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Key role for mast cells in nonatopic asthma
S34 A b s t r a c t s
J ALLERGYCLIN IMMUNOL JANUARY 2002
6 Th2 Cytokine Expression by Bronchoalveolar Lavage T-Lymphocytes in Asthma and Eosinophilic BronchitisWithout Asthma Christopher Edward Brightling, Fiona A Symon, Surinder S Birring, lan D Pavord, Andrew J Wardlaw Institute for Lung Health, Leicester, UK Eosinophilic bronchitis (EB) is a common cause of chronic cough characterised by a sputum eosinophilia. In contrast to asthma there is no variable airflow obstruction or airway hyperresponsiveness. Eosinophilic airway inflammation in asthma is under the control ofTh2 cytokines, whereas the cytokine regulation of the inflammatory response in eosinophilic bronchitis is unknown. We hypothesised that the differences in airway pathophysiology between these two conditions may be related to cytokine expression by airway T-cells. From 10 subjects with EB, 9 with asthma and 9 normal controls 20ml of venous blood was taken and the subjects underwent a fibreoptic bronchoscopy and 180ml bronchoalveolar lavage (BAL) to the middle lobe. The peripheral blood mononuclear cell (PBMC) fraction was obtained by centrifugation on Ficoll. After washing PBMC and BAL cells were either stimulated with PMA, calcium ionophore and Brefeldin A or incubated in culture medium alone (resting) for 4hr at 37°C. The cells were fixed, incubated with CD3-F1TC/RPE and CD8-PerCP, permeabilised and labelled with IL-4-RPE and IFN-y-FITC or isotypic controls and analysed using three-colour flow cytometry. The median (range) % of stimulated CD4+ PBMC expressing IFN-y were increased in those subjects with EB 14 (2.8-52) and asthma 15 (7-30) compared to normals 3.3 (0.1-12) (Kruskal-Wallis p=0.024). Expression of 1L-4 was increased in EB and asthma compared to normals in both the resting (p=0.03) and stimulated (p=0.045) CD4+ BAL cells (Table). There were no between group differences in IL-4 expression in PBMC cells or IFN-yin BAL cells. In conclusion, increased Th2 cytokine expression by BAL T-cells is a feature of EB and asthma suggesting that they play a role in the airway inflammation observed in EB but that release of Th2 cytokines is not important in the development of disordered airway physiology in asthma. Median (range) % CD4+ BAL T-cells expressing intracellular cytokines
BAL resting IL-4 EB Asthma Normals
BAL stimulated IL-4
7.2 (2-13) 11.4 (4.5-16) 5.3 (1.5-14.7) 5.5 (3.6-27) 2.6 (0-7.6) 3.8 (0-17.4)
BAL resting IFN-~
BAL stimulated IFN-7
1.8 (0.4-9) 26.8 (8.2-74) 2.2 (0-14.4) 28.3 (8.5-65) 1.1 (0.7-4.3) 20.2 (6.1-48)
7 Key Role for Mast Cells in Nonatopic Asthma Anneke H Van Houwelingen, Aletta D Kraneveld, Hanneke P Van der Kleij, Mirjam Keel, Frank A Redegeld, Frans P Nijkamp Faculty of Pharmacy, Utrecht University, Utrecht, Netherlands The mechanisms involved in nonatopic asthma are poorly defined. In particular, the importance of mast cells in the development of nonatopic asthma is not clear. In the mouse, pulmonary hypersensitivity reaction induced by skin sensitization with the low molecular weight compound dinitrofluorobenzene (DNFB) followed by an intra-airway application of the hapten has been featured as a model for nonatopic asthma.This pulmonary hypersensitivity reaction is not associated with an elevated haptenspecific serum IcE. In present study we employed the model to examine the role of mast cells in the pathogenesis of nonatopic asthma. Firstly, the effect of DNFB sensitization and intra-airway challenge with dinitrobenzene sulphonic acid (DNS) on mast cell activation was monitored during the early phase of the reaction in BALB/c mice. Secondly, mast cell-deficient W./W~ and SI/SI d mice and their respective normal (+/+) littermate mice and mast cell-reconstituted W/Wv mice (BMMC-+W/WD were used. Early phase mast cell activation was found, which was maximal 30 rain after DNS challenge in DNFB-sensitized BALB/c, +/+ mice, but not in mast cell-deficient mice. An acute bronchoconstriction and increase in vascular permeability accompanied the early phase mast cell activation. BALB/c, +/+ and
BMMC--)W/'W~ mice sensitized with DNFB and DNS-challenged exhibited tracheal hyperreactivity 24 and 48 h after the challenge when compared to vehicle-treated mice. Mucosal exudation and an infiltration of neutrophils in BAL fluid associated this late phase response. Both mast celldeficient strains failed to show any features of this hypersensitivity response. Our findings show that mast cells play a key role in the regulation of pulmonary hypersensitivity responses in this murine model for nonatopic asthma. Sponsored by a research grant of the Netherlands Asthma Foundation (NAF94.34) and by Glaxo/Welcome, the Netherlands.
8 Elevated Plasma and Mononuclear Cell Culture Supernate Eetaxin Levels in Asthmatic Children E/ham Mohamed Hossny, MH Ezzat, S1 Bakr, MM Soliman Ain Shams University, Cairo, Egypt The eosinophil specific chemokine, eotaxin, was assayed in plasma and mononuclear cell culture supernatant fluid samples from 14 asthmatic children. 6-12 years old, during an asthma exacerbation and after complete remission of symptoms and physical signs. The results were compared to those of 10 age and sex matched healthy children as a control group. The plasma eotaxin levels studied during asthma exacerbation [median = 335 pg/ml, mean (SD) = 364 (277) pg/ml] were statistically comparable to those of the same patients taken in-between attacks [median = 215 pg/ml, mean (SD) = 288.6 (252.7) pg/ml]. However, the healthy children had much lower plasma eotaxin levels [median = 50 pg/ml, mean (SD) = 59 (39.8) pg/ml] as compared to the patients' levels whether during asthma exacerbation or quiescence. Eotaxin concentrations in mononuclear cell culture supemate during asthma exacerbations [median = 7.2 pg/ml, mean (SD) = 7.6 (2.2) pg/ml[ were significantly higher than the corresponding values in-between exacerbations [median = 5.4 pg/ml, mean (SD) = 5.6 (1.3) pg/ml] and the control values [median = 4 pg/ml, mean (SD) = 4.3 (1.1) pg/ml]. Children enrolled with acute severe asthma had a significantly higher mean plasma eotaxin level as compared to those with mild to moderate attacks. Our findings reinforce the concept that eotaxin is implicated in the pathogenesis of bronchial asthma and may represent a biomarker of allergic inflammation. This may pave the way for eotaxin antagonism among the adjuvant therapeutic strategies.
A New Mechanism for Asthma: Immunoglobulin Light Chain
~Jl Induces Bronchoconstrictionand Airway Inflammation in Mice 41[~
A/eLLa D Kraneveld, Frank A Redegeld, Mirjam Keel, Frans P Nijkamp Faculty of Pharmacy, Utrecht University, Utrecht, Netherlands The current opinion that IcE plays a dominant role in allergic responses eg asthma, has recently been questioned. In IcE-deficient mice immediate hypersensitivity reactions were still evident. Recently, we have identified immunoglobulin light chain (IgLC) as an antigen-specific factor capable of inducing hypersensitivity reactions. Here, we present a new mechanism for immediate hypersensitivity-like reactions in the airways, that could possibly be involved in asthma. IgLC was isolated after reduction and alkylation of TNP-specific IgG. BALB/c mice were iv injected with IgLC, whole IgGl or vehicle (sterile saline) and after 30 min challenged intranasally with hapten. Challenge with picryl sulfonic acid (PSA, the water soluble form of PC1), but not with the non-related hapten oxazolone, induced a profound in vivo hronchoconstriction in IgLC-sensitized mice compared to vehicle-sensitized animals 0 to 15 min after challenge (body plethysmographic chamber). IgGl-sensitization did not influence lung function after PSA challenge. An enhanced mucosal exudation into the BAL was found 1 h after PSA challenge in mice sensitized with IgLC (Evans blue accumulation in BAL fluid). These airway responses were associated with in vivo mast cell activation in IgLC-sensitized and PSA-challenged mice (mMCP EL1SA and histology). In addition, the bronchoconstriction was absent in mast cell-deficient (W/'W~') mice, however, reconstitution of mast cells restored the response. Our results indicate that hapten-specific kappa IgLC is the biological active component of lymphocyte-derived factors, that is essential for the development of this mast cell dependent pulmonary hypersensitivity reaction. Sponsored by Fornix Biosciences NV, the Netherlands.
J ALLERGYCLIN IMMUNOL JANUARY 2002
6 Th2 Cytokine Expression by Bronchoalveolar Lavage T-Lymphocytes in Asthma and Eosinophilic BronchitisWithout Asthma Christopher Edward Brightling, Fiona A Symon, Surinder S Birring, lan D Pavord, Andrew J Wardlaw Institute for Lung Health, Leicester, UK Eosinophilic bronchitis (EB) is a common cause of chronic cough characterised by a sputum eosinophilia. In contrast to asthma there is no variable airflow obstruction or airway hyperresponsiveness. Eosinophilic airway inflammation in asthma is under the control ofTh2 cytokines, whereas the cytokine regulation of the inflammatory response in eosinophilic bronchitis is unknown. We hypothesised that the differences in airway pathophysiology between these two conditions may be related to cytokine expression by airway T-cells. From 10 subjects with EB, 9 with asthma and 9 normal controls 20ml of venous blood was taken and the subjects underwent a fibreoptic bronchoscopy and 180ml bronchoalveolar lavage (BAL) to the middle lobe. The peripheral blood mononuclear cell (PBMC) fraction was obtained by centrifugation on Ficoll. After washing PBMC and BAL cells were either stimulated with PMA, calcium ionophore and Brefeldin A or incubated in culture medium alone (resting) for 4hr at 37°C. The cells were fixed, incubated with CD3-F1TC/RPE and CD8-PerCP, permeabilised and labelled with IL-4-RPE and IFN-y-FITC or isotypic controls and analysed using three-colour flow cytometry. The median (range) % of stimulated CD4+ PBMC expressing IFN-y were increased in those subjects with EB 14 (2.8-52) and asthma 15 (7-30) compared to normals 3.3 (0.1-12) (Kruskal-Wallis p=0.024). Expression of 1L-4 was increased in EB and asthma compared to normals in both the resting (p=0.03) and stimulated (p=0.045) CD4+ BAL cells (Table). There were no between group differences in IL-4 expression in PBMC cells or IFN-yin BAL cells. In conclusion, increased Th2 cytokine expression by BAL T-cells is a feature of EB and asthma suggesting that they play a role in the airway inflammation observed in EB but that release of Th2 cytokines is not important in the development of disordered airway physiology in asthma. Median (range) % CD4+ BAL T-cells expressing intracellular cytokines
BAL resting IL-4 EB Asthma Normals
BAL stimulated IL-4
7.2 (2-13) 11.4 (4.5-16) 5.3 (1.5-14.7) 5.5 (3.6-27) 2.6 (0-7.6) 3.8 (0-17.4)
BAL resting IFN-~
BAL stimulated IFN-7
1.8 (0.4-9) 26.8 (8.2-74) 2.2 (0-14.4) 28.3 (8.5-65) 1.1 (0.7-4.3) 20.2 (6.1-48)
7 Key Role for Mast Cells in Nonatopic Asthma Anneke H Van Houwelingen, Aletta D Kraneveld, Hanneke P Van der Kleij, Mirjam Keel, Frank A Redegeld, Frans P Nijkamp Faculty of Pharmacy, Utrecht University, Utrecht, Netherlands The mechanisms involved in nonatopic asthma are poorly defined. In particular, the importance of mast cells in the development of nonatopic asthma is not clear. In the mouse, pulmonary hypersensitivity reaction induced by skin sensitization with the low molecular weight compound dinitrofluorobenzene (DNFB) followed by an intra-airway application of the hapten has been featured as a model for nonatopic asthma.This pulmonary hypersensitivity reaction is not associated with an elevated haptenspecific serum IcE. In present study we employed the model to examine the role of mast cells in the pathogenesis of nonatopic asthma. Firstly, the effect of DNFB sensitization and intra-airway challenge with dinitrobenzene sulphonic acid (DNS) on mast cell activation was monitored during the early phase of the reaction in BALB/c mice. Secondly, mast cell-deficient W./W~ and SI/SI d mice and their respective normal (+/+) littermate mice and mast cell-reconstituted W/Wv mice (BMMC-+W/WD were used. Early phase mast cell activation was found, which was maximal 30 rain after DNS challenge in DNFB-sensitized BALB/c, +/+ mice, but not in mast cell-deficient mice. An acute bronchoconstriction and increase in vascular permeability accompanied the early phase mast cell activation. BALB/c, +/+ and
BMMC--)W/'W~ mice sensitized with DNFB and DNS-challenged exhibited tracheal hyperreactivity 24 and 48 h after the challenge when compared to vehicle-treated mice. Mucosal exudation and an infiltration of neutrophils in BAL fluid associated this late phase response. Both mast celldeficient strains failed to show any features of this hypersensitivity response. Our findings show that mast cells play a key role in the regulation of pulmonary hypersensitivity responses in this murine model for nonatopic asthma. Sponsored by a research grant of the Netherlands Asthma Foundation (NAF94.34) and by Glaxo/Welcome, the Netherlands.
8 Elevated Plasma and Mononuclear Cell Culture Supernate Eetaxin Levels in Asthmatic Children E/ham Mohamed Hossny, MH Ezzat, S1 Bakr, MM Soliman Ain Shams University, Cairo, Egypt The eosinophil specific chemokine, eotaxin, was assayed in plasma and mononuclear cell culture supernatant fluid samples from 14 asthmatic children. 6-12 years old, during an asthma exacerbation and after complete remission of symptoms and physical signs. The results were compared to those of 10 age and sex matched healthy children as a control group. The plasma eotaxin levels studied during asthma exacerbation [median = 335 pg/ml, mean (SD) = 364 (277) pg/ml] were statistically comparable to those of the same patients taken in-between attacks [median = 215 pg/ml, mean (SD) = 288.6 (252.7) pg/ml]. However, the healthy children had much lower plasma eotaxin levels [median = 50 pg/ml, mean (SD) = 59 (39.8) pg/ml] as compared to the patients' levels whether during asthma exacerbation or quiescence. Eotaxin concentrations in mononuclear cell culture supemate during asthma exacerbations [median = 7.2 pg/ml, mean (SD) = 7.6 (2.2) pg/ml[ were significantly higher than the corresponding values in-between exacerbations [median = 5.4 pg/ml, mean (SD) = 5.6 (1.3) pg/ml] and the control values [median = 4 pg/ml, mean (SD) = 4.3 (1.1) pg/ml]. Children enrolled with acute severe asthma had a significantly higher mean plasma eotaxin level as compared to those with mild to moderate attacks. Our findings reinforce the concept that eotaxin is implicated in the pathogenesis of bronchial asthma and may represent a biomarker of allergic inflammation. This may pave the way for eotaxin antagonism among the adjuvant therapeutic strategies.
A New Mechanism for Asthma: Immunoglobulin Light Chain
~Jl Induces Bronchoconstrictionand Airway Inflammation in Mice 41[~
A/eLLa D Kraneveld, Frank A Redegeld, Mirjam Keel, Frans P Nijkamp Faculty of Pharmacy, Utrecht University, Utrecht, Netherlands The current opinion that IcE plays a dominant role in allergic responses eg asthma, has recently been questioned. In IcE-deficient mice immediate hypersensitivity reactions were still evident. Recently, we have identified immunoglobulin light chain (IgLC) as an antigen-specific factor capable of inducing hypersensitivity reactions. Here, we present a new mechanism for immediate hypersensitivity-like reactions in the airways, that could possibly be involved in asthma. IgLC was isolated after reduction and alkylation of TNP-specific IgG. BALB/c mice were iv injected with IgLC, whole IgGl or vehicle (sterile saline) and after 30 min challenged intranasally with hapten. Challenge with picryl sulfonic acid (PSA, the water soluble form of PC1), but not with the non-related hapten oxazolone, induced a profound in vivo hronchoconstriction in IgLC-sensitized mice compared to vehicle-sensitized animals 0 to 15 min after challenge (body plethysmographic chamber). IgGl-sensitization did not influence lung function after PSA challenge. An enhanced mucosal exudation into the BAL was found 1 h after PSA challenge in mice sensitized with IgLC (Evans blue accumulation in BAL fluid). These airway responses were associated with in vivo mast cell activation in IgLC-sensitized and PSA-challenged mice (mMCP EL1SA and histology). In addition, the bronchoconstriction was absent in mast cell-deficient (W/'W~') mice, however, reconstitution of mast cells restored the response. Our results indicate that hapten-specific kappa IgLC is the biological active component of lymphocyte-derived factors, that is essential for the development of this mast cell dependent pulmonary hypersensitivity reaction. Sponsored by Fornix Biosciences NV, the Netherlands.