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Alternaria alternata Induces Mast Cell Activation in an IgE-Independent Fashion
Abstracts AB239
J ALLERGY CLIN IMMUNOL VOLUME 135, NUMBER 2
IgE Is Necessary for Pulmonary Vascular Leak during a Respiratory Viral Infection Brian T. Kelly, MD, MA, Desire Hunter, Jennifer L. Santoro, BS, Mitchell H. Grayson, MD FAAAAI; Medical College of Wisconsin, Milwaukee, WI. RATIONALE: Childhood respiratory viral infections are associated with increased risk of asthma. Severe paramyxoviral (Sendai virus, SeV) respiratory infection in mice translates into post-viral atopic disease. In this model, IgE is critical for development of post-viral atopic disease. This pathway appears operative in humans, as we and others have shown antiviral IgE is part of respiratory antiviral immune responses. Why IgE is part of the antiviral immune response is not known; therefore, we hypothesized it is needed to generate pulmonary vascular leak. METHODS: C57BL6 (WT) or IgE–/– mice were inoculated with SeV. At different days post inoculation (PI) SeV, Evans Blue Dye (EBD) was injected i.v. one hour before euthanasia. After flushing the pulmonary vasculature with PBS, EBD concentration in lung homogenates was determined by spectrophotometry and reported as fold increase in SeV-infected versus uninfected mice. RESULTS: In WT mice, SeV infection induced pulmonary vascular leak (1.7460.41, p50.15 versus uninfected; 2.5460.33, p50.015; 3.2460.17, p50.0001; 2.8960.43, p50.016; mean6SEM fold EBD concentration at days 4, 6, 8, and 10 PI SeV, n52-6). IgE–/– mice failed to demonstrate increased vascular leak at all days PI SeV (1.3160.40, 0.9760.20, 1.3460.27, 1.3960.28, respectively; n53). Compared to WT mice vascular leak at days 6, 8, and 10 PI SeV was significantly reduced in IgE–/– mice (p50.0037, 0.0009, 0.0283, respectively, n53-5). CONCLUSIONS: IgE is required for development of pulmonary vascular leak during a severe paramyxoviral respiratory infection. Further studies will identify cell(s) responsible for the leak and the specificity of the IgE response.
772
Rhinovirus Infection Modulates the Activation Status of Circulating Basophils and Dendritic Cells Rachana Agrawal, PhD1, Peter W. Heymann, MD1, Thomas A. E. PlattsMills, MD, PhD, FAAAAI, FRS2, Judith A. Woodfolk, MBChB, PhD, FAAAAI1; 1Division of Asthma, Allergy & Immunology, University of Virginia Health System, Charlottesville, VA, 2Division of Asthma, Allergy and Immunology, University of Virginia Health System, Charlottesville, VA. RATIONALE: Rhinovirus (RV) infection is a major risk factor for asthma exacerbations in atopic subjects; however, the cellular mechanisms involved remain unclear. Given the pivotal role of basophils and myeloid dendritic cells (DCs) in promoting Th2-driven inflammation, we aimed to explore how RV infection impacts circulating basophils and DCs using an experimental challenge model. METHODS: Atopic asthmatics and non-atopic controls were challenged intranasally with human rhinovirus 16. Circulating basophils and DCs in whole blood were immunophenotyped by flow cytometry in fresh whole blood obtained immediately before inoculation (day 0), and during acute (day 4) and convalescent (day 21) phases post-inoculation. RESULTS: Within both groups of subjects, basophils displayed increased expression of FcεRIa and CD63, as well as increased intracellular Syk (both total and phosphorylated) during acute infection. Moreover, this phenotype was maintained at day 21. Basophils from those subjects who had a high percentage of IL-4 positivity on day 0 (>50%) showed a decrease in IL-4 positivity by day 4. In contrast to basophils, FcεRIa expression decreased on DCs during acute infection in all subjects, whereas Syk levels (total and phosphorylated) increased, but only among asthmatics. Additionally, DCs showed a marked increase in expression of the prostaglandin D2 receptor, CRTH2. CONCLUSIONS: The activation status of circulating basophils and DCs is modulated in a dynamic fashion by rhinovirus infection, irrespective of concomitant asthma. Upregulation of the Th2 cell-associated receptor CRTH2, on DCs, points to a heretofore unrecognized role for the CRTH2 pathway in DCs during RV infection.
773
Alternaria alternata Induces Mast Cell Activation in an IgEIndependent Fashion Lora G. Bankova, MD1, Li Li1, Joshua A. Boyce, MD, FAAAAI2, K. Frank Austen, MD, FAAAAI1, Yoshihide Kanaoka, MD, PhD1, Nora A. Barrett, MD, FAAAAI1; 1Brigham and Women’s Hospital, Division of Rheumatology, Immunology and Allergy, Boston, MA, 2Brigham and Women’s Hospital, Division of Rheumatology, Immunology, and Allergy, Boston, MA. RATIONALE: Alternaria alternata is an environmental fungus implicated in acute exacerbations of asthma and was recently demonstrated to trigger cysteinyl leukotriene (cys-LT) production in the lung of naive mice. As mast cells (MCs) have the capacity to generate significant quantities of cys-LTs, we sought to determine whether A. alternata could induce MC cys-LT production and the pathway that might be involved. METHODS: Human MCs were grown from peripheral blood. Murine bone marrow-derived MCs (BMMCs) were generated from wild type (WT), leukotriene C4 synthase (LTC4S)-deficient, and FcεR1g-deficient mice. A. alternata-induced cys-LTs were measured by ELISA. Degranulation was assessed by release of beta-hexosaminidase. WT BMMCs were activated with A. alternata in the presence or absence of inhibitors of spleen tyrosine kinase (Syk), phosphoinositide 3-kinase (PI3K), and protein kinase C (PKC) and pertussis toxin. Cutaneous MC degranulation and ear swelling after intradermal injection of A. alternata was assessed. RESULTS: A. alternata triggered cys-LT generation and degranulation of human peripheral blood MCs and murine BMMCs. Cys-LT production was absent in LTC4S-deficient BMMCs. Cys-LT production was FcεR1g-independent and was not reduced by inhibitors of Syk, PI3K, PKCa, PKCb, or pertussis toxin. Intradermal ear injection of A. alternata in both BALB/c and C57BL/6 mice led to dose-dependent ear edema associated with MC degranulation. CONCLUSIONS: A. alternata triggers cys-LT generation and degranulation in human and murine MCs through a mechanism that is independent of IgE receptor signaling. This demonstrates an innate pathway by which environmental allergens can trigger pathologic MC activation.
MONDAY
771
J ALLERGY CLIN IMMUNOL VOLUME 135, NUMBER 2
IgE Is Necessary for Pulmonary Vascular Leak during a Respiratory Viral Infection Brian T. Kelly, MD, MA, Desire Hunter, Jennifer L. Santoro, BS, Mitchell H. Grayson, MD FAAAAI; Medical College of Wisconsin, Milwaukee, WI. RATIONALE: Childhood respiratory viral infections are associated with increased risk of asthma. Severe paramyxoviral (Sendai virus, SeV) respiratory infection in mice translates into post-viral atopic disease. In this model, IgE is critical for development of post-viral atopic disease. This pathway appears operative in humans, as we and others have shown antiviral IgE is part of respiratory antiviral immune responses. Why IgE is part of the antiviral immune response is not known; therefore, we hypothesized it is needed to generate pulmonary vascular leak. METHODS: C57BL6 (WT) or IgE–/– mice were inoculated with SeV. At different days post inoculation (PI) SeV, Evans Blue Dye (EBD) was injected i.v. one hour before euthanasia. After flushing the pulmonary vasculature with PBS, EBD concentration in lung homogenates was determined by spectrophotometry and reported as fold increase in SeV-infected versus uninfected mice. RESULTS: In WT mice, SeV infection induced pulmonary vascular leak (1.7460.41, p50.15 versus uninfected; 2.5460.33, p50.015; 3.2460.17, p50.0001; 2.8960.43, p50.016; mean6SEM fold EBD concentration at days 4, 6, 8, and 10 PI SeV, n52-6). IgE–/– mice failed to demonstrate increased vascular leak at all days PI SeV (1.3160.40, 0.9760.20, 1.3460.27, 1.3960.28, respectively; n53). Compared to WT mice vascular leak at days 6, 8, and 10 PI SeV was significantly reduced in IgE–/– mice (p50.0037, 0.0009, 0.0283, respectively, n53-5). CONCLUSIONS: IgE is required for development of pulmonary vascular leak during a severe paramyxoviral respiratory infection. Further studies will identify cell(s) responsible for the leak and the specificity of the IgE response.
772
Rhinovirus Infection Modulates the Activation Status of Circulating Basophils and Dendritic Cells Rachana Agrawal, PhD1, Peter W. Heymann, MD1, Thomas A. E. PlattsMills, MD, PhD, FAAAAI, FRS2, Judith A. Woodfolk, MBChB, PhD, FAAAAI1; 1Division of Asthma, Allergy & Immunology, University of Virginia Health System, Charlottesville, VA, 2Division of Asthma, Allergy and Immunology, University of Virginia Health System, Charlottesville, VA. RATIONALE: Rhinovirus (RV) infection is a major risk factor for asthma exacerbations in atopic subjects; however, the cellular mechanisms involved remain unclear. Given the pivotal role of basophils and myeloid dendritic cells (DCs) in promoting Th2-driven inflammation, we aimed to explore how RV infection impacts circulating basophils and DCs using an experimental challenge model. METHODS: Atopic asthmatics and non-atopic controls were challenged intranasally with human rhinovirus 16. Circulating basophils and DCs in whole blood were immunophenotyped by flow cytometry in fresh whole blood obtained immediately before inoculation (day 0), and during acute (day 4) and convalescent (day 21) phases post-inoculation. RESULTS: Within both groups of subjects, basophils displayed increased expression of FcεRIa and CD63, as well as increased intracellular Syk (both total and phosphorylated) during acute infection. Moreover, this phenotype was maintained at day 21. Basophils from those subjects who had a high percentage of IL-4 positivity on day 0 (>50%) showed a decrease in IL-4 positivity by day 4. In contrast to basophils, FcεRIa expression decreased on DCs during acute infection in all subjects, whereas Syk levels (total and phosphorylated) increased, but only among asthmatics. Additionally, DCs showed a marked increase in expression of the prostaglandin D2 receptor, CRTH2. CONCLUSIONS: The activation status of circulating basophils and DCs is modulated in a dynamic fashion by rhinovirus infection, irrespective of concomitant asthma. Upregulation of the Th2 cell-associated receptor CRTH2, on DCs, points to a heretofore unrecognized role for the CRTH2 pathway in DCs during RV infection.
773
Alternaria alternata Induces Mast Cell Activation in an IgEIndependent Fashion Lora G. Bankova, MD1, Li Li1, Joshua A. Boyce, MD, FAAAAI2, K. Frank Austen, MD, FAAAAI1, Yoshihide Kanaoka, MD, PhD1, Nora A. Barrett, MD, FAAAAI1; 1Brigham and Women’s Hospital, Division of Rheumatology, Immunology and Allergy, Boston, MA, 2Brigham and Women’s Hospital, Division of Rheumatology, Immunology, and Allergy, Boston, MA. RATIONALE: Alternaria alternata is an environmental fungus implicated in acute exacerbations of asthma and was recently demonstrated to trigger cysteinyl leukotriene (cys-LT) production in the lung of naive mice. As mast cells (MCs) have the capacity to generate significant quantities of cys-LTs, we sought to determine whether A. alternata could induce MC cys-LT production and the pathway that might be involved. METHODS: Human MCs were grown from peripheral blood. Murine bone marrow-derived MCs (BMMCs) were generated from wild type (WT), leukotriene C4 synthase (LTC4S)-deficient, and FcεR1g-deficient mice. A. alternata-induced cys-LTs were measured by ELISA. Degranulation was assessed by release of beta-hexosaminidase. WT BMMCs were activated with A. alternata in the presence or absence of inhibitors of spleen tyrosine kinase (Syk), phosphoinositide 3-kinase (PI3K), and protein kinase C (PKC) and pertussis toxin. Cutaneous MC degranulation and ear swelling after intradermal injection of A. alternata was assessed. RESULTS: A. alternata triggered cys-LT generation and degranulation of human peripheral blood MCs and murine BMMCs. Cys-LT production was absent in LTC4S-deficient BMMCs. Cys-LT production was FcεR1g-independent and was not reduced by inhibitors of Syk, PI3K, PKCa, PKCb, or pertussis toxin. Intradermal ear injection of A. alternata in both BALB/c and C57BL/6 mice led to dose-dependent ear edema associated with MC degranulation. CONCLUSIONS: A. alternata triggers cys-LT generation and degranulation in human and murine MCs through a mechanism that is independent of IgE receptor signaling. This demonstrates an innate pathway by which environmental allergens can trigger pathologic MC activation.
MONDAY
771