Downregulation of a disintegrin and metalloproteinase 33 by IFN-γ in human airway smooth muscle cells Isao Ito, MD, PhD, Johanne D. Laporte, PhD, Pierre.

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Downregulation of a disintegrin and metalloproteinase 33 by IFN-γ in human airway smooth muscle cells Isao Ito, MD, PhD, Johanne D. Laporte, PhD, Pierre O. Fiset, PhD, Kazuhisa Asai, MD, PhD, Yasuhiro Yamauchi, MD, James G. Martin, MD, Qutayba Hamid, MD, PhD Journal of Allergy and Clinical Immunology Volume 119, Issue 1, Pages 89-97 (January 2007) DOI: 10.1016/j.jaci.2006.08.038 Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 In situ hybridization study of ADAM33 using bronchial biopsy specimens from patients with asthma (A) and normal subjects (B; ×400). Positive signal is indicated as black granules. Black arrows, positive cells; gray arrows, negative cells. C, The ratio of SM cells with positive signals to total SM cells. ASM cells from patients with asthma showed more positive cells compared with controls (∗P = .002). Box-bar graph indicates ranges, first/third quarters, and medians. Journal of Allergy and Clinical Immunology 2007 119, 89-97DOI: (10.1016/j.jaci.2006.08.038) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 A-D, LCM for SM cells. A, Before dissection; B, after shooting; C, after dissection; and D, captured SM cells (×200). E, RT-PCR amplicons from laser-captured SM cells were loaded to 3% agarose gel. ADAM33 protein detected as brown by immunohistochemistry was stronger in patients with asthma (F) than control subjects (G). SM layers are shown between arrows. Note increased SM mass in subjects with asthma (F). posi con, Positive control. Journal of Allergy and Clinical Immunology 2007 119, 89-97DOI: (10.1016/j.jaci.2006.08.038) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Effect of IL-4 and IL-13 (A) and IFN-γ (B) on ADAM33 mRNA expression by quantitative RT-PCR. Ratio of ADAM33 to S9 of ASM cells treated with vehicle in each experiment was set as 1, and relative values of cytokine-stimulated/vehicle-treated are expressed. Means ± SEMs of 3 experiments performed in duplicate are shown (same in Figs 3-6). ∗P = .01; ∗∗P < .01; ∗∗∗P < .05. Journal of Allergy and Clinical Immunology 2007 119, 89-97DOI: (10.1016/j.jaci.2006.08.038) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Western blot analysis showing activation of ERK1/2 by IFN-γ (10 ng/mL; A) and its abrogation by MEK inhibitor U0126 (10 μmol/L; B). PC, Positive control. C, Effect of U0126 on the downregulation of ADAM33 by IFN-γ. Vehicle contains 1 × 10−5% BSA for IFN-γ and 0.1% DMSO for U0126. White and grey bars express ADAM33α and ADAM33β. ∗P < .05; ∗∗P < .01; ∗∗∗P = .07. p-ERK, Phosphorylated-ERK; t-ERK, total-ERK. Journal of Allergy and Clinical Immunology 2007 119, 89-97DOI: (10.1016/j.jaci.2006.08.038) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Effect of IFN-γ on decay kinetics of ADAM33 mRNA in ASM cells quantified by real-time RT-PCR. After 24-hour starvation, cells were treated with actinomycin D (Act-D; 2.5 μg/mL), IFN-γ (10 ng/mL), or both for the indicated time. Act-D was given 1 hour before IFN-γ. Values are indicated as the ratio to the baseline. Journal of Allergy and Clinical Immunology 2007 119, 89-97DOI: (10.1016/j.jaci.2006.08.038) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 A-D, Immunofluorescent study of ADAM33 in cultured ASM cells (×200). ADAM33 antibody (A and B) and preimmune serum (negative control; C and D). Nuclear staining with Hoechst33342 (B and D). E and F, Western blot analysis of ADAM33 expression in ASM cells. ∗Nonspecific bands (E). Treatment with IFN-γ for 48 hours decreased the amount of ADAM33 protein compared with the vehicle-treated sample (∗P < .05; F). Journal of Allergy and Clinical Immunology 2007 119, 89-97DOI: (10.1016/j.jaci.2006.08.038) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions