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Eosinophils contribute to the resolution of lung-allergic responses following repeated allergen challenge Katsuyuki Takeda, MD, PhD, Yoshiki Shiraishi, PhD, Shigeru Ashino, PhD, Junyan Han, PhD, Yi Jia, MD, PhD, Meiqin Wang, MD, PhD, Nancy A. Lee, PhD, James J. Lee, PhD, Erwin W. Gelfand, MD Journal of Allergy and Clinical Immunology Volume 135, Issue 2, Pages e5 (February 2015) DOI: /j.jaci Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Fig 1 Experimental protocol for sensitization and challenge with OVA. A, PHIL mice and WT littermates were sensitized to OVA on days 0 and 14 followed by aerosolized OVA challenges 7 times (OVA/OVA-7) or 11 times (OVA/OVA-11). Control mice received sham sensitization (PBS/OVA-7 and PBS/OVA-11). B, Mice were sensitized with OVA in the same manner as described above. One week after the last sensitization, mice received bone marrow cells (5 × 106) from WT or IL-10−/− mice. OVA challenges were initiated 30 days after bone marrow cell transfer (BMT). Controls received vehicle only. Journal of Allergy and Clinical Immunology , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Fig 2 Changes in airway function and inflammation after repeated OVA challenges in WT and PHIL mice. A, (a) Changes in RL after 7 OVA challenges. (b) BAL cell composition. (c) Airway responsiveness after 11 OVA challenges. (d) BAL cell composition. n = 9. #P < .05 vs PBS/OVA-7 or PBS/OVA-11 groups. B, Goblet cell metaplasia. Representative photomicrographs of lung tissue from (a) WT mice after sham sensitization and 11 OVA challenges, (b) PHIL mice after sham sensitization and 11 OVA challenges, (c) WT mice after sensitization and 11 OVA challenges, and (d) PHIL mice after sensitization and 11 OVA challenges. (e) Quantitation of PAS-positive areas along the airways. C, BAL cytokine levels in WT and PHIL mice. BAL fluid samples were collected 48 hours after 7 or 11 OVA challenges. n = 9 in all experiments except panel B-e (n = 6). BM, Bone marrow; Eo, eosinophils; Ly, lymphocytes; Mac, macrophages; Nt, neutrophils. #P < .05 vs PBS/OVA-7 and PBS/OVA-11. *P < .05 vs PHIL OVA/OVA-11. Journal of Allergy and Clinical Immunology , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Fig 2 Changes in airway function and inflammation after repeated OVA challenges in WT and PHIL mice. A, (a) Changes in RL after 7 OVA challenges. (b) BAL cell composition. (c) Airway responsiveness after 11 OVA challenges. (d) BAL cell composition. n = 9. #P < .05 vs PBS/OVA-7 or PBS/OVA-11 groups. B, Goblet cell metaplasia. Representative photomicrographs of lung tissue from (a) WT mice after sham sensitization and 11 OVA challenges, (b) PHIL mice after sham sensitization and 11 OVA challenges, (c) WT mice after sensitization and 11 OVA challenges, and (d) PHIL mice after sensitization and 11 OVA challenges. (e) Quantitation of PAS-positive areas along the airways. C, BAL cytokine levels in WT and PHIL mice. BAL fluid samples were collected 48 hours after 7 or 11 OVA challenges. n = 9 in all experiments except panel B-e (n = 6). BM, Bone marrow; Eo, eosinophils; Ly, lymphocytes; Mac, macrophages; Nt, neutrophils. #P < .05 vs PBS/OVA-7 and PBS/OVA-11. *P < .05 vs PHIL OVA/OVA-11. Journal of Allergy and Clinical Immunology , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Fig 3 Consequences of eosinophil restoration after transfer of bone marrow cells. A, (a) RL after bone marrow cell transfer (BMT) and 7 OVA challenges. (b) BAL cell composition. (c) RL after bone marrow cell transfer and 11 OVA challenges. (d) BAL cell composition after 11 OVA challenges. B, Goblet cell metaplasia. Representative photomicrographs in the lung tissue from (a) vehicle-treated WT mice after sensitization and 11 OVA challenges, (b) vehicle-treated PHIL mice after sensitization and 11 OVA challenges, (c) PHIL mice that received bone marrow cells from WT mice after sensitization and 11 OVA challenges, and (d) PHIL mice that received bone marrow cells from IL-10−/− mice after sensitization and 11 OVA challenges. (e) Quantitation of PAS-positive areas along the airways. C, BAL fluid cytokine levels after bone marrow cell transfer and 11 OVA challenges. n = 9 in all experiments except panel B-e (n = 6). BM, bone marrow; Eo, eosinophils; Ly, lymphocytes; Mac, macrophages. *P < .05 vs PHIL OVA/OVA-7 + vehicle group, #P < .05 vs WT OVA/OVA-11 + vehicle and PHIL OVA/OVA-11 + WT-BM. Journal of Allergy and Clinical Immunology , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Fig 3 Consequences of eosinophil restoration after transfer of bone marrow cells. A, (a) RL after bone marrow cell transfer (BMT) and 7 OVA challenges. (b) BAL cell composition. (c) RL after bone marrow cell transfer and 11 OVA challenges. (d) BAL cell composition after 11 OVA challenges. B, Goblet cell metaplasia. Representative photomicrographs in the lung tissue from (a) vehicle-treated WT mice after sensitization and 11 OVA challenges, (b) vehicle-treated PHIL mice after sensitization and 11 OVA challenges, (c) PHIL mice that received bone marrow cells from WT mice after sensitization and 11 OVA challenges, and (d) PHIL mice that received bone marrow cells from IL-10−/− mice after sensitization and 11 OVA challenges. (e) Quantitation of PAS-positive areas along the airways. C, BAL fluid cytokine levels after bone marrow cell transfer and 11 OVA challenges. n = 9 in all experiments except panel B-e (n = 6). BM, bone marrow; Eo, eosinophils; Ly, lymphocytes; Mac, macrophages. *P < .05 vs PHIL OVA/OVA-7 + vehicle group, #P < .05 vs WT OVA/OVA-11 + vehicle and PHIL OVA/OVA-11 + WT-BM. Journal of Allergy and Clinical Immunology , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Fig 4 Intracellular IL-10 staining of airway eosinophils. BAL leukocytes from OVA-sensitized and 7-challenged WT mice were activated with PMA/ionomysin and then stained with anti-mouse CCR3-FITC, anti-mouse Siglec-F PE, and anti-mouse IL-10-APC or APC-conjugated isotype control antibody. To identify the source of IL-10 in BAL fluid, live cells were first gated (region 1, R1) on forward scatter (FSC) and side scatter (SSC) (A). Cells in R1 were separated into Siglec-F– and CCR3–double-positive cells (R2) or negative cells (R3) (B). IL-10–positive cells in R2 or R3 were expressed as histograms (C and D) and the values for MFI were analyzed and expressed as the ratio to MFIs of isotype control staining in each sample (E and F). n = 6. *P < .001 and #P < .01 vs isotype control antibody group. MFI, Mean fluorescence intensity; PMA, phorbol 12-myristate 13-acetate. Journal of Allergy and Clinical Immunology , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Fig E1 The number of IL-4–, IL-5–, or IL-13–positive CD4+ T cells in lungs. To identify the source of TH2 cytokines in sensitized and 7 times OVA-challenged WT or PHIL mice, intracellular cytokine staining was carried out. WT mice that received sham sensitization followed by 7 times OVA challenge served as controls. Isolated lung leukocytes were activated with PMA/ionomycin and then stained with anti-CD4 followed by intracellular cytokine staining. The number of IL-4–, IL-5–, or IL-13–positive CD4+ T cells in lungs was calculated and expressed as histograms. n = 5. *P < .05 vs WT PBS/OVA-7 group. PMA, Phorbol 12-myristate 13-acetate. Journal of Allergy and Clinical Immunology , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Fig E2 Circulating eosinophil numbers in WT and IL-10−/− mice were shown to be similar by either cell differential assessment of white blood cells after Wright's-Giemsa staining (A) or by ELISA assessments of EPO levels (arbitrary EPO units/1 × 106 of eosinophils) in unfractionated white blood cell pellets (n = 5 mice/group) (B). Eos, Eosinophils. Journal of Allergy and Clinical Immunology , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Fig E3 Localization of eosinophils in the lung tissue after bone marrow cell transfer (BMT). Lung tissues were stained with anti-mouse major basic protein (MBP) antibody. A, Representative photomicrographs of lung tissue from (a) vehicle-treated WT mice after sensitization and 7 OVA challenges, (b) vehicle-treated PHIL mice after sensitization and 7 OVA challenges, (c) PHIL mice that received bone marrow cells from WT mice after sensitization and 7 OVA challenges, and (d) PHIL mice that received bone marrow cells from IL-10−/− mice after sensitization and 7 OVA challenges. B, Number of MBP-positive cells. n = 6. *P < .05 vs PHIL OVA/OVA-7 + vehicle group. Journal of Allergy and Clinical Immunology , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Fig E4 Characterization of bone marrow–derived eosinophils. After stimulation with PMA/ionomycin, levels of IL-10 (ng/mL) (A) or EPO (B) (arbitrary EPO units/mL) in cell culture supernatants from eosinophils derived from WT and IL-10−/− mice were measured by using ELISA (n = 5 mice/group). PMA, Phorbol 12-myristate 13-acetate. *P < .05. Journal of Allergy and Clinical Immunology , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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