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CLC3 Transcript Variants in the Activation and Migration of Eosinophils in Allergic Asthmatics
Abstracts AB123
J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 2
CLC3 Transcript Variants in the Activation and Migration of Eosinophils in Allergic Asthmatics Rohit Gaurav1, Min-Jung Kim1, Againdra Bewtra2, Devendra K. Agrawal1; 1Center for Clinical and Translational Science and Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE, 2Department of Medicine, Division of Allergy and Immunology, Creighton University Medical Center, Omaha, NE. RATIONALE: Migration and activation of eosinophils play a major role in the pathophysiology of allergic asthma. Recently, we reported the role of CLC3 in TGF-b-induced migration of eosinophils, increased expression of CLC3b and CLC3e transcript variants on eosinophil membranes in response to TGF-b and eotaxin. Here, we focused on the function of CLC3 in eosinophils of allergic asthmatics. METHODS: Human peripheral blood eosinophils were isolated (>99% pure >98% viable) with negative selection from healthy and mild-tomoderate allergic asthmatic donors. Cells were stimulated with TGF-b1, eotaxin-1, eotaxin-3, DIDS and NPPB. QPCR, chemotaxis and whole-cell patch were used to obtain outcome measures. RESULTS: CLC3 and NOX2 (gp91phox) mRNA levels were 5-9 fold and 5-25 fold, respectively, higher in asthmatics compared to healthy individuals (n54, p<0.05). Also, CLC3b and CLC3e transcript variants were 7-11 fold and 13-21 fold, respectively, higher in asthmatics than in controls (n54, p<0.05). Significantly higher current due to CLC3 and greater migration of eosinophils were observed in the eosinophils of asthmatic compared to healthy individuals. CONCLUSIONS: Significantly greater increase in CLC3e transcript variant compared to CLC3b in asthmatic eosinophils suggests it’s imperative role in allergic asthma. Greater migratory potential of eosinophils and higher activity of CLC3 channel in asthmatics support its importance in allergic asthmatics. NADPH oxidase is vital for oxidative burst, and to compensate the resultant charge imbalance, CLC3 works in conjunction with NADPH oxidase. Higher level of NOX2 mRNA in asthmatics suggests that the cells are in an activated state with active participation of both CLC3 and NADPH oxidase.
445
Eosinophils Through the CXCR4 Chemokine Receptor Christof Straub1, Konrad Pazdrak, MD, PhD2, Alexander Kurosky, PhD3; 1University of Texas Medical Branch, Galveston, TX, 2 Univ. of TX Medical Branch, Galveston, TX, 3University of Texas Medical Branch, League City, TX. RATIONALE: HMGB1, a DAMP protein, has recently been implicated in asthma. HMGB1 is secreted by necrosis or through active release following stimulation, and it can subsequently act as a proinflammatory mediator with cytokine-like properties. The protein is expressed by resident and infiltrating airway cells and it can bind to any cell that expresses one of its target receptors (e.g. RAGE, TLR4, CXCR4). We have investigated the chemoattractant role of HMGB1 on eosinophils based on previous reports that implicate HMGB1 in chemotaxis of other cell types. METHODS: We purified bacterially-expressed, recombinant HMGB1 as well as HMGB1 secreted by stimulated Eol-1 cells. The two HMGB1 proteins were assessed for chemotactic properties in assays with human peripheral blood eosinophils (EosCD16-/CD3-/CD235a-) using a TranswellÒ system (Corning; 5 mm pore size). RESULTS: Both recombinant and secreted HMGB1 exert chemotactic properties on human peripheral blood eosinophils, with stronger chemotactic potencies exerted by the secreted form. Similar results were observed with human neutrophils. Pretreatment of eosinophils with the CXCR4specific inhibitor AMD3100 blocked chemotaxis toward HMGB1. Blockage of RAGE had no effects on eosinophil chemotaxis toward HMGB1. CONCLUSIONS: These results implicate HMGB1 as a novel, potent chemotactic mediator for eosinophils and neutrophils. Since HMGB1 has been found in the airways of asthmatics, this data points to an important role of HMGB1 in asthma that is exerted via eosinophil- and neutrophilassociated infiltration and inflammation.
446
Correlations Between Eosinophil Markers in Allergic Disorders Mahsheed Taeb, DO1, Katrina Elio, DO1, Oral Alpan, MD2,3; 1Inova Fairfax Hospital, 2Amerimmune, LLC, VA, 3O&O ALPAN, LLC. RATIONALE: CD69 is an activation marker present on eosinophils, shown to be a marker of activation. On the other hand, FceRI at the surface of eosinophils endow these cells with both effector and regulatory function such as the production of IL-10. We describe 3 cases (ages 3, 12 and 28, all males) with asthma and allergic rhinitis, presenting with a flare up of their disease and peripheral blood eosinophilia in which we asked whether the expression of CD69 correlates with the expression of high affinity IgE receptor (FceRI) on eosinophils. METHODS: Whole blood was stained with antibodies to CD9, FceRI, CD16, CD69. Samples were analyzed on a Accuri flow cytometer. Eosinophils were separated from granulocytes by their characteristic high side-scatter, dim staining for CD16 and CD9 positivity. RESULTS: Eosinophil CD69 expression was 11.7, 8 and 5.5% and FceRI expression was 3, 4.5 and 3.9%. To our surprise, eosinophils did not coexpress FceRI and CD69. All patients had IgE mediated allergies based on skin prick and serum allergen specific IgE levels. CONCLUSIONS: We show for the first time, the distinct expression profile of CD69 and FceRI on eosinophils. The non-coexisting expression of CD69 and FceRI on eosinophils suggests distinct roles for each requiring further work in this field.
447
Effect of Sensitization and Exposure to Mold On Asthma Morbidity in Inner-City Children Jean Curtin-Brosnan, MA1, Patrick J. Lenehan2, Patrick Breysse, Ph.D3, Gregory B. Diette, MD, MHS1, Elizabeth Matsui, MD1,4; 1Johns Hopkins University, Baltimore, MD, 2Johns Hopkins University, 3Johns Hopkins School of Public Health, 4Johns Hopkins University School of Medicine, Baltimore, MD. RATIONALE: To examine the relationship between sensitization and exposure to mold and asthma morbidity in inner-city children. METHODS: 144 children (5-17 years old, 57% male and 91% black) with persistent asthma were studied for one year. At baseline, subjects underwent skin testing to Alternaria, Aspergillus, and Cladosporium. A net _3mm was considered positive. Home assessments were conducted wheal > every three months. Mold exposure was defined as any signs of mold: moisture or water damage, musty smells or visible mildew. Symptoms and asthma-related health care use were assessed by questionnaire every three months. Participants were grouped into four categories: not sensitized to either of the two indoor molds and not exposed; sensitized/not exposed; exposed/not sensitized; and sensitized and exposed. Generalized linear mixed-effects models and Wilcoxon rank-sum tests were run to determine associations between group and measures of asthma morbidity. RESULTS: At baseline, 51% of subjects were SPT+ to one or more of the 3 tested molds (Alternaria 34%, Aspergillus 29%, and Cladosporium 7%).Twenty percent had moisture or water damage, musty smells or visible mildew in bedrooms/family rooms. Sensitization to mold was not associated with any asthma-related symptoms or acute care visits. After controlling for gender, age, and total IgE, sensitization and exposure to mold was associated with emergency department visits (OR, 2.06;CI 1.10-3.87), days of slowed activity (OR, 1.8;CI 0.96-3.56) and exercise-related symptoms (OR, 2.0;CI 0.98-3.95), although the symptoms associations were not statistically significant. CONCLUSIONS: Sensitization to mold was not associated with asthmarelated symptoms, but sensitization and exposure to mold was associated with symptoms and ED visits.
SUNDAY
444
J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 2
CLC3 Transcript Variants in the Activation and Migration of Eosinophils in Allergic Asthmatics Rohit Gaurav1, Min-Jung Kim1, Againdra Bewtra2, Devendra K. Agrawal1; 1Center for Clinical and Translational Science and Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE, 2Department of Medicine, Division of Allergy and Immunology, Creighton University Medical Center, Omaha, NE. RATIONALE: Migration and activation of eosinophils play a major role in the pathophysiology of allergic asthma. Recently, we reported the role of CLC3 in TGF-b-induced migration of eosinophils, increased expression of CLC3b and CLC3e transcript variants on eosinophil membranes in response to TGF-b and eotaxin. Here, we focused on the function of CLC3 in eosinophils of allergic asthmatics. METHODS: Human peripheral blood eosinophils were isolated (>99% pure >98% viable) with negative selection from healthy and mild-tomoderate allergic asthmatic donors. Cells were stimulated with TGF-b1, eotaxin-1, eotaxin-3, DIDS and NPPB. QPCR, chemotaxis and whole-cell patch were used to obtain outcome measures. RESULTS: CLC3 and NOX2 (gp91phox) mRNA levels were 5-9 fold and 5-25 fold, respectively, higher in asthmatics compared to healthy individuals (n54, p<0.05). Also, CLC3b and CLC3e transcript variants were 7-11 fold and 13-21 fold, respectively, higher in asthmatics than in controls (n54, p<0.05). Significantly higher current due to CLC3 and greater migration of eosinophils were observed in the eosinophils of asthmatic compared to healthy individuals. CONCLUSIONS: Significantly greater increase in CLC3e transcript variant compared to CLC3b in asthmatic eosinophils suggests it’s imperative role in allergic asthma. Greater migratory potential of eosinophils and higher activity of CLC3 channel in asthmatics support its importance in allergic asthmatics. NADPH oxidase is vital for oxidative burst, and to compensate the resultant charge imbalance, CLC3 works in conjunction with NADPH oxidase. Higher level of NOX2 mRNA in asthmatics suggests that the cells are in an activated state with active participation of both CLC3 and NADPH oxidase.
445
Eosinophils Through the CXCR4 Chemokine Receptor Christof Straub1, Konrad Pazdrak, MD, PhD2, Alexander Kurosky, PhD3; 1University of Texas Medical Branch, Galveston, TX, 2 Univ. of TX Medical Branch, Galveston, TX, 3University of Texas Medical Branch, League City, TX. RATIONALE: HMGB1, a DAMP protein, has recently been implicated in asthma. HMGB1 is secreted by necrosis or through active release following stimulation, and it can subsequently act as a proinflammatory mediator with cytokine-like properties. The protein is expressed by resident and infiltrating airway cells and it can bind to any cell that expresses one of its target receptors (e.g. RAGE, TLR4, CXCR4). We have investigated the chemoattractant role of HMGB1 on eosinophils based on previous reports that implicate HMGB1 in chemotaxis of other cell types. METHODS: We purified bacterially-expressed, recombinant HMGB1 as well as HMGB1 secreted by stimulated Eol-1 cells. The two HMGB1 proteins were assessed for chemotactic properties in assays with human peripheral blood eosinophils (EosCD16-/CD3-/CD235a-) using a TranswellÒ system (Corning; 5 mm pore size). RESULTS: Both recombinant and secreted HMGB1 exert chemotactic properties on human peripheral blood eosinophils, with stronger chemotactic potencies exerted by the secreted form. Similar results were observed with human neutrophils. Pretreatment of eosinophils with the CXCR4specific inhibitor AMD3100 blocked chemotaxis toward HMGB1. Blockage of RAGE had no effects on eosinophil chemotaxis toward HMGB1. CONCLUSIONS: These results implicate HMGB1 as a novel, potent chemotactic mediator for eosinophils and neutrophils. Since HMGB1 has been found in the airways of asthmatics, this data points to an important role of HMGB1 in asthma that is exerted via eosinophil- and neutrophilassociated infiltration and inflammation.
446
Correlations Between Eosinophil Markers in Allergic Disorders Mahsheed Taeb, DO1, Katrina Elio, DO1, Oral Alpan, MD2,3; 1Inova Fairfax Hospital, 2Amerimmune, LLC, VA, 3O&O ALPAN, LLC. RATIONALE: CD69 is an activation marker present on eosinophils, shown to be a marker of activation. On the other hand, FceRI at the surface of eosinophils endow these cells with both effector and regulatory function such as the production of IL-10. We describe 3 cases (ages 3, 12 and 28, all males) with asthma and allergic rhinitis, presenting with a flare up of their disease and peripheral blood eosinophilia in which we asked whether the expression of CD69 correlates with the expression of high affinity IgE receptor (FceRI) on eosinophils. METHODS: Whole blood was stained with antibodies to CD9, FceRI, CD16, CD69. Samples were analyzed on a Accuri flow cytometer. Eosinophils were separated from granulocytes by their characteristic high side-scatter, dim staining for CD16 and CD9 positivity. RESULTS: Eosinophil CD69 expression was 11.7, 8 and 5.5% and FceRI expression was 3, 4.5 and 3.9%. To our surprise, eosinophils did not coexpress FceRI and CD69. All patients had IgE mediated allergies based on skin prick and serum allergen specific IgE levels. CONCLUSIONS: We show for the first time, the distinct expression profile of CD69 and FceRI on eosinophils. The non-coexisting expression of CD69 and FceRI on eosinophils suggests distinct roles for each requiring further work in this field.
447
Effect of Sensitization and Exposure to Mold On Asthma Morbidity in Inner-City Children Jean Curtin-Brosnan, MA1, Patrick J. Lenehan2, Patrick Breysse, Ph.D3, Gregory B. Diette, MD, MHS1, Elizabeth Matsui, MD1,4; 1Johns Hopkins University, Baltimore, MD, 2Johns Hopkins University, 3Johns Hopkins School of Public Health, 4Johns Hopkins University School of Medicine, Baltimore, MD. RATIONALE: To examine the relationship between sensitization and exposure to mold and asthma morbidity in inner-city children. METHODS: 144 children (5-17 years old, 57% male and 91% black) with persistent asthma were studied for one year. At baseline, subjects underwent skin testing to Alternaria, Aspergillus, and Cladosporium. A net _3mm was considered positive. Home assessments were conducted wheal > every three months. Mold exposure was defined as any signs of mold: moisture or water damage, musty smells or visible mildew. Symptoms and asthma-related health care use were assessed by questionnaire every three months. Participants were grouped into four categories: not sensitized to either of the two indoor molds and not exposed; sensitized/not exposed; exposed/not sensitized; and sensitized and exposed. Generalized linear mixed-effects models and Wilcoxon rank-sum tests were run to determine associations between group and measures of asthma morbidity. RESULTS: At baseline, 51% of subjects were SPT+ to one or more of the 3 tested molds (Alternaria 34%, Aspergillus 29%, and Cladosporium 7%).Twenty percent had moisture or water damage, musty smells or visible mildew in bedrooms/family rooms. Sensitization to mold was not associated with any asthma-related symptoms or acute care visits. After controlling for gender, age, and total IgE, sensitization and exposure to mold was associated with emergency department visits (OR, 2.06;CI 1.10-3.87), days of slowed activity (OR, 1.8;CI 0.96-3.56) and exercise-related symptoms (OR, 2.0;CI 0.98-3.95), although the symptoms associations were not statistically significant. CONCLUSIONS: Sensitization to mold was not associated with asthmarelated symptoms, but sensitization and exposure to mold was associated with symptoms and ED visits.
SUNDAY
444