Lipoteichoic acid downregulates FcεRI expression on human mast cells through Toll-like receptor 2 Mino Yoshioka, MSc, Nobuyuki Fukuishi, PhD, Sayuri.

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Lipoteichoic acid downregulates FcεRI expression on human mast cells through Toll-like receptor 2 Mino Yoshioka, MSc, Nobuyuki Fukuishi, PhD, Sayuri Iriguchi, BSc, Kanae Ohsaki, BSc, Hiroyuki Yamanobe, BSc, Asumi Inukai, BSc, Daisuke Kurihara, BSc, Naoki Imajo, PhD, Yumiko Yasui, MSc, Nobuaki Matsui, PhD, Tadayuki Tsujita, PhD, Akihiro Ishii, PhD, Tsukasa Seya, MD, PhD, Makoto Takahama, MD, PhD, Masaaki Akagi, PhD Journal of Allergy and Clinical Immunology Volume 120, Issue 2, Pages 452-461 (August 2007) DOI: 10.1016/j.jaci.2007.03.027 Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Expression of TLRs by LAD2 MCs. A, TLR mRNA expression was analyzed by means of RT-PCR. For the negative control, the reverse transcription step was omitted. B, TLR protein expression was analyzed by means of Western blotting. Isotype antibody was used as a negative control. C, Activation of LAD2 cells by TLR ligands. LAD2 cells were treated without or with LTA (30 μg/mL), PGN (100 μg/mL), LPS (1 ng/mL), flagellin (30 ng/mL), or 3CpG-ODN (1 μg/mL) for the indicated times, and phosphorylated ERK1/2 (p-ERK1/2) levels were analyzed by means of Western blotting. Membranes were then stripped and reprobed with an antibody against total ERK1. The data shown are representative of 3 independent experiments. Journal of Allergy and Clinical Immunology 2007 120, 452-461DOI: (10.1016/j.jaci.2007.03.027) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 LTA and PGN decrease the expression of FcεRI on LAD2 MCs. MCs were stimulated with LTA (30 μg/mL), PGN (100 μg/mL), LPS (1 ng/mL), flagellin (30 ng/mL), or 3CpG-ODN (1 μg/mL). A, FcεRI surface expression on LAD2 cells treated without (solid area) or with (open area) TLR ligands was determined by using flow cytometry. B, C, and D, Effect of TLR ligands on the level of FcεRIα (Fig 2, B) FcεRIβ (Fig 2, C), and FcεRIγ (Fig 2, D) protein was measured by using Western blotting. Culture medium was treated as a negative control for LTA, PGN, LPS, flagellin, and 3CpG-ODN. The data shown are representative of 3 independent experiments. Journal of Allergy and Clinical Immunology 2007 120, 452-461DOI: (10.1016/j.jaci.2007.03.027) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 LTA and PGN decrease the FcεRI mRNA in LAD2 MCs. LAD2 cells were stimulated without or with LTA (30 μg/mL) or PGN (100 μg/mL) for 4 or 24 hours. RNA was extracted from the cells, purified, and used to generate cDNA by means of RT. FcεRIα, FcεRIβ, or FcεRIγ mRNA expression was assessed by using real-time PCR. Results represent the means ± SEMs of 3 (FcεRIβ and FcεRIγ) to 4 (FcεRIα) independent experiments. Journal of Allergy and Clinical Immunology 2007 120, 452-461DOI: (10.1016/j.jaci.2007.03.027) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 LTA and PGN dose-dependently reduce the expression of FcεRI and inhibit β-hex release. A and B, LAD2 cells were incubated for 48 hours with the indicated concentrations of LTA (Fig 4, A) or PGN (Fig 4, B). FcεRI surface expression was determined by means of flow cytometry. The extent of β-hex release was compared between cells treated with and without TLR2 ligands. C and D, The dose-response curves of the NP-BSA antigen with or without TLR2 ligands were investigated. ∗P < .05 and ∗∗P < .01 versus no added ligand. #P < .05 and ##P < .01 versus the same concentration of antigen without TLR2 ligand. Data represent the means ± SEMs of 3 independent experiments. Journal of Allergy and Clinical Immunology 2007 120, 452-461DOI: (10.1016/j.jaci.2007.03.027) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Effect of a TLR2-neutralizing antibody on LTA- and PGN-induced downregulation of FcεRI in LAD2 cells. A, The expression of FcεRI was determined by means of flow cytometry. Data shown are representative of 3 independent experiments. B, The effect of a TLR2-neutralizing antibody on LTA-treated LAD2 cells was evaluated on the basis of β-hex release. Data represent the means ± SEM of 3 independent experiments. C, The effect of a TLR2-neutralizing antibody on PGN-treated MCs was evaluated on the basis of β-hex release. Data represent the means ± SEMs of 3 independent experiments. Ab, Antibody. Journal of Allergy and Clinical Immunology 2007 120, 452-461DOI: (10.1016/j.jaci.2007.03.027) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Effect of LTA and PGN on FcεRI expression and β-hex release by freshly isolated puMCs. A, The expression of TLR2 on naive human puMCs was determined by using Western blotting. B, The purity of isolated human MCs was determined by using flow cytometry. C, Naive puMCs (solid area) were treated with LTA (30 μg/mL) or PGN (100 μg/mL) for 48 hours (solid line), and the expression of FcεRI was determined by means of flow cytometry. An isotype control was used as a negative control (thin line and dotted line). D, After incubation of puMCs with LTA or PGN, FcεRI was aggregated with IgE-NP and NP-BSA, and β-hex release was measured. ∗P < .05 and ∗∗P < .01. Data represent the means ± SEMs of 3 independent experiments. Journal of Allergy and Clinical Immunology 2007 120, 452-461DOI: (10.1016/j.jaci.2007.03.027) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions