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Human type 1 CD4 T cell cytokine responses are selectively dependent on multidrug resistance protein1
Abstracts S255
J ALLERGY CLIN IMMUNOL VOLUME 115, NUMBER 2
Association Between Cytokine Gene Polymorphisms and Otitis Media (OM) in Children With Acute Respiratory Syncytial Virus (RSV) Infection D. Skoner1, D. Gentile1, E. Kairis1, K. Barnoski1, C. Buzzard1, A. Patel1, W. Doyle2; 1Department of Pediatrics, Allegheny General Hospital, Pittsburgh, PA, 2University of Pittsburgh, Pittsburgh, PA. RATIONALE: Complications of viral respiratory infections may be influenced by specific cytokines. Cytokine gene polymorphisms moderate illness severity in a variety of inflammatory disorders. The purpose of this study was to determine whether certain cytokine gene polymorphisms are associated with the development of OM during RSV infection. METHODS: Cytokine genotyping was performed on 42 children aged eighteen months to three years (57% Caucasian; 57% male) who were prospectively evaluated for the development of OM during RSV season. DNA was extracted from buccal brushings and assayed for TNF-, IFN, IL-6, IL-10 and TGF-1 genotypes using PCR-SSP. RSV infection was confirmed by rapid EIA testing of nasal secretions and OM was diagnosed by validated pneumatic otoscopy. RESULTS: Twenty-five subjects had confirmed RSV infection. Among those subjects, TNF- and IFN- genotypes were related to the development of OM during RSV infection. The frequencies of the genotype associated with high production of TNF- were 38% versus 15% in subjects with and without OM, respectively (p<0.05). The frequencies of the genotype associated with high production of IFN- were 15% versus 33% in subjects with and without OM, respectively (p<0.05). There were no associations between IL-6, IL-10 and TGF- genotypes and any of the outcome parameters. CONCLUSIONS: These results suggest a role for certain cytokine genotypes in the pathogenesis of OM during RSV infection. If confirmed by future studies, cytokine genotyping may become a tool for identifying “at risk” children who may benefit from the selective use of preventative and/or early intervention treatments for these disorders. Funding: NIH
1015
Role of CCR4 and CCR8 in Antigen Specific T Helper 2 Cell Trafficking Into Allergic Lung M. Fukui1,2, A. M. Tager3,2, A. D. Luster1,2; 1Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Boston, MA, 2Center for Immunology and Inflammatory diseases, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, 3Pulmonary and Critical Care Unit, Massachusetts General Hospital, Boston, MA. RATIONALE: Infiltration of CD4+ T helper (Th) 2 cells into the airway is one of the characteristic features of atopic asthma. CC chemokine receptors such as CCR4 and CCR8 are highly expressed on Th2 cells and their expression is upregulated in the asthmatic lung. However, the significance of CCR4 and CCR8 expression in antigen specific trafficking of Th2 cells to the site of allergic inflammation is controversial and not well understood. METHODS: To address the specific functional role of CCR4 and of CCR8 for the in vivo recruitment of antigen specific Th2 cells in a murine model of asthma, we have generated antigen (OVA) specific Th2 cells defective in CCR4 or CCR8. We then generated and transferred highly polarized antigen specific wild-type, CCR4 KO and CCR8 KO Th2 cells into naïve recipient mice. After airway antigen challenge, trafficking of these cells was analyzed by flow cytometry. RESULTS: We have found that expression of CCR4 on Th2 cells but not CCR8 was important for optimum trafficking of antigen specific Th2 cells to the site of allergic inflammation, including thoracic lymph nodes, lung interstitium and the airway lumen, but not into peripheral lymph nodes or spleen. CONCLUSIONS: Our study supports the important role of CCR4 rather than CCR8 in regulating antigen specific trafficking of Th2 cells into asthmatic bronchi and thoracic lymph nodes. Funding: NIH
1016
Human Type 1 CD4 T Cell Cytokine Responses Are Selectively Dependent on Multidrug Resistance Protein1 J. Zhang1, M. A. Alston1, H. Huang1, R. A. Houghtling2, R. W. Pastor1, R. L. Rabin1; 1Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD, 2National Institute of Human Genome Research, Bethesda, MD. RATIONALE: Multidrug Resistance Protein1 (MRP1) belongs to subfamily C of the ATP-binding cassette transporters, and exports organic anions. We observed that leukocytes from patients with an autoimmune disease exported indo-1 at a higher rate than controls. This observation prompted the hypothesis that MRP1 contributes to the function of activated T cells. METHODS: PBMC were treated with MRP1 specific inhibitor MK571 for 30 min, and activated by superantigen. RNA expression was determined with RT-PCR or Real-Time PCR. Cytokine secretion was analyzed with Flow cytometry or ELISA. Cells were loaded with Indo-1, and calcium flux was analyzed with Flow Cytometry. For MRP1 suppression, Jurkat cells were transfected with MRP1 siRNA using a BTX ElectroSquarePorater. RESULTS: We found that MRP1 is expressed on resting memory but not naïve CD4 and CD8 T cells, but is expressed at high levels by cord blood CD4 T cells after activation through the T cell receptor. Blockade of MRP1 abrogated superantigen induced expression of IFN-, TNF-, IL10, IL-2, and CD69 by T cells. By contrast, expression of IL-4 was unaffected. Furthermore, MRP1 blockade and siRNA “gene knockdown” abrogated calcium flux in response to T cell receptor stimulation. CONCLUSIONS: Human type 1 CD4 T cell cytokine responses are selectively dependent on MRP1.
