Glandular mast cells with distinct phenotype are highly elevated in chronic rhinosinusitis with nasal polyps Tetsuji Takabayashi, MD, Atsushi Kato, PhD,

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Glandular mast cells with distinct phenotype are highly elevated in chronic rhinosinusitis with nasal polyps Tetsuji Takabayashi, MD, Atsushi Kato, PhD, Anju T. Peters, MD, Lydia A. Suh, BSc, Roderick Carter, BSc, James Norton, MS, Leslie C. Grammer, MD, Bruce K. Tan, MD, Rakesh K. Chandra, MD, David B. Conley, MD, Robert C. Kern, MD, Shigeharu Fujieda, MD, Robert P. Schleimer, PhD Journal of Allergy and Clinical Immunology Volume 130, Issue 2, Pages 410-420.e5 (August 2012) DOI: 10.1016/j.jaci.2012.02.046 Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Increased expression of tryptase in NPs. The gene expression of tryptase in UT and NPs (A) and nasal scrapings (B) was measured by using real-time PCR. The concentration of tryptase in tissue homogenates of UT and NPs (C) and nasal lavage (D) was measured by using ELISA. Tryptase concentration was normalized to the concentration of total protein. ∗P < .05, ∗∗P < .001, ∗∗∗P < .0001. Journal of Allergy and Clinical Immunology 2012 130, 410-420.e5DOI: (10.1016/j.jaci.2012.02.046) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Immunohistochemical detection of tryptase was performed by using antihuman tryptase mAb. Representative immunostaining for tryptase within the epithelium of UT from a control subject (A), a patient with CRSsNP (B), a patient with CRSwNP (C), and an NP (D). Tryptase-positive cells in the glands of UT from a control subject (E) and an NP (F). Magnification ×400. Journal of Allergy and Clinical Immunology 2012 130, 410-420.e5DOI: (10.1016/j.jaci.2012.02.046) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Quantitation of mast cell protease-positive cells in nasal mucosal tissue from a control subject and patients with CRS and distribution in epithelium, submucosa, and glands. Immunostaining for tryptase (A), chymase (B), and CPA3 (C). ∗P < .05, ∗∗P < .001, ∗∗∗P < .0001. Journal of Allergy and Clinical Immunology 2012 130, 410-420.e5DOI: (10.1016/j.jaci.2012.02.046) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 The expression of chymase in NPs. The gene expression of chymase in tissue (A) and nasal scrapings (B) was measured by using real-time PCR. Representative immunostaining for chymase within the epithelium of UT from a control subject (C) and an NP (D) and within the glands of UT from a control subject (E) and an NP (F). Magnification ×400. ∗P < .05. Journal of Allergy and Clinical Immunology 2012 130, 410-420.e5DOI: (10.1016/j.jaci.2012.02.046) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 The expression of CPA3 in NPs. The gene expression of CPA3 in tissue (A) and nasal scrapings (B) was measured by using real-time PCR. Representative immunostaining for CPA3 within the epithelium of UT from a control subject (C) and an NP (D) and within the gland of UT from a control subject (E) and an NP (F). Magnification ×400. ∗∗P < .001, ∗∗∗P < .0001. Journal of Allergy and Clinical Immunology 2012 130, 410-420.e5DOI: (10.1016/j.jaci.2012.02.046) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Immunofluorescence of mast cell proteases in NPs. Immunofluorescence assay was performed with antitryptase (green fluorescence) and antichymase (red fluorescence) (A), and anti-CPA3 (red fluorescence) (B) in epithelium and antitryptase (green fluorescence), and antichymase (red fluorescence) (C), and CPA3 (red fluorescence) (D) in glands. Nuclei ware counterstained with 4′,6-diamidino-2-phenylindole (blue fluorescence). The results are representative of 5 separate subjects. Journal of Allergy and Clinical Immunology 2012 130, 410-420.e5DOI: (10.1016/j.jaci.2012.02.046) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 The relationship of tryptase and ECP in UT was evaluated by using ELISA. The correlation was assessed by using the Spearman rank correlation test. Journal of Allergy and Clinical Immunology 2012 130, 410-420.e5DOI: (10.1016/j.jaci.2012.02.046) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Negative control antibody staining in NPs tissue from a patient with CRSwNP. A, Normal rabbit IgG. B, Anti-human CPA3 antibody. Journal of Allergy and Clinical Immunology 2012 130, 410-420.e5DOI: (10.1016/j.jaci.2012.02.046) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Correlation of tryptase with candidate mediators for elevation of mast cells. Total RNA was extracted from UT from control subjects (n = 16), patients with CRSsNP (n = 26), patients with CRSwNP (n = 33), and NPs (n = 34). A, The expression of tryptase and candidates for mast cell chemoattractant, SCF, eotaxin-1, eotaxin-2, and eotaxin-3, was analyzed by using real-time PCR. B, The correlations in nasal tissues were assessed by using Spearman rank correlation. ∗∗P < .001, ∗∗∗P < .0001. Journal of Allergy and Clinical Immunology 2012 130, 410-420.e5DOI: (10.1016/j.jaci.2012.02.046) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions