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Detection of Influenza Specific T-Cell Response in Allergic and Healthy Controls
Abstracts S123
J ALLERGY CLIN IMMUNOL VOLUME 121, NUMBER 2
Detection of Influenza Specific T-Cell Response in Allergic and Healthy Controls N. Kim, B. Foster, C. Prussin; Laboratory of Allergic Diseases, NIAID/ NIH, Bethesda, MD. RATIONALE: Asthma is associated with a Th2 cytokine rich milieu that may affect the generation of immune responses to pathogens and vaccines. We hypothesized that the T-cell response to influenza may have a distinct cytokine pattern in allergic asthmatic subjects relative to non-atopic healthy controls. METHODS: Carboxyfluorescein succinimidyl ester (CFSE), intracellular cytokine staining and polychromatic 9-color flow cytometry were used in a 7-day culture to detect influenza specific CD41 T cell proliferation and IL4, IL-5, IFN-g, and TNF-a expression in 7 allergic asthmatic and 5 healthy control subjects. RESULTS: The frequency of influenza (New Caledonia strain) specific T cells producing IL-4 (2.8% vs. 4.5%), IFN-g (72% vs. 78%) and TNF (53% vs. 66%) in allergic asthmatic and non-atopic control subjects, respectively, were not significantly different. IL-5 was not detectable (<0.1%) in either group. The ratio of IL-4: IFN-g was 0.4 and 0.5 respectively, which were not significantly different. Influenza Panama antigen as well as a hemagglutinin peptide pool also showed no difference between the two groups. CONCLUSIONS: Influenza specific Th2 cytokines are reproducibly detectable and quantifiable in CD4 T cells. Influenza specific T cells from allergic asthmatic subjects did not demonstrate a pattern of cytokine expression significantly different from healthy controls. Funding: NIAID, Division of Intramural Research
475
IL-5 and IFN-g Levels And Spirometric Parameters In Induced Sputum Of Patients With Asma, Allergic Rhinitis And Controls G. R. S. Segundo, S. M. G. Marra, R. Alves, D. A. O. Silva, E. A. Taketomi; Universidade Federal de Uberlaˆndia, Uberlaˆndia, BRAZIL. RATIONALE: Asthma and rinithis are frequently associated, suggesting the concept of ’’one airway, one disease’’. The aims of this study were to analyze spirometrics parameters and IFN-g and IL-5 levels in the induced sputum from patients with asthma or allergic rhinitis (AR) and non-atopic subjects. METHODS: Thirty-three subjects aged 18 to 60 years, both sexes, were analyzed. From these, eight were asthmatics and 16 had allergic rhinitis, and both groups had positive skin prick test (SPT) to aeroallergens. The nine remaining subjects were healthy non-atopics with negative SPT to aeroallergens. Spirometry was performed through evaluating the FVC, FEV1, and FEF25-75% pre- and post-bronchodilators. Induced sputum samples were collected and IFN-g and IL-5 levels were quantified by immunoassays. RESULTS: Significant pre- and post-bronchodilator variation was observed only for FEV1 with higher values in asthmatics compared to patients with AR or non-atopics. There was no significant difference in pre- and post-bronchodilator parameters among the three groups, although there was a tendency for pre-bronchodilator lower FEF25-75% values in asthmatic patients. IFN-g levels in the induced sputum showed no significant difference between the groups but, IL-5 levels were higher in patients with asthma or AR compared to non-atopics. CONCLUSIONS: Spirometric parameters have shown no implications in AR patients, but higher IL-5 levels in the induced sputum in asthma and AR patients reinforce the role of the Th2-type immune response in low respiratory airways that could contribute to the concept of ’’one airway, one disease’’. Funding: Fundac xa˜o de Amparo a` Pesquisa do Estado de Minas Gerais (FAPEMIG), Coordenac xa˜o de Aperfeic xoamento de Pessoal de Nı´vel Superior (CAPES), and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPQ)
476
Involvement Of VEGF In Nasal Inflammation Induced By Allergen G. Choi, H. Park, S. Choi, S. Shin, G. Hur, S. Kim, D. Nahm, H. Park; Ajou University School of Medicine, Suwon, REPUBLIC OF KOREA. RATIONALE: Increased vessel number and vascular permeability with vasodilatation are important characteristics of allergic rhinitis (AR). VGEF is one of the most potent proangiogenic cytokine to increase vascular permeability. Eosinophil is the major effector cell in nasal secretion of allergic rhinitis patients during early and late responses induced by allergen challenge. To evaluate the involvement of VEGF in nasal allergic inflammation, we observed the changes of VEGF and IL-8 levels in relationship with eosinophil cationic protein (ECP) and specific antibody responses in nasal lavage fluid of 52 AR patients after housed dust mite (HDM) challege. METHODS: 52 AR patients sensitized to HDM were enrolled; 30 had responses. Nasal lavage fluids were collected before and 10,30, and 60 minutes, 3, 6, and 24 hours after NPTwith monitoring symptom score. ECP levels were measured by CAP system. Specific IgA antibodies to HDM, VEGF, and IL-8 were measured by ELISA. RESULTS: Early responses with elevated ECP levels were observed between 10 to 30 min, while late responses were noted between 6 to 24 hrs with increased ECP level. Increased production of VEGF was noted in early and late phase of AR, but IL-8 level was not significantly changed with allergen exposure. Significant correlations were found between VEGF level, and ECP or specific IgA to HDM (p < 0.05, respectively). CONCLUSIONS: VEGF production was noted in nasal secretion after the allergen exposure which could augment eosinophilic inflammation in mucosa of AR patients. Funding: the Korean Health 21 R&D project, Ministry of Health & Welfare, ROK
