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CCL17/thymus and activation-regulated chemokine induces calcitonin gene–related peptide in human airway epithelial cells through CCR4  Kandace Bonner,

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Presentation on theme: "CCL17/thymus and activation-regulated chemokine induces calcitonin gene–related peptide in human airway epithelial cells through CCR4  Kandace Bonner,"— Presentation transcript:

1 CCL17/thymus and activation-regulated chemokine induces calcitonin gene–related peptide in human airway epithelial cells through CCR4  Kandace Bonner, PhD, James E. Pease, PhD, Christopher J. Corrigan, MD, PhD, Peter Clark, PhD, A. Barry Kay, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 132, Issue 4, Pages e3 (October 2013) DOI: /j.jaci Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Induction of CGRP by CCL17 in the bronchial epithelial cell line BEAS-2B. A and B, Time course of α-CGRP mRNA (Fig 1, A) and peptide product (Fig 1, B) expression measured by using an enzyme immunoassay. C and D, Concentration response of α-CGRP mRNA (Fig 1, C) and cell-associated peptide (Fig 1, D) expression measured by using immunofluorescence. Results shown are means ± SEMs of 5 independent experiments. *P < .05, **P < .01, and ***P < .001 compared with untreated cells or between treated samples, as indicated. MFU, Mean fluorescence units. E and F Confocal microscopic images of CGRP immunoreactivity in BEAS-2B cells cultured with medium alone (Fig 1, E) or medium with 10 ng/mL CCL17 (Fig 1, F). G, Control serum. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 CCL17-induced CGRP expression in primary human bronchial epithelial cells. Bronchial epithelial cells from 3 donors were cultured with increasing concentrations of CCL17 for 24 hours, and CGRP expression in supernatants was measured by using enzyme immunoassay. Results shown are means ± SEMs of triplicate samples. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Time course of CGRP mRNA (upper panel) and peptide product (lower panel) expression by bronchial epithelial cells stimulated with CCL17, IL-13, or a mixture of proinflammatory cytokines (CM; IL-1β, TNF-α, and GM-CSF, all at 10 ng/mL). Results shown are means ± SEMs of 5 independent experiments. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 A, C, and E, Confocal microscopic images showing CCR4 expression in BEAS-2B cells (Fig 4, A), A549 cells (Fig 4, C), H9 cells (Fig 4, E). B, D, and F, Respective isotype controls. G-I, Surface expression of CCR4 is shown by using flow cytometry in BEAS-2B cells (Fig 4, G), A549 cells (Fig 4, H), and H9 cells (Fig 4, I) with an anti-CCR4 mAb (blue line), with an isotype control (purple line), or unlabeled (purple shaded area). Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Internalization of CCR4 in BEAS-2B cells (A), A549 cells (B), and H9 cells (C) after stimulation with CCL17 for 30 minutes. Bars represent the mean percentage of CCR4 expression in treated versus untreated cells (n = 3). *P < .05, **P < .01, and ***P < Representative flow cytometric tracings are also shown. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Inhibition of CCL17-induced chemotaxis and CGRP release by an anti-CCR4 blocking antibody. Blockade of CCL17-induced chemotaxis of HUT78 cells by anti-CCR4 (10E7) is shown in the inset. Inhibition of CGRP release from BEAS-2B cells was investigated, as described in the Methods section. Anti-CCR3 was used as a negative (irrelevant antibody) control. Experiments were performed in triplicate. Results are the mean of 3 experiments. Pooled data were analyzed by using the Mann-Whitney test: *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig 7 CCR4 expression in the bronchial epithelium of healthy subjects and asthmatic patients. Confocal microscopic images showing CCR4 expression (green) in the bronchial epithelium of a nonasthmatic subject (top left), an asthmatic patient at baseline (top middle), and an asthmatic patient challenged with an allergen-derived peptide (top right). Nuclei are stained blue. Isotype control antibody staining is shown in the lower panels. Images are representative of 3 subjects in each group. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E1 CCL17 induced a time- and concentration-dependent increase in β-CGRP mRNA expression in BEAS-2B cells (A and B) and CCL17-induced α-CGRP and β-CGRP mRNA expression and secretion of CGRP peptide by using A549 cells (C-E). *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E2 CCL17 induces CGRP more potently than CCL22. A, Time course of α-CGRP mRNA expression in response to CCL22. B, Comparison of CCL17- and CCL22-induced α-CGRP mRNA expression after 24 hours of stimulation expressed as the fold change from untreated cells. C and D, Time course of CGRP release into supernatants of BEAS-2B cells cultured with or without 10 ng/mL CCL22 for up to 24 hours (Fig E2, C) and compared with CCL17 (10 ng/mL; Fig E2, D; n = 3-5 independent experiments). **P < .01 and ***P < .001 compared with untreated cells or between treated samples, as indicated. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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