and IL-3. For coincubation experiments, primary human conjunctival epithelial cells were cultured on transwell filters in 24 well plates. Eosinophils were added either apically or basally (and to epithelial cell free, control wells) and challenged with TSLP. RESULTS: Eosinophils pre-treated with TNFa and IL-3 had enhanced sensitivity to TSLP-mediated degranulation with approximately 80-fold less TSLP concentration needed to achieve comparable degranulation. Coincubation of eosinophils with conjunctival epithelial cells reduced TSLPmediated degranulation in both apical and basal compartments, suggesting that the response was not contact dependent. CONCLUSIONS: Eosinophils exhibit enhanced responsiveness to TSLP when exposed to cytokines present in allergic inflammation. Conversely, epithelial cells may provide a stabilizing influence on TSLP-mediated eosinophil degranulation.
J ALLERGY CLIN IMMUNOL FEBRUARY 2012
443
Eosinophil Granular Proteins Specifically Major Basic Protein Damage Bronchial Epithelial Cells Infected with Respiratory Syncytial Virus M. Kato1, T. Ishioka2, H. Kita3, K. Kozawa2, Y. Hayashi4, H. Kimura5; 1 Department of Allergy and Immunology, Gunma Children’s Medical Center, Shibukawa, Gunma, JAPAN, 2Gunma Prefectural Institute of Public Health and Environmental Sciences, Maebashi, Gunma, JAPAN, 3Department of Internal Medicine, Mayo Clinic, Rochester, MN, 4Department of Hematology and Oncology, Gunma Children’s Medical Center, Shibukawa, Gunma, JAPAN, 5National Institute of Infectious Diseases, Musashimurayama, Tokyo, JAPAN. RATIONALE: Respiratory syncytial (RS) virus is an important inducer of wheezing in infancy, and plays a role in the development of asthma. However, the precise mechanisms responsible for viral infection-induced exacerbations of asthma are uncertain. To elucidate the role of eosinophilic inflammation in the pathogenesis of virus-induced asthma, we investigated the effects of eosinophil granule proteins on bronchial epithelial cell infected with RS virus. METHODS: Morphological changes and cytopathic effects in human type II pulmonary alveolar epithelial cells (A549) infected with RS virus and/or eosinophil granule proteins including major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN) were observed by microscopy. Apoptosis/necrosis was evaluated by trypan blue exclusion test. We also measured 8 types of phosphorylated proteins in MBP-treated A549 cells infected with RS virus. RESULTS: Although RS virus alone did not affect cytopathic effects of A549 cells, high concentrations of MBP or combination of four granule proteins resulted in cytopathic effects. MBP or EPO, but not ECP or EDN, induced cytotoxicity and necrosis of A549 cells infected with RS virus. Furthermore, MBP induced the phosphorylation of the extracellular signalregulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (MAPK), Jun-N-terminal protein kinase (JNK), and signal transducer and activator of transcription (STAT) 3 in A549 cells infected with RS virus. CONCLUSIONS: Eosinophil granule proteins specifically MBP damage bronchial epithelial cells infected with RS virus and that MAPK family are involved in these responses, indicating that eosinophilic inflammation might be associated with pathophysiology of RS virus-induced acute exacerbations of asthma.
441
SUNDAY
Induced-Tolerogenic Dendritic Cells That Promote Tolerance And De Novo Differentiation Of Regulatory T Cells R. A. Maldonado1,2, L. C. Perdomo2, F. Vascotto2, L. Francisco2, A. T. Bauquet3, V. K. Kuchroo3, A. H. Sharpe2, U. H. von Andrian2; 1Selecta Biosciences Inc, Watertown, MA, 2Harvard Medical School, Boston, MA, 3Center for Neurologic Diseases and Brigham and Womens Hospital, Boston, MA. RATIONALE: We hypothesized that incubation of Dendritic Cells (DC) with tolerogenic factors may induce tolerogenic function artificially in order to use these cells as research and therapeutic tools. METHODS: several combinations of compounds were tested for their effect on the tolerogenic function of DC. The capacity of these cells to induce deletion of antigen-specific cells and induce the differentiation of na€ıve CD4+ T cells (Tn) into Treg was surveyed in vitro as well as their capacity to ameliorate autoimmune conditions in vivo. RESULTS: mouse splenic and bone marrow-derived DC and human monocyte-derived DC treated in vitro with rapamycin and TGFb induce Treg differentiation. The resulting adaptive Treg (aTreg) are phenotypically and functionally equivalent to natural-occurring Treg (nTreg) in vitro. These induced-tolerogenic DC (itDC) cotreated with toll-like receptor (TLR) agonists remain tolerogenic, required direct itDC-Tn contact, TCR stimulation, but no cell division or IL-6, IL-10, TGFb, IDO1, FasL or EBI3 to stimulate Treg differentiation. Further, a marked reduction in the numbers of Tn in the first two days could be observed previous to Treg differentiation. Using the Experimental Autoimmune Encephalomyelitis model (EAE) we demonstrate that infusions of itDC blocked the progress of the disease while significantly diminishing the number of effector T cells and increasing Treg. CONCLUSIONS: itDC can shape the repertoire of antigen specific T cells through the elimination of antigen specific cells and/or the induction aTreg that suppress autoimmune responses in vivo.
442
Modulation of Eosinophil Responsiveness to TSLP-Mediated Degranulation E. B. Cook, J. L. Stahl, E. A. Schwantes, N. P. Barney, S. A. Mathur; University Wisconsin, Madison, WI. RATIONALE: Eosinophils and TSLP are increased during allergic inflammation in the epithelium of the respiratory tract, skin, and on the ocular surface. Our previous studies demonstrated that eosinophils respond to high concentrations of TSLP with degranulation. The purpose of these studies was to examine whether priming with TNFa and IL-3 (previously shown to upregulate eosinophil TSLP receptor expression) or co-incubation with primary human conjunctival epithelial cells could affect the responsiveness of eosinophils to TSLP. METHODS: Eosinophils were stimulated with various concentrations of TSLP and evaluated for release of eosinophil derived neurotoxin by ELISA. For priming experiments, degranulation was evaluated in the presence and absence of pre-treatment with TNFa and IL-3. For coincubation experiments, primary human conjunctival epithelial cells were cultured on transwell filters in 24 well plates. Eosinophils were added either apically or basally (and to epithelial cell free, control wells) and challenged with TSLP. RESULTS: Eosinophils pre-treated with TNFa and IL-3 had enhanced sensitivity to TSLP-mediated degranulation with approximately 80-fold less TSLP concentration needed to achieve comparable degranulation. Coincubation of eosinophils with conjunctival epithelial cells reduced TSLPmediated degranulation in both apical and basal compartments, suggesting that the response was not contact dependent. CONCLUSIONS: Eosinophils exhibit enhanced responsiveness to TSLP when exposed to cytokines present in allergic inflammation. Conversely, epithelial cells may provide a stabilizing influence on TSLP-mediated eosinophil degranulation.
J ALLERGY CLIN IMMUNOL FEBRUARY 2012
443
Eosinophil Granular Proteins Specifically Major Basic Protein Damage Bronchial Epithelial Cells Infected with Respiratory Syncytial Virus M. Kato1, T. Ishioka2, H. Kita3, K. Kozawa2, Y. Hayashi4, H. Kimura5; 1 Department of Allergy and Immunology, Gunma Children’s Medical Center, Shibukawa, Gunma, JAPAN, 2Gunma Prefectural Institute of Public Health and Environmental Sciences, Maebashi, Gunma, JAPAN, 3Department of Internal Medicine, Mayo Clinic, Rochester, MN, 4Department of Hematology and Oncology, Gunma Children’s Medical Center, Shibukawa, Gunma, JAPAN, 5National Institute of Infectious Diseases, Musashimurayama, Tokyo, JAPAN. RATIONALE: Respiratory syncytial (RS) virus is an important inducer of wheezing in infancy, and plays a role in the development of asthma. However, the precise mechanisms responsible for viral infection-induced exacerbations of asthma are uncertain. To elucidate the role of eosinophilic inflammation in the pathogenesis of virus-induced asthma, we investigated the effects of eosinophil granule proteins on bronchial epithelial cell infected with RS virus. METHODS: Morphological changes and cytopathic effects in human type II pulmonary alveolar epithelial cells (A549) infected with RS virus and/or eosinophil granule proteins including major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN) were observed by microscopy. Apoptosis/necrosis was evaluated by trypan blue exclusion test. We also measured 8 types of phosphorylated proteins in MBP-treated A549 cells infected with RS virus. RESULTS: Although RS virus alone did not affect cytopathic effects of A549 cells, high concentrations of MBP or combination of four granule proteins resulted in cytopathic effects. MBP or EPO, but not ECP or EDN, induced cytotoxicity and necrosis of A549 cells infected with RS virus. Furthermore, MBP induced the phosphorylation of the extracellular signalregulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (MAPK), Jun-N-terminal protein kinase (JNK), and signal transducer and activator of transcription (STAT) 3 in A549 cells infected with RS virus. CONCLUSIONS: Eosinophil granule proteins specifically MBP damage bronchial epithelial cells infected with RS virus and that MAPK family are involved in these responses, indicating that eosinophilic inflammation might be associated with pathophysiology of RS virus-induced acute exacerbations of asthma.