IL-17E upregulates the expression of proinflammatory cytokines in lung fibroblasts Séverine Létuvé, PhD, Stéphane Lajoie-Kadoch, MSc, Séverine Audusseau,

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IL-17E upregulates the expression of proinflammatory cytokines in lung fibroblasts Séverine Létuvé, PhD, Stéphane Lajoie-Kadoch, MSc, Séverine Audusseau, MSc, Marc E. Rothenberg, MD, PhD, Pierre-Olivier Fiset, BSc, Mara S. Ludwig, MD, Qutayba Hamid, MD, PhD Journal of Allergy and Clinical Immunology Volume 117, Issue 3, Pages 590-596 (March 2006) DOI: 10.1016/j.jaci.2005.10.025 Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 1 Expression of IL-17BR in human airway fibroblasts. A, IL-17BR immunofluorescent staining in cultured lung fibroblasts (left panel). Negative control experiment using an isotype-matched control primary antibody is shown in right panel. Magnification: 200×. B, Detection of IL-17BR expression by Western blot in fibroblast lysates (lane 2) in parallel with the purified control fusion protein Fc/IL-17BR (lane 1). Results are representative of a minimum of 3 experiments. C, Expression of IL-17BR mRNA, as quantified by real-time PCR in fibroblasts stimulated with vehicle, TNF-α, or TGF-β1. IL-17BR levels were normalized over those of the housekeeping gene S9, and values are expressed as a fold increase compared with vehicle-treated cells. Values are means ± SEMs of 3 to 5 experiments. ∗P ≤ .05 compared with vehicle-treated fibroblasts at the same time point. Journal of Allergy and Clinical Immunology 2006 117, 590-596DOI: (10.1016/j.jaci.2005.10.025) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 2 Effect of IL-17E on the expression of fibroblast-derived mediators. Serum-deprived lung fibroblasts were stimulated with IL-17E or its vehicle and CCL-11 (A), CCL-5 (B), GM-CSF (C), and CXCL-8 (D) mRNA levels were evaluated. Results are presented as a fold increase compared with vehicle-treated cells at the same time point. Values are means ± SEMs of 6 experiments. ∗P ≤ .05 compared with vehicle-treated fibroblasts. Journal of Allergy and Clinical Immunology 2006 117, 590-596DOI: (10.1016/j.jaci.2005.10.025) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 3 Synergistic effect of TNF-α and IL-17E on the expression of cytokines in lung fibroblasts. Fibroblasts were stimulated with IL-17E or its vehicle, alone or in combination with TNF-α, and CCL-11 (A), CCL-5 (B), GM-CSF (C), and CXCL-8 (D) mRNA levels were quantified. Results are expressed as a fold increase, compared with the value obtained in vehicle-treated cells at the same time point. Values are means ± SEMs of 3 to 7 experiments. ∗P ≤ .05 compared with IL-17E–treated cells at the same time point. Journal of Allergy and Clinical Immunology 2006 117, 590-596DOI: (10.1016/j.jaci.2005.10.025) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 4 Effect of the combination of TGF-β1 and IL-17E on the expression of cytokine expression in fibroblasts. Fibroblasts were stimulated with IL-17E or its vehicle, alone or in combination with TGF-β1, and mRNA levels of CCL-11 (A), CCL-5 (B), GM-CSF (C), and CXCL-8 (D) were quantified. Results are expressed as a percentage of the value obtained in vehicle-treated cells at the same time point. Values are means ± SEMs of 3 to 6 experiments. ∗P ≤ .05 compared with IL-17E–stimulated cells and †P ≤ .05 compared with TGF-β1–treated cells at the same time point. Journal of Allergy and Clinical Immunology 2006 117, 590-596DOI: (10.1016/j.jaci.2005.10.025) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 5 Synergistic effect of IL-17E and TNF-α on cytokine release by fibroblasts. Fibroblasts were stimulated for 48 hours with IL-17E or its vehicle, with or without TNF-α. GM-CSF (A) and CXCL-8 (B) were quantified in supernatants. Values are expressed as means ± SEMs of 3 to 6 experiments. ∗P ≤ .05 compared with vehicle and †P ≤ .05 compared with IL-17E–treated cells. Journal of Allergy and Clinical Immunology 2006 117, 590-596DOI: (10.1016/j.jaci.2005.10.025) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 6 Expression of IL-17E in asthma. Immunohistochemistry for IL-17E (A) and MBP (B) was performed on bronchial biopsies of subjects with asthma. Representative staining experiments are shown. Journal of Allergy and Clinical Immunology 2006 117, 590-596DOI: (10.1016/j.jaci.2005.10.025) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions