Maria B. Sukkar, PhD, Shaoping Xie, PhD, Nadia M

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Toll-like receptor 2, 3, and 4 expression and function in human airway smooth muscle Maria B. Sukkar, PhD, Shaoping Xie, PhD, Nadia M. Khorasani, BSc, Onn Min Kon, MD, Rex Stanbridge, MD, Razao Issa, PhD, Kian Fan Chung, MD Journal of Allergy and Clinical Immunology Volume 118, Issue 3, Pages 641-648 (September 2006) DOI: 10.1016/j.jaci.2006.05.013 Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 1 TLR mRNA expression in ASMCs. TLR1 through TLR10 mRNA expression under basal conditions (A) or after stimulation with IL-1β or TNF-α (10 ng/mL) for 24 hours (B) is shown. Note that the scale for threshold cycle (CT) is inverted, so that higher histogram bars represent higher levels of mRNA expression. Bars represent means ± SEM of 3 ASMC donors. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. Journal of Allergy and Clinical Immunology 2006 118, 641-648DOI: (10.1016/j.jaci.2006.05.013) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 2 Effects of cytokines, dsRNA, and dexamethasone (Dex) on TLR2 (A), TLR3 (B), and TLR4 (C) mRNA. Cells were treated with cytokines (10 ng/mL each) or dsRNA (25 μg/mL) ± dsRNA (25 μg/mL) or dexamethasone (1 μM) for 24 hours. Bars represent means ± SEM of 8 ASMC donors. ∗P < .05 versus unstimulated control; ∗∗P < .05 versus dsRNA only; ∗∗∗P < .05 versus stimulated cells in the absence of dexamethasone; ∞P < .05 versus cells treated with IFN-γ or TNF-α only; ∂P < .05 versus cells treated with TNF-α only. Journal of Allergy and Clinical Immunology 2006 118, 641-648DOI: (10.1016/j.jaci.2006.05.013) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 3 Effect of TLR ligands on TLR2, TLR3, and TLR4 mRNA. Cells were treated with PGN (10 μg/mL), zymosan (ZYM; 100 μg/mL), dsRNA (25 μg/mL), LPS (10 μg/mL), or CpG (3 μg/mL) for 24 hours. Bars represent means ± SEM of 6 to 10 ASMC donors. ∗P < .05 versus control. Journal of Allergy and Clinical Immunology 2006 118, 641-648DOI: (10.1016/j.jaci.2006.05.013) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 4 Flow cytometric analysis of TLR2 (A) and TLR3 (B) expression in ASMCs. Cells were treated with IFN-γ and TNF-α (10 ng/mL each) or dsRNA (25 μg/mL) ± dexamethasone (Dex) for 48 hours. Bars represent means ± SEM of 7 to 9 ASMC donors. C, Representative histograms: thin lines represent cells stained with isotype control antibodies, and bold lines represent cells stained with TLR2 or TLR3 antibodies. ∗P < .05 versus appropriate control (Con); ∗∗P < .05 versus appropriate control in the absence of dexamethasone. MFI, Mean fluorescence intensity. Journal of Allergy and Clinical Immunology 2006 118, 641-648DOI: (10.1016/j.jaci.2006.05.013) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 5 Effect of cytokines and TLR ligands on IL-8 (A) and eotaxin (B) release. Cells were treated with IL-1β (10 ng/mL), TNF-α (10 ng/mL), PGN (10 μg/mL), dsRNA (25 μg/mL), or LPS (10 μg/mL) ± dexamethasone (Dex; 1 μM) for 24 hours. Bars represent means ± SEM of 6 to 8 ASMC donors. ∗P < .05 versus unstimulated cells. ∗∗P < .05 versus cells in the absence of dexamethasone. Journal of Allergy and Clinical Immunology 2006 118, 641-648DOI: (10.1016/j.jaci.2006.05.013) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 6 Effects of dsRNA on cytokine-induced IL-8 (A) and eotaxin (B) release. Cells were treated with IL-1β (10 ng/mL), TNF-α (10 ng/mL) or IFN-γ (10 ng/mL) ± dsRNA (25 μg/mL) for 24 hours. Bars represent means ± SEM of 6 to 8 ASMC donors. ∗P < .05 versus unstimulated cells; ∗∗P < .05 versus stimulated cells in the absence of dsRNA; ∗∗∗P < .05 versus cells stimulated with dsRNA only. Journal of Allergy and Clinical Immunology 2006 118, 641-648DOI: (10.1016/j.jaci.2006.05.013) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions