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Yilin Qi, MSc, Darwin J. Operario, PhD, Christopher M

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1 Human basophils express amphiregulin in response to T cell–derived IL-3 
Yilin Qi, MSc, Darwin J. Operario, PhD, Christopher M. Oberholzer, MD, James J. Kobie, PhD, R. John Looney, MD, Steve N. Georas, MD, Tim R. Mosmann, PhD  Journal of Allergy and Clinical Immunology  Volume 126, Issue 6, Pages e4 (December 2010) DOI: /j.jaci Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Human basophils express amphiregulin (AR). A, PBMCs were stimulated with anti-CD3/CD28, stained, and gated as shown. B, AR expression on unstimulated (shaded) and stimulated (line) CD4−CD8−CD123+ cells. C, Sorted CD4−CD8−CD14−CD19−7AAD−AR+ cells were stained with Wright-Giemsa. D, Stimulation-induced AR mRNA changes were measured in CD4+CD8−CD14−CD19−7AAD−CD123− (“CD4”) and CD4−CD8−CD14−CD19−7AAD−CD123+ (“CD123”) cells sorted from anti-CD3/CD28–stimulated PBMCs (4 subjects; 16 hours). Results are representative of at least 3 experiments. Max, Maximum. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 IL-3 is necessary and sufficient for induction of amphiregulin (AR) expression on basophils by activated T cells. A, Surface antigens on basophils (CD4−CD8−CD14−CD19−7AAD−CD203c+) were analyzed by flow cytometry in unstimulated (shaded) or stimulated (line) PBMCs. B, PBMCs were treated with rhIL-3 (left, 6 hours; right, 5 ng/mL). Means of triplicate cultures are shown. C, Sorted basophils treated for 16 hours as indicated were analyzed by flow cytometry. Results are representative of at least 3 experiments. FSC, Forward scatter; hIL, human interleukin; mIgG1, normal mouse IgG1; rhIL-3, recombinant human IL-3; SSC; side scatter. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Amphiregulin (AR) expression in human basophils is induced more strongly by IL-3 than IgE cross-linking. Enriched basophils were stimulated as indicated. A, Histamine release and surface AR expression (4 hours). Symbols represent different individuals. B, Surface AR on basophils from 5 individuals (16 hours). C, Supernatant AR after stimulation with or without anti–IL-3Rα or TAPI-1 (24 hours). con., Concentration; ns, not significant. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 IL-3 and IgE cross-linking induce different activation profiles in human basophils. Enriched basophils were stimulated as indicated, and mRNA was measured by qPCR. Kinetics of amphiregulin (AR), IL-4, IL-13, and HB-EGF mRNA expression in response to IL-3 or anti-IgE stimulation (expressed as the fold change relative to unstimulated cells) are shown. Similar results were obtained in at least 4 experiments. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Mouse basophils express amphiregulin (AR) in response to IL-3 activation. Mouse blood leukocytes from C57BL/6.PL (B6/PL) or AR−/− mice were treated as indicated and stained for surface markers and intracellular IL-4 and AR expression. Basophils (CD4−CD19−Gr-1−FcεRI+) were gated and then analyzed for IL-4 versus AR. Results are representative of 3 experiments. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Kinetics of amphiregulin (AR) mRNA, cell surface, and soluble form expression in response to IL-3. Enriched basophils were treated with medium alone or rhIL-3 (10 ng/mL) for indicated times. A, AR mRNA was measured by RT-PCR. Data are shown as the fold change relative to 1 hour unstimulated cells. B, Surface AR expression on basophils was analyzed by flow cytometry. AR MFI of basophils and the percentage of AR+ cells are shown. C, Soluble AR expression was measured by ELISA. Results are representative of at least 3 experiments. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Soluble amphiregulin (AR) expression in response to IL-3 or IgE cross-linking. Enriched basophils were treated with medium alone, rhIL-3 (5 ng/mL), or anti-IgE (1 ng/mL to 1 μg/mL). Histamine release (at 4 hours) and soluble AR (at 24 hours) were measured by ELISA. Results are representative of 3 experiments. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Synergy between IL-3 and anti-IgE for soluble amphiregulin (AR) expression. Enriched basophils were treated with medium alone, IL-3 (5 ng/mL), anti-IgE (10 ng/mL), or IL-3 (10 ng/mL) + anti-IgE (10 ng/mL) for 24 hours. Soluble AR was measured by ELISA. Representative of 3 experiments. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Increased amphiregulin (AR)–expressing basophils in subjects with asthma. This study was approved by the institutional review board of the University of Rochester Medical Center, and written informed consent was obtained from all participating subjects. Eleven subjects with allergic asthma (age, years; 5 women, 6 men), 17 subjects with allergic rhinitis but no asthma (age, years; 9 women, 8 men), and 11 normal subjects without allergy (age, years; 5 women, 6 men) were enrolled for the study. The screening and classification of subjects into 3 groups were based on the results from skin tests, RAST, and lung function tests. Exclusion criteria included a history of cigarette smoking or drug or alcohol abuse, significant occupational exposure to respiratory irritants or toxins, chronic use of medications (other than medications for allergies/asthma and oral contraceptives), cardiac or severe lung disease, history of allergen immunotherapy or underlying illnesses that may alter lung function, and pregnancy. Peripheral blood was drawn from participating subjects with no recent exacerbations. PBMCs were isolated as described in Methods. Cells were stimulated with medium alone (No), 5 ng/mL rhIL-3, 5 μg/mL anti-CD3 + 1 μg/mL anti-CD28, or 1 μg/mL SEB. Anti-CD3/CD28 and SEB stimulations were also performed in the presence of anti–IL-3 (aIL-3, 10 μg/mL). After 16 hours, the cells were stained with antibodies specific for CD4, CD8, CD14, FcεRIα, CD123, CD203c, CD69, AR, and 7AAD and analyzed by flow cytometry. All analysis of flow cytometry was performed by a blind investigator. Basophils expressing AR (FcεRIα+CD123+AR+) are expressed as the numbers per million PBMCs. Statistical comparisons were made by 1-sided Mann-Whitney test. No, No treatment; aIL, anti-IL-3. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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