1017
TUESDAY
Effect of Glucocorticoid on the Expression of RANTES and MIP-1 in the Bronchus of Mice With Asthma W. Zhang, C. Li, R. Wu, Z. Zhang, Y. Luo, M. Li, L. Dong, X. Cai; Division of Pulmonology, Yuying Children’s Hospital,Wenzhou Medical College, Wenzhou City, Zhejiang, P.R. China., CHINA. RATIONALE: To study the effect of glucocorticoid on the expression of RANTES and MIP-1 in murine asthma. METHODS: Thirty BALB/C mice were randomly divided into control, asthma, and dexamethasone treated group. Asthma was established by the ovalbumin (OVA) sensitization/challenge method. Bronchial expression of RANTES and MIP-1 was detected by immunohistochemistry and in situ hybridization at protein and mRNA levels. RESULTS: Immunohistochemistry showed that the percentage of positive cells that express RANTES and MIP-1 (23.8 ± 4.8 %, 26.4 ± 6.2% ) were significantly elevated in asthma group as compared to control (9.5 ± 1.9%, 10.3 ± 2.5% , P<0.01), with airway epithelial cells as main source. However, their expressions (15.7 ± 2.9 %, 17.5±3.7%) was significantly lower in dexamethasone treated group (P<0.01 comparing to asthma group). mRNA expression by in situ hybridization showed the same fashion that asthma group (22.8 ± 4.8%, 23.9 ± 4.2%) was higher than control (7.9 ± 1.1%, 8.7 ± 1.8% , P<0.01). After treatment with dexamethasone, the expression in treated group fell below asthma group (12.7 ± 2.7%, 14.5 ± 3.3%, P<0.01). CONCLUSIONS: RANTES and MIP-1 are over-expressed by bronchial epithelial cells in murine asthma. Glucocorticoid’s anti-inflammatory effects are partially mediated through the downregulation of expressions of RANTES and MIP-1. Funding: Zhejiang provincial educational foundation,P.R. China
1014
J ALLERGY CLIN IMMUNOL VOLUME 115, NUMBER 2
Association Between Cytokine Gene Polymorphisms and Otitis Media (OM) in Children With Acute Respiratory Syncytial Virus (RSV) Infection D. Skoner1, D. Gentile1, E. Kairis1, K. Barnoski1, C. Buzzard1, A. Patel1, W. Doyle2; 1Department of Pediatrics, Allegheny General Hospital, Pittsburgh, PA, 2University of Pittsburgh, Pittsburgh, PA. RATIONALE: Complications of viral respiratory infections may be influenced by specific cytokines. Cytokine gene polymorphisms moderate illness severity in a variety of inflammatory disorders. The purpose of this study was to determine whether certain cytokine gene polymorphisms are associated with the development of OM during RSV infection. METHODS: Cytokine genotyping was performed on 42 children aged eighteen months to three years (57% Caucasian; 57% male) who were prospectively evaluated for the development of OM during RSV season. DNA was extracted from buccal brushings and assayed for TNF-, IFN, IL-6, IL-10 and TGF-1 genotypes using PCR-SSP. RSV infection was confirmed by rapid EIA testing of nasal secretions and OM was diagnosed by validated pneumatic otoscopy. RESULTS: Twenty-five subjects had confirmed RSV infection. Among those subjects, TNF- and IFN- genotypes were related to the development of OM during RSV infection. The frequencies of the genotype associated with high production of TNF- were 38% versus 15% in subjects with and without OM, respectively (p<0.05). The frequencies of the genotype associated with high production of IFN- were 15% versus 33% in subjects with and without OM, respectively (p<0.05). There were no associations between IL-6, IL-10 and TGF- genotypes and any of the outcome parameters. CONCLUSIONS: These results suggest a role for certain cytokine genotypes in the pathogenesis of OM during RSV infection. If confirmed by future studies, cytokine genotyping may become a tool for identifying “at risk” children who may benefit from the selective use of preventative and/or early intervention treatments for these disorders. Funding: NIH
1015
Role of CCR4 and CCR8 in Antigen Specific T Helper 2 Cell Trafficking Into Allergic Lung M. Fukui1,2, A. M. Tager3,2, A. D. Luster1,2; 1Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Boston, MA, 2Center for Immunology and Inflammatory diseases, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, 3Pulmonary and Critical Care Unit, Massachusetts General Hospital, Boston, MA. RATIONALE: Infiltration of CD4+ T helper (Th) 2 cells into the airway is one of the characteristic features of atopic asthma. CC chemokine receptors such as CCR4 and CCR8 are highly expressed on Th2 cells and their expression is upregulated in the asthmatic lung. However, the significance of CCR4 and CCR8 expression in antigen specific trafficking of Th2 cells to the site of allergic inflammation is controversial and not well understood. METHODS: To address the specific functional role of CCR4 and of CCR8 for the in vivo recruitment of antigen specific Th2 cells in a murine model of asthma, we have generated antigen (OVA) specific Th2 cells defective in CCR4 or CCR8. We then generated and transferred highly polarized antigen specific wild-type, CCR4 KO and CCR8 KO Th2 cells into naïve recipient mice. After airway antigen challenge, trafficking of these cells was analyzed by flow cytometry. RESULTS: We have found that expression of CCR4 on Th2 cells but not CCR8 was important for optimum trafficking of antigen specific Th2 cells to the site of allergic inflammation, including thoracic lymph nodes, lung interstitium and the airway lumen, but not into peripheral lymph nodes or spleen. CONCLUSIONS: Our study supports the important role of CCR4 rather than CCR8 in regulating antigen specific trafficking of Th2 cells into asthmatic bronchi and thoracic lymph nodes. Funding: NIH
1016
Human Type 1 CD4 T Cell Cytokine Responses Are Selectively Dependent on Multidrug Resistance Protein1 J. Zhang1, M. A. Alston1, H. Huang1, R. A. Houghtling2, R. W. Pastor1, R. L. Rabin1; 1Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD, 2National Institute of Human Genome Research, Bethesda, MD. RATIONALE: Multidrug Resistance Protein1 (MRP1) belongs to subfamily C of the ATP-binding cassette transporters, and exports organic anions. We observed that leukocytes from patients with an autoimmune disease exported indo-1 at a higher rate than controls. This observation prompted the hypothesis that MRP1 contributes to the function of activated T cells. METHODS: PBMC were treated with MRP1 specific inhibitor MK571 for 30 min, and activated by superantigen. RNA expression was determined with RT-PCR or Real-Time PCR. Cytokine secretion was analyzed with Flow cytometry or ELISA. Cells were loaded with Indo-1, and calcium flux was analyzed with Flow Cytometry. For MRP1 suppression, Jurkat cells were transfected with MRP1 siRNA using a BTX ElectroSquarePorater. RESULTS: We found that MRP1 is expressed on resting memory but not naïve CD4 and CD8 T cells, but is expressed at high levels by cord blood CD4 T cells after activation through the T cell receptor. Blockade of MRP1 abrogated superantigen induced expression of IFN-, TNF-, IL10, IL-2, and CD69 by T cells. By contrast, expression of IL-4 was unaffected. Furthermore, MRP1 blockade and siRNA “gene knockdown” abrogated calcium flux in response to T cell receptor stimulation. CONCLUSIONS: Human type 1 CD4 T cell cytokine responses are selectively dependent on MRP1.
1017
TUESDAY
Effect of Glucocorticoid on the Expression of RANTES and MIP-1 in the Bronchus of Mice With Asthma W. Zhang, C. Li, R. Wu, Z. Zhang, Y. Luo, M. Li, L. Dong, X. Cai; Division of Pulmonology, Yuying Children’s Hospital,Wenzhou Medical College, Wenzhou City, Zhejiang, P.R. China., CHINA. RATIONALE: To study the effect of glucocorticoid on the expression of RANTES and MIP-1 in murine asthma. METHODS: Thirty BALB/C mice were randomly divided into control, asthma, and dexamethasone treated group. Asthma was established by the ovalbumin (OVA) sensitization/challenge method. Bronchial expression of RANTES and MIP-1 was detected by immunohistochemistry and in situ hybridization at protein and mRNA levels. RESULTS: Immunohistochemistry showed that the percentage of positive cells that express RANTES and MIP-1 (23.8 ± 4.8 %, 26.4 ± 6.2% ) were significantly elevated in asthma group as compared to control (9.5 ± 1.9%, 10.3 ± 2.5% , P<0.01), with airway epithelial cells as main source. However, their expressions (15.7 ± 2.9 %, 17.5±3.7%) was significantly lower in dexamethasone treated group (P<0.01 comparing to asthma group). mRNA expression by in situ hybridization showed the same fashion that asthma group (22.8 ± 4.8%, 23.9 ± 4.2%) was higher than control (7.9 ± 1.1%, 8.7 ± 1.8% , P<0.01). After treatment with dexamethasone, the expression in treated group fell below asthma group (12.7 ± 2.7%, 14.5 ± 3.3%, P<0.01). CONCLUSIONS: RANTES and MIP-1 are over-expressed by bronchial epithelial cells in murine asthma. Glucocorticoid’s anti-inflammatory effects are partially mediated through the downregulation of expressions of RANTES and MIP-1. Funding: Zhejiang provincial educational foundation,P.R. China
1014