477
Effects Of CUF2 On Cytokine Release From Airway Cells Of Asthmatic Children Y. O. Wong, R. Y. T. Sung, A. M. Li, Y. Zhao, T. F. Leung, R. Wong, K. Li, K. P. Fung; The Chinese University of Hong Kong, Hong Kong, HONG KONG. RATIONALE: Our previous study showed that the Chinese herbal formula CUF2 is effective in reducing inflammatory responses in a rat model of asthma. In the present study, we investigated the effect of CUF2 on cytokine release from the airway cells of asthmatic patients. METHODS: Children aged 7-15 years with chronic asthma were recruited. Induced sputum cell samples were obtained within one hour of collection for the study of cytokine release from these airway cells. The endotoxin level in the CUF2 extract was measured by the limulus amebocytes lysate (LAL) method. Airway cells were activated with phorbol 12-myristate 13-acetate (PMA) in the presence or absence of CUF2. The cytotoxic effect of CUF2 on the viability of airway cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Concentrations of cytokines in the culture supernatants were measured by enzyme-linked immunoabsorbent assay (ELISA). RESULTS: The amount of endotoxin present in final working concentration of CUF2 was negligible (<0.25 EU/mL). No IL-4 release from the airway cell was detected. A small amount of IL-8, TNF-a and GM-CSF production was observed in resting airway cells. IL-8 and TNF-a production increased slightly in airway cells following PMA stimulation (P > 0.05). GM-CSF production in activated sputum cells were increased significantly compared with resting cells (P 5 0.000) and markedly decreased with addition of 1 mg/mL CUF2 (P < 0.05). CONCLUSIONS: We demonstrated that airway cells in sputum are partly active and only some cytokines (such as GM-CSF) responded to further stimulation, and the results suggest a possible anti-allergic role of CUF2 by regulating the production of GM-CSF. Funding: Funded by an Area of Excellence grant from the University Grants Committee of the Hong Kong Special Adminstrative Region for the Project ’’Chinese Medicine Research and Further Development’’
SUNDAY
474
J ALLERGY CLIN IMMUNOL VOLUME 121, NUMBER 2
Detection of Influenza Specific T-Cell Response in Allergic and Healthy Controls N. Kim, B. Foster, C. Prussin; Laboratory of Allergic Diseases, NIAID/ NIH, Bethesda, MD. RATIONALE: Asthma is associated with a Th2 cytokine rich milieu that may affect the generation of immune responses to pathogens and vaccines. We hypothesized that the T-cell response to influenza may have a distinct cytokine pattern in allergic asthmatic subjects relative to non-atopic healthy controls. METHODS: Carboxyfluorescein succinimidyl ester (CFSE), intracellular cytokine staining and polychromatic 9-color flow cytometry were used in a 7-day culture to detect influenza specific CD41 T cell proliferation and IL4, IL-5, IFN-g, and TNF-a expression in 7 allergic asthmatic and 5 healthy control subjects. RESULTS: The frequency of influenza (New Caledonia strain) specific T cells producing IL-4 (2.8% vs. 4.5%), IFN-g (72% vs. 78%) and TNF (53% vs. 66%) in allergic asthmatic and non-atopic control subjects, respectively, were not significantly different. IL-5 was not detectable (<0.1%) in either group. The ratio of IL-4: IFN-g was 0.4 and 0.5 respectively, which were not significantly different. Influenza Panama antigen as well as a hemagglutinin peptide pool also showed no difference between the two groups. CONCLUSIONS: Influenza specific Th2 cytokines are reproducibly detectable and quantifiable in CD4 T cells. Influenza specific T cells from allergic asthmatic subjects did not demonstrate a pattern of cytokine expression significantly different from healthy controls. Funding: NIAID, Division of Intramural Research
475
IL-5 and IFN-g Levels And Spirometric Parameters In Induced Sputum Of Patients With Asma, Allergic Rhinitis And Controls G. R. S. Segundo, S. M. G. Marra, R. Alves, D. A. O. Silva, E. A. Taketomi; Universidade Federal de Uberlaˆndia, Uberlaˆndia, BRAZIL. RATIONALE: Asthma and rinithis are frequently associated, suggesting the concept of ’’one airway, one disease’’. The aims of this study were to analyze spirometrics parameters and IFN-g and IL-5 levels in the induced sputum from patients with asthma or allergic rhinitis (AR) and non-atopic subjects. METHODS: Thirty-three subjects aged 18 to 60 years, both sexes, were analyzed. From these, eight were asthmatics and 16 had allergic rhinitis, and both groups had positive skin prick test (SPT) to aeroallergens. The nine remaining subjects were healthy non-atopics with negative SPT to aeroallergens. Spirometry was performed through evaluating the FVC, FEV1, and FEF25-75% pre- and post-bronchodilators. Induced sputum samples were collected and IFN-g and IL-5 levels were quantified by immunoassays. RESULTS: Significant pre- and post-bronchodilator variation was observed only for FEV1 with higher values in asthmatics compared to patients with AR or non-atopics. There was no significant difference in pre- and post-bronchodilator parameters among the three groups, although there was a tendency for pre-bronchodilator lower FEF25-75% values in asthmatic patients. IFN-g levels in the induced sputum showed no significant difference between the groups but, IL-5 levels were higher in patients with asthma or AR compared to non-atopics. CONCLUSIONS: Spirometric parameters have shown no implications in AR patients, but higher IL-5 levels in the induced sputum in asthma and AR patients reinforce the role of the Th2-type immune response in low respiratory airways that could contribute to the concept of ’’one airway, one disease’’. Funding: Fundac xa˜o de Amparo a` Pesquisa do Estado de Minas Gerais (FAPEMIG), Coordenac xa˜o de Aperfeic xoamento de Pessoal de Nı´vel Superior (CAPES), and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPQ)
476
Involvement Of VEGF In Nasal Inflammation Induced By Allergen G. Choi, H. Park, S. Choi, S. Shin, G. Hur, S. Kim, D. Nahm, H. Park; Ajou University School of Medicine, Suwon, REPUBLIC OF KOREA. RATIONALE: Increased vessel number and vascular permeability with vasodilatation are important characteristics of allergic rhinitis (AR). VGEF is one of the most potent proangiogenic cytokine to increase vascular permeability. Eosinophil is the major effector cell in nasal secretion of allergic rhinitis patients during early and late responses induced by allergen challenge. To evaluate the involvement of VEGF in nasal allergic inflammation, we observed the changes of VEGF and IL-8 levels in relationship with eosinophil cationic protein (ECP) and specific antibody responses in nasal lavage fluid of 52 AR patients after housed dust mite (HDM) challege. METHODS: 52 AR patients sensitized to HDM were enrolled; 30 had responses. Nasal lavage fluids were collected before and 10,30, and 60 minutes, 3, 6, and 24 hours after NPTwith monitoring symptom score. ECP levels were measured by CAP system. Specific IgA antibodies to HDM, VEGF, and IL-8 were measured by ELISA. RESULTS: Early responses with elevated ECP levels were observed between 10 to 30 min, while late responses were noted between 6 to 24 hrs with increased ECP level. Increased production of VEGF was noted in early and late phase of AR, but IL-8 level was not significantly changed with allergen exposure. Significant correlations were found between VEGF level, and ECP or specific IgA to HDM (p < 0.05, respectively). CONCLUSIONS: VEGF production was noted in nasal secretion after the allergen exposure which could augment eosinophilic inflammation in mucosa of AR patients. Funding: the Korean Health 21 R&D project, Ministry of Health & Welfare, ROK
477
Effects Of CUF2 On Cytokine Release From Airway Cells Of Asthmatic Children Y. O. Wong, R. Y. T. Sung, A. M. Li, Y. Zhao, T. F. Leung, R. Wong, K. Li, K. P. Fung; The Chinese University of Hong Kong, Hong Kong, HONG KONG. RATIONALE: Our previous study showed that the Chinese herbal formula CUF2 is effective in reducing inflammatory responses in a rat model of asthma. In the present study, we investigated the effect of CUF2 on cytokine release from the airway cells of asthmatic patients. METHODS: Children aged 7-15 years with chronic asthma were recruited. Induced sputum cell samples were obtained within one hour of collection for the study of cytokine release from these airway cells. The endotoxin level in the CUF2 extract was measured by the limulus amebocytes lysate (LAL) method. Airway cells were activated with phorbol 12-myristate 13-acetate (PMA) in the presence or absence of CUF2. The cytotoxic effect of CUF2 on the viability of airway cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Concentrations of cytokines in the culture supernatants were measured by enzyme-linked immunoabsorbent assay (ELISA). RESULTS: The amount of endotoxin present in final working concentration of CUF2 was negligible (<0.25 EU/mL). No IL-4 release from the airway cell was detected. A small amount of IL-8, TNF-a and GM-CSF production was observed in resting airway cells. IL-8 and TNF-a production increased slightly in airway cells following PMA stimulation (P > 0.05). GM-CSF production in activated sputum cells were increased significantly compared with resting cells (P 5 0.000) and markedly decreased with addition of 1 mg/mL CUF2 (P < 0.05). CONCLUSIONS: We demonstrated that airway cells in sputum are partly active and only some cytokines (such as GM-CSF) responded to further stimulation, and the results suggest a possible anti-allergic role of CUF2 by regulating the production of GM-CSF. Funding: Funded by an Area of Excellence grant from the University Grants Committee of the Hong Kong Special Adminstrative Region for the Project ’’Chinese Medicine Research and Further Development’’
SUNDAY
474