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Jagged1, a Ligand for Notch Receptor Expressed on Dendritic Cells, Regulates Airway Hyperresponsiveness and Inflammation by Initiating Th2 Responses
Abstracts S153
J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 2
584
585
STAT1 Negatively Regulates Basophil IL-4 Expression Induced by Respiratory Syncytial Vrus Infection M. L. Moore1, V. V. Parekh1, L. Van Kaer1, R. D. Collins1, K. Goleniewska1, M. H. Chi1, D. Mitchell1, J. E. Durbin2, R. S. Peebles, Jr.1; 1Vanderbilt University Medical Center, Nashville, TN, 2The Ohio State University, Columbus, OH. RATIONALE: BALB/c and STAT12/2 mice provide models of Th1 and enhanced Th2 inflammation in respiratory syncytial virus (RSV) infection, respectively. We analyzed the cellular source of IL-4 in RSV-infected BALB/c and STAT12/2 mice. METHODS: Mice were infected with RSV. Lungs were harvested at time points and homogenized. IL-41 cells were identified by intracellular cytokine staining and flow cytometry, sorted, and stained with Wright-Giemsa. IL-41 cells were also identified using BALB/c IL4 GPF reporter mice (4get) and STAT12/24get mice. Natural killer T (NKT) cells as a source of IL-4 was investigated using CD1d tetramer analyses and by using NKT cell-deficient CD1d2/2STAT12/2 mice. RESULTS: CD3-CD49b1 cells, not T cells, were the predominant source of IL-4 in the lungs of RSV-infected BALB/c and STAT12/2 mice. CD3-CD49b1IL-41 cells sorted from the lungs of RSV-infected STAT12/2 mice and stained with Wright-Giemsa were granulocytes. Using 4get and STAT12/24get mice, we identified these cells as basophils (CD32CD49b1FceRI1c-kit2). RSV infection resulted in more IL-4-expressing basophils in the lungs of STAT12/2 mice than in wild-type BALB/c mice.We ruled out NK cells, NKT cells, mast cells, and eosinophils as IL-4 expressors in RSV infection by flow cytometry. We also ruled out NKT cells as IL-4 expressors in RSV-infected STAT12/2 mice using CD1d2/2STAT12/2 mice. Basophils and, to a lesser extent, T cells, expressed IL-4 in RSV-infected STAT12/2 mice. Basophils were the only cells that expressed IL-4 in the lungs of RSV-infected BALB/c mice. CONCLUSIONS: Basophils were the major source of IL-4 in the lung in primary RSV infection. STAT1 modulated RSV-induced basophilia.
586
Jagged1, a Ligand for Notch Receptor Expressed on Dendritic Cells, Regulates Airway Hyperresponsiveness and Inflammation by Initiating Th2 Responses M. Okamoto, K. Takeda, H. Matsuda, A. Joetham, J. Domenico, J. J. Lucas, E. W. Gelfand; National Jewish Health, Denver, CO. RATIONALE: Lipopolysaccaride (LPS) is reported to induce opposing immune responses, Th1 vs. Th2. Activation of a Th2 phenotype is pivotal in development of airway hyperresponsiveness (AHR) and inflammation. We hypothesized that Th2 differentiation was induced through the activation of DCs by antigen and LPS, increasing Jagged1 expression and signaling and differentiating Th2 cells through Notch. METHODS: Bone marrow-derived dendritic cells (BMDC) were cultured with purified LPS-free ovalbumin (OVA) with/without added LPS and analyzed for Notch ligand expression. To assess the importance of Jagged1 in AHR and inflammation after transfer of OVA-pulsed BMDCs, the following mice were challenged to OVA: (1) IL-4-deficient recipients of naive CD4 1 T cells treated with the Notch inhibitor gamma secretase inhibitor (GSI); (2) wild-type mice, recipients of BMDC treated with LPS-free OVA, treated with Jagged1-Fc; (3) IL-4-deficient recipients of naive CD4 1 T cells and OVA-pulsed, Jagged1-siRNA transfected BMDC. RESULTS: Jagged1 was markedly induced on BMDC pulsed with purified OVA and low levels of LPS but not with LPS-free OVA. (1) IL-4-deficient recipients of GSI-treated compared to non-treated CD4 1 T cells developed significantly lower levels of AHR and BAL eosinophilia. (2) Treatment of wild-type mice with Jagged1-Fc enhanced AHR and airway inflammation whereas (3) IL-4-deficient recipients of OVA-pulsed, Jagged1-siRNA transfected BMDC failed to develop AHR or airway inflammation. CONCLUSIONS: Following upregulation of Jagged1 on DCs by antigen and LPS, Jagged1-Notch signaling plays an important role in Th2 differentiation and in development of allergen-induced AHR and inflammation.
SUNDAY
CD41CD45RBLow T Cells Are Critical In Regulating Esophageal Eosinophilia In Mice X. Zhu, M. Wang, C. H. Crump, M. E. Rothenberg, A. Mishra; Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. RATIONALE: Eosinophilic esophagitis (EE) is an emerging disease and we previously demonstrated that adaptive T cell immunity is critical in the disease pathogenesis. However, the specific subsets of T cells that promote esophageal eosinophilia are not yet understood. Therefore, we focused our studies to examine the role of CD45RB T subsets, as the pathogenic and regulatory activity of T cells depends on the expression levels of CD45RB. METHODS: A flow cytometric analysis was performed to examine the frequency of CD45RBhigh and CD45RBlow T cell subsets in the mouse esophagus. A real time PCR analysis was performed to examine the pathogenic and anti-inflammatory characteristics of both subsets of T cells. In addition, we adoptively transferred these purified cell subsets in mice with experimental EE and examined their role in the disease pathogenesis. RESULTS: An increase of CD41CD45RBhigh and a decrease of CD41CD45RBlow cells were observed in mouse esophagus following the induction of experimental EE. A relatively high transcript level of IL-5 and IL-13 in CD41CD45RBhigh and IL-10 and TGF-b1 in CD41CD45RBlow spleen purified cells was observed. Further, our initial experimentation revealed that mice adoptively transferred with 4X105 CD41CD45RBlow T cells/week were protected from the induction of esophageal eosinophilia compared to control mice that did not received adoptively transferred cells. Mice receiving CD41CD45RBhigh T cell had no significant change in the number of eosinophils in the esophagus compared to control mice following allergen challenge. CONCLUSIONS: CD41CD45RBlow T cells have a critical role in downregulating allergen-induced eosinophil recruitment to the mouse esophagus.
J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 2
584
585
STAT1 Negatively Regulates Basophil IL-4 Expression Induced by Respiratory Syncytial Vrus Infection M. L. Moore1, V. V. Parekh1, L. Van Kaer1, R. D. Collins1, K. Goleniewska1, M. H. Chi1, D. Mitchell1, J. E. Durbin2, R. S. Peebles, Jr.1; 1Vanderbilt University Medical Center, Nashville, TN, 2The Ohio State University, Columbus, OH. RATIONALE: BALB/c and STAT12/2 mice provide models of Th1 and enhanced Th2 inflammation in respiratory syncytial virus (RSV) infection, respectively. We analyzed the cellular source of IL-4 in RSV-infected BALB/c and STAT12/2 mice. METHODS: Mice were infected with RSV. Lungs were harvested at time points and homogenized. IL-41 cells were identified by intracellular cytokine staining and flow cytometry, sorted, and stained with Wright-Giemsa. IL-41 cells were also identified using BALB/c IL4 GPF reporter mice (4get) and STAT12/24get mice. Natural killer T (NKT) cells as a source of IL-4 was investigated using CD1d tetramer analyses and by using NKT cell-deficient CD1d2/2STAT12/2 mice. RESULTS: CD3-CD49b1 cells, not T cells, were the predominant source of IL-4 in the lungs of RSV-infected BALB/c and STAT12/2 mice. CD3-CD49b1IL-41 cells sorted from the lungs of RSV-infected STAT12/2 mice and stained with Wright-Giemsa were granulocytes. Using 4get and STAT12/24get mice, we identified these cells as basophils (CD32CD49b1FceRI1c-kit2). RSV infection resulted in more IL-4-expressing basophils in the lungs of STAT12/2 mice than in wild-type BALB/c mice.We ruled out NK cells, NKT cells, mast cells, and eosinophils as IL-4 expressors in RSV infection by flow cytometry. We also ruled out NKT cells as IL-4 expressors in RSV-infected STAT12/2 mice using CD1d2/2STAT12/2 mice. Basophils and, to a lesser extent, T cells, expressed IL-4 in RSV-infected STAT12/2 mice. Basophils were the only cells that expressed IL-4 in the lungs of RSV-infected BALB/c mice. CONCLUSIONS: Basophils were the major source of IL-4 in the lung in primary RSV infection. STAT1 modulated RSV-induced basophilia.
586
Jagged1, a Ligand for Notch Receptor Expressed on Dendritic Cells, Regulates Airway Hyperresponsiveness and Inflammation by Initiating Th2 Responses M. Okamoto, K. Takeda, H. Matsuda, A. Joetham, J. Domenico, J. J. Lucas, E. W. Gelfand; National Jewish Health, Denver, CO. RATIONALE: Lipopolysaccaride (LPS) is reported to induce opposing immune responses, Th1 vs. Th2. Activation of a Th2 phenotype is pivotal in development of airway hyperresponsiveness (AHR) and inflammation. We hypothesized that Th2 differentiation was induced through the activation of DCs by antigen and LPS, increasing Jagged1 expression and signaling and differentiating Th2 cells through Notch. METHODS: Bone marrow-derived dendritic cells (BMDC) were cultured with purified LPS-free ovalbumin (OVA) with/without added LPS and analyzed for Notch ligand expression. To assess the importance of Jagged1 in AHR and inflammation after transfer of OVA-pulsed BMDCs, the following mice were challenged to OVA: (1) IL-4-deficient recipients of naive CD4 1 T cells treated with the Notch inhibitor gamma secretase inhibitor (GSI); (2) wild-type mice, recipients of BMDC treated with LPS-free OVA, treated with Jagged1-Fc; (3) IL-4-deficient recipients of naive CD4 1 T cells and OVA-pulsed, Jagged1-siRNA transfected BMDC. RESULTS: Jagged1 was markedly induced on BMDC pulsed with purified OVA and low levels of LPS but not with LPS-free OVA. (1) IL-4-deficient recipients of GSI-treated compared to non-treated CD4 1 T cells developed significantly lower levels of AHR and BAL eosinophilia. (2) Treatment of wild-type mice with Jagged1-Fc enhanced AHR and airway inflammation whereas (3) IL-4-deficient recipients of OVA-pulsed, Jagged1-siRNA transfected BMDC failed to develop AHR or airway inflammation. CONCLUSIONS: Following upregulation of Jagged1 on DCs by antigen and LPS, Jagged1-Notch signaling plays an important role in Th2 differentiation and in development of allergen-induced AHR and inflammation.
SUNDAY
CD41CD45RBLow T Cells Are Critical In Regulating Esophageal Eosinophilia In Mice X. Zhu, M. Wang, C. H. Crump, M. E. Rothenberg, A. Mishra; Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. RATIONALE: Eosinophilic esophagitis (EE) is an emerging disease and we previously demonstrated that adaptive T cell immunity is critical in the disease pathogenesis. However, the specific subsets of T cells that promote esophageal eosinophilia are not yet understood. Therefore, we focused our studies to examine the role of CD45RB T subsets, as the pathogenic and regulatory activity of T cells depends on the expression levels of CD45RB. METHODS: A flow cytometric analysis was performed to examine the frequency of CD45RBhigh and CD45RBlow T cell subsets in the mouse esophagus. A real time PCR analysis was performed to examine the pathogenic and anti-inflammatory characteristics of both subsets of T cells. In addition, we adoptively transferred these purified cell subsets in mice with experimental EE and examined their role in the disease pathogenesis. RESULTS: An increase of CD41CD45RBhigh and a decrease of CD41CD45RBlow cells were observed in mouse esophagus following the induction of experimental EE. A relatively high transcript level of IL-5 and IL-13 in CD41CD45RBhigh and IL-10 and TGF-b1 in CD41CD45RBlow spleen purified cells was observed. Further, our initial experimentation revealed that mice adoptively transferred with 4X105 CD41CD45RBlow T cells/week were protected from the induction of esophageal eosinophilia compared to control mice that did not received adoptively transferred cells. Mice receiving CD41CD45RBhigh T cell had no significant change in the number of eosinophils in the esophagus compared to control mice following allergen challenge. CONCLUSIONS: CD41CD45RBlow T cells have a critical role in downregulating allergen-induced eosinophil recruitment to the mouse esophagus.