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Basophils In Peripheral Blood can be Sensitized in Local Allergic Rhinitis
Abstracts AB209
J ALLERGY CLIN IMMUNOL VOLUME 129, NUMBER 2
Basophils In Peripheral Blood can be Sensitized in Local Allergic Rhinitis E. Gomez1, C. Rondon2, P. Campo2, L. Galindo Reyes1, J. A. Huertas2, L. Melendez1, J. Garcia Campos2, M. J. Torres2, M. Blanca2; 1Research Laboratory, Carlos Haya Hospital-Fundacion IMABIS, Malaga, SPAIN, 2 Allergy Service, Carlos Haya Hospital, Malaga, SPAIN. RATIONALE: Local allergic rhinitis (LAR) is a new phenotype of rhinitis characterized by local production of sIgE, a Th2 nasal inflammatory pattern and positive response to nasal allergen provocation test (NAPT) with negative skin prick test and serum specific-IgE. The presence of surface-bound IgE to basophils has not been investigated yet. In this study we evaluated the specific activation of basophil in patients with LAR to D. pteronyssinus (DP). METHODS: The study group was 10 LAR patients to DP. Two controls groups were included: 11 allergic rhinitis (AR) patients to DP and 10 healthy controls. Skin prick test, serum specific-IgE, NAPT-DP, and BAT with DP at different concentrations (5, 20, 50 ng/ml) were performed. RESULTS: NAPT-DP was positive in all LAR and AR patients and negative in healthy controls. BAT was positive in 5 LAR patients (50%), 8 AR patients (72.7%), and 2 healthy controls (20%). BAT results in AR patients and healthy controls showed a sensitivity of 72.2%, a specificity of 80%, and a significant concordance with NAPT (kappa index 0.525, p50.016). CONCLUSIONS: In this study the 50% of LAR patients to DP showed specific basophil activation, indicating that at least half of the patients with LAR to DP can be diagnosed by this approach. These results suggest that after local production of specific-IgE, basophils may be the first target cells for specific-IgE, prior to the detection of free serum specific-IgE and skin mast-cells sensitization.
786
Evaluation of Thymic Stromal Lymphopoietin (TSLP) and its Receptor in Adults with Allergic Rhinitis Following Controlled Allergen Challenge in the Environmental Exposure Unit (EEU) T. Batool1, J. Thiele2, A. K. Ellis2; 1McMaster University, Hamilton, ON, CANADA, 2Queen‘s University, Kingston, ON, CANADA. RATIONALE: Thymic stromal lymphopoietin(TSLP) is an epithelialderived IL-7 like cytokine, and a potent inducer of Th2 response deregulation. We hypothesized that active allergic rhinitis (AR) inflammation may induce a systemic inflammatory cascade leading to the detection of TSLP or changes in TSLP-receptor(TSLPR) mRNA levels in peripheral blood(PB). METHODS: 44 subjects with ragweed-induced AR underwent a 3hr controlled ragweed pollen challenge in the EEU to develop AR symptoms. Serum and PB mononuclear cells(MNCs) were collected before and after allergen challenge. ELISA was performed on serum samples from 12 subjects for the detection of human TSLP. The cytokine profile was also analyzed utilizing Luminex technology(Human 17-Plex Assay). TSLPR mRNA expression in PBMNCs was evaluated using qPCR with ACTB as the reference gene. RESULTS: TSLP was undetectable in all 12 subject serum samples examined, either before or after allergen challenge. The cytokine profile analysis confirmed that other cytokines were detectable in the subject samples, eliminating sample storage as a potential cause for the lack of TSLP detection. Additionally, we were unable to detect the presence of TSLPR mRNA either before or after allergen challenge, despite appropriate reference gene detection. CONCLUSIONS: TSLP does not appear to be detectable in the systemic circulation of adults with active allergic rhinitis inflammation using currently available ELISA techniques. Furthermore, our results suggest that active AR inflammation does not induce detectable changes of TSLP or TSLPR. The lack of TSLP and TSLPR mRNA in these PB samples may indicate that this cytokine is of greater importance at the level of epithelial interface.
787
Group V sPLA2 is Required in Alternative Activated Macrophages for the Development of Allergic Pulmonary Inflammation S. Ohta, G. Giannattasio, W. Xing, J. A. Boyce, B. Balestrieri; Brigham and Women’s Hospital / Harvard Medical School, Boston, MA. RATIONALE: Expression of group V secretory phospholipase A2 sPLA2) by both dendritic cells (DC) and yet-to-be identified lung cell type(s) is required for development of pulmonary inflammation by an extract of house dust mite Dermatophagoides farinae (Df). Because group V sPLA2 is strongly expressed by alternative activated macrophages (AAMacs), prominent effector cells in allergic inflammation, we investigated the role of group V sPLA2 in AAMacs in pulmonary inflammation in vivo. METHODS: WT and group V sPLA2 (Pla2g5)-null mice were challenged intranasally with Df (1, 3 or 6 doses). Relm-a expression in lung was evaluated by qPCR and immunohistochemistry. T-cells were isolated from lung and intracellular cytokines measured by FACS. WT and Pla2g5-null mice were sensitized by intratracheal transfer of Df-pulsed WT-DC, and intravenously transferred with WT or Pla2g5-null IL-4-stimulated bone-marrow macrophages (BM-IL-4-Macs) (surrogate of AAMacs). RESULTS: Relm-a was dose-dependently induced in lungs of WT mice, but almost absent in Pla2g5-null mice. The number of lung T-cells dosedependently increased in WT but not Pla2g5-null mice. Compared to WT controls, Df-treated Pla2g5-null mice completely lacked IL-5, IL-4, and IL-17 producing lung T-cells. WT-BM-IL-4-Macs, but not Pla2g5null-BM-IL-4-Macs, transferred into sensitized WT mice increased BAL cellularity and eosinophilia. BM-IL-4-Mac transfers from either genotype failed to increase inflammation in sensitized Pla2g5-null hosts. CONCLUSIONS: Endogenous expression of group V sPLA2 by AAMacs amplifies their effector function in Df-induced pulmonary inflammation, but its expression is also required in other cell types for development of the full spectrum of inflammation. Group V sPLA2 may be an important target for treatment of allergic disorders.
788
Thioredoxin is Positively Associated with Eosinophils in Induced Sputum from Patients with Asthma J. M. Hartman, A. Hastie, W. C. Moore, B. Rector, S. P. Peters, E. R. Bleecker, M. S. Dykewicz; NHLBI Severe Asthma Research Program, Wake Forest University School of Medicine, Winston-Salem, NC. RATIONALE: Thioredoxin (TRX) is a multifunctional protein actively secreted by various airway cells including activated lymphocytes. TRX reduces chemotaxis of human eosinophils, and reduces eosinophils in BAL of TRX-treated ovalbumin-sensitized mice, potentially suppressing airway inflammation. We hypothesized increased TRX concentrations would inversely associate with decreased eosinophils in induced sputum from asthma patients. METHODS: Asthma patients (N562) enrolled in an IRB-approved protocol in the Wake Forest Severe Asthma Research Program (SARP) were comprehensively characterized including spirometry, serology and induced sputum. Sputum supernatants were assayed for TRX by commercially available ELISA. Statistical analysis was performed using the student’s t test, ANOVA, and linear regression. RESULTS: TRX levels did not significantly differ by gender (p50.88), or corticosteroid use/non-use (p50.19), but showed trends toward higher levels in Caucasians than in African Americans (228 + 18 vs. 172 + 25, respectively, p50.07) and in severe compared with nonsevere asthma (medians 279 vs 169, respectively, p50.08). Elevated TRX levels were associated with increased eosinophil numbers in induced sputum samples (R5 0.32, p5 0.013). Additionally, increased sputum lymphocytes (R5 0.36, P5 0.004) and macrophages (R5 0.43, p< 0.001) were associated with increased TRX. However, there was no significant association of serum inflammatory cells with sputum TRX. CONCLUSIONS: Our results demonstrate a significant positive association between sputum TRX levels and sputum eosinophil levels in human asthma, in contrast to observations in murine models. Moreover, significant associations of TRX with sputum lymphocytes and macrophages indicate complex regulatory mechanisms related to TRX expression and release in airway inflammation.
MONDAY
785
J ALLERGY CLIN IMMUNOL VOLUME 129, NUMBER 2
Basophils In Peripheral Blood can be Sensitized in Local Allergic Rhinitis E. Gomez1, C. Rondon2, P. Campo2, L. Galindo Reyes1, J. A. Huertas2, L. Melendez1, J. Garcia Campos2, M. J. Torres2, M. Blanca2; 1Research Laboratory, Carlos Haya Hospital-Fundacion IMABIS, Malaga, SPAIN, 2 Allergy Service, Carlos Haya Hospital, Malaga, SPAIN. RATIONALE: Local allergic rhinitis (LAR) is a new phenotype of rhinitis characterized by local production of sIgE, a Th2 nasal inflammatory pattern and positive response to nasal allergen provocation test (NAPT) with negative skin prick test and serum specific-IgE. The presence of surface-bound IgE to basophils has not been investigated yet. In this study we evaluated the specific activation of basophil in patients with LAR to D. pteronyssinus (DP). METHODS: The study group was 10 LAR patients to DP. Two controls groups were included: 11 allergic rhinitis (AR) patients to DP and 10 healthy controls. Skin prick test, serum specific-IgE, NAPT-DP, and BAT with DP at different concentrations (5, 20, 50 ng/ml) were performed. RESULTS: NAPT-DP was positive in all LAR and AR patients and negative in healthy controls. BAT was positive in 5 LAR patients (50%), 8 AR patients (72.7%), and 2 healthy controls (20%). BAT results in AR patients and healthy controls showed a sensitivity of 72.2%, a specificity of 80%, and a significant concordance with NAPT (kappa index 0.525, p50.016). CONCLUSIONS: In this study the 50% of LAR patients to DP showed specific basophil activation, indicating that at least half of the patients with LAR to DP can be diagnosed by this approach. These results suggest that after local production of specific-IgE, basophils may be the first target cells for specific-IgE, prior to the detection of free serum specific-IgE and skin mast-cells sensitization.
786
Evaluation of Thymic Stromal Lymphopoietin (TSLP) and its Receptor in Adults with Allergic Rhinitis Following Controlled Allergen Challenge in the Environmental Exposure Unit (EEU) T. Batool1, J. Thiele2, A. K. Ellis2; 1McMaster University, Hamilton, ON, CANADA, 2Queen‘s University, Kingston, ON, CANADA. RATIONALE: Thymic stromal lymphopoietin(TSLP) is an epithelialderived IL-7 like cytokine, and a potent inducer of Th2 response deregulation. We hypothesized that active allergic rhinitis (AR) inflammation may induce a systemic inflammatory cascade leading to the detection of TSLP or changes in TSLP-receptor(TSLPR) mRNA levels in peripheral blood(PB). METHODS: 44 subjects with ragweed-induced AR underwent a 3hr controlled ragweed pollen challenge in the EEU to develop AR symptoms. Serum and PB mononuclear cells(MNCs) were collected before and after allergen challenge. ELISA was performed on serum samples from 12 subjects for the detection of human TSLP. The cytokine profile was also analyzed utilizing Luminex technology(Human 17-Plex Assay). TSLPR mRNA expression in PBMNCs was evaluated using qPCR with ACTB as the reference gene. RESULTS: TSLP was undetectable in all 12 subject serum samples examined, either before or after allergen challenge. The cytokine profile analysis confirmed that other cytokines were detectable in the subject samples, eliminating sample storage as a potential cause for the lack of TSLP detection. Additionally, we were unable to detect the presence of TSLPR mRNA either before or after allergen challenge, despite appropriate reference gene detection. CONCLUSIONS: TSLP does not appear to be detectable in the systemic circulation of adults with active allergic rhinitis inflammation using currently available ELISA techniques. Furthermore, our results suggest that active AR inflammation does not induce detectable changes of TSLP or TSLPR. The lack of TSLP and TSLPR mRNA in these PB samples may indicate that this cytokine is of greater importance at the level of epithelial interface.
787
Group V sPLA2 is Required in Alternative Activated Macrophages for the Development of Allergic Pulmonary Inflammation S. Ohta, G. Giannattasio, W. Xing, J. A. Boyce, B. Balestrieri; Brigham and Women’s Hospital / Harvard Medical School, Boston, MA. RATIONALE: Expression of group V secretory phospholipase A2 sPLA2) by both dendritic cells (DC) and yet-to-be identified lung cell type(s) is required for development of pulmonary inflammation by an extract of house dust mite Dermatophagoides farinae (Df). Because group V sPLA2 is strongly expressed by alternative activated macrophages (AAMacs), prominent effector cells in allergic inflammation, we investigated the role of group V sPLA2 in AAMacs in pulmonary inflammation in vivo. METHODS: WT and group V sPLA2 (Pla2g5)-null mice were challenged intranasally with Df (1, 3 or 6 doses). Relm-a expression in lung was evaluated by qPCR and immunohistochemistry. T-cells were isolated from lung and intracellular cytokines measured by FACS. WT and Pla2g5-null mice were sensitized by intratracheal transfer of Df-pulsed WT-DC, and intravenously transferred with WT or Pla2g5-null IL-4-stimulated bone-marrow macrophages (BM-IL-4-Macs) (surrogate of AAMacs). RESULTS: Relm-a was dose-dependently induced in lungs of WT mice, but almost absent in Pla2g5-null mice. The number of lung T-cells dosedependently increased in WT but not Pla2g5-null mice. Compared to WT controls, Df-treated Pla2g5-null mice completely lacked IL-5, IL-4, and IL-17 producing lung T-cells. WT-BM-IL-4-Macs, but not Pla2g5null-BM-IL-4-Macs, transferred into sensitized WT mice increased BAL cellularity and eosinophilia. BM-IL-4-Mac transfers from either genotype failed to increase inflammation in sensitized Pla2g5-null hosts. CONCLUSIONS: Endogenous expression of group V sPLA2 by AAMacs amplifies their effector function in Df-induced pulmonary inflammation, but its expression is also required in other cell types for development of the full spectrum of inflammation. Group V sPLA2 may be an important target for treatment of allergic disorders.
788
Thioredoxin is Positively Associated with Eosinophils in Induced Sputum from Patients with Asthma J. M. Hartman, A. Hastie, W. C. Moore, B. Rector, S. P. Peters, E. R. Bleecker, M. S. Dykewicz; NHLBI Severe Asthma Research Program, Wake Forest University School of Medicine, Winston-Salem, NC. RATIONALE: Thioredoxin (TRX) is a multifunctional protein actively secreted by various airway cells including activated lymphocytes. TRX reduces chemotaxis of human eosinophils, and reduces eosinophils in BAL of TRX-treated ovalbumin-sensitized mice, potentially suppressing airway inflammation. We hypothesized increased TRX concentrations would inversely associate with decreased eosinophils in induced sputum from asthma patients. METHODS: Asthma patients (N562) enrolled in an IRB-approved protocol in the Wake Forest Severe Asthma Research Program (SARP) were comprehensively characterized including spirometry, serology and induced sputum. Sputum supernatants were assayed for TRX by commercially available ELISA. Statistical analysis was performed using the student’s t test, ANOVA, and linear regression. RESULTS: TRX levels did not significantly differ by gender (p50.88), or corticosteroid use/non-use (p50.19), but showed trends toward higher levels in Caucasians than in African Americans (228 + 18 vs. 172 + 25, respectively, p50.07) and in severe compared with nonsevere asthma (medians 279 vs 169, respectively, p50.08). Elevated TRX levels were associated with increased eosinophil numbers in induced sputum samples (R5 0.32, p5 0.013). Additionally, increased sputum lymphocytes (R5 0.36, P5 0.004) and macrophages (R5 0.43, p< 0.001) were associated with increased TRX. However, there was no significant association of serum inflammatory cells with sputum TRX. CONCLUSIONS: Our results demonstrate a significant positive association between sputum TRX levels and sputum eosinophil levels in human asthma, in contrast to observations in murine models. Moreover, significant associations of TRX with sputum lymphocytes and macrophages indicate complex regulatory mechanisms related to TRX expression and release in airway inflammation.
MONDAY
785