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Expression of Mas-related gene X2 on mast cells is upregulated in the skin of patients with severe chronic urticaria  Daisuke Fujisawa, MD, Jun-ichi Kashiwakura,

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Presentation on theme: "Expression of Mas-related gene X2 on mast cells is upregulated in the skin of patients with severe chronic urticaria  Daisuke Fujisawa, MD, Jun-ichi Kashiwakura,"— Presentation transcript:

1 Expression of Mas-related gene X2 on mast cells is upregulated in the skin of patients with severe chronic urticaria  Daisuke Fujisawa, MD, Jun-ichi Kashiwakura, PhD, Hirohito Kita, MD, PhD, Yusuke Kikukawa, MSc, Yasushi Fujitani, PhD, Tomomi Sasaki-Sakamoto, BSc, Kazumichi Kuroda, PhD, Satoshi Nunomura, PhD, Koremasa Hayama, MD, PhD, Tadashi Terui, MD, PhD, Chisei Ra, MD, PhD, Yoshimichi Okayama, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 134, Issue 3, Pages e9 (September 2014) DOI: /j.jaci Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Cell-surface expression of MrgX2 on skin-derived cultured MCs. A, MrgX2 mRNA expression levels of human MCs and fibroblasts. The expression level of MrgX2 mRNA in LAD2 cells was designated as 1. The data are shown as means ± SEMs of 3 different donors (lung-derived cultured MCs [LMC], skin-derived cultured MCs [SMC], CB-derived cultured MCs [CBMC], and fibroblasts [Fibro]) and 3 different experiments (LAD2 cells). N.D., Not detected. B, Cell-surface expression levels of MrgX2 on human MCs and fibroblasts. Solid lines indicate histograms of cells treated with anti-MrgX2 mAb. Isotype immunoglobulin-treated MCs are shown as gray filled histograms. Right upper values indicate mean fluorescence intensity ratios. Data are representative of similar results obtained from 2 independent experiments. C, LAD2 cells were immunostained with anti-MrgX2 antibody (red, a). As controls, cells stained with rabbit IgG are shown (c). As a negative control, fibroblasts were immunostained with anti-MrgX2 antibody (b). The cells were stained with 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI; blue). Bar = 10 μm. D, Cell-surface expression levels of NK-1R on skin- and lung-derived cultured MCs and Jurkat cells. Solid lines indicate histograms of cells treated with anti–NK-1R antibody. Isotype immunoglobulin-treated MCs are shown as gray filled histograms. Right upper values indicate mean fluorescence intensity ratios. Data are representative of similar results obtained from 2 independent experiments. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 MrgX2 expression is significantly upregulated on human skin MCs of patients with CU. A and B, Representative images of MrgX2+ MCs (tryptase-positive cells) in skin tissues from a patient with CU (Fig 2, B) and an NC subject (Fig 2, A). Immunochemical staining of skin specimens with anti-tryptase mAb (green, a), anti-MrgX2 antibody (red, b), and 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI; blue, c). White arrows indicate tryptase-MrgX2 double-positive cells. Bar = 50 μm. C, As negative controls, skin specimens from patients with CU were stained with mouse IgG1 or rabbit IgG. D-F, A comparison of numbers of MCs (Fig 2, D), numbers of MrgX2+ MCs (Fig 2, E), and percentages of MrgX2+ MCs among all MCs (Fig 2, F) in skin tissues from NC subjects and patients with CU. Triangles, squares, and circles indicate skin biopsy sites at the arms, legs, and trunk, respectively. Data are expressed as medians and interquartile ranges. ***P < N.S., Not significant. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 SP-induced histamine release and PGD2 production from skin-derived cultured MCs through MrgX2 but not through NK-1R. A and B, MrgX2 (Fig 3, A) and FcεRIα (Fig 3, B) expression on skin-derived cultured MCs with or without transduction of nontargeted shRNA or MrgX2 shRNA. Black lines indicate histograms of skin-derived cultured MCs treated with anti-MrgX2 or anti-FcεRIα mAb. Isotype immunoglobulin-treated MCs are shown as gray filled histograms. Right upper values indicate mean fluorescence intensity ratios. C, Statistical analysis of expression levels shown in Fig 3, A. Expression levels of MrgX2 shown on the vertical axis are mean fluorescence intensity (MFI) ratios (n = 3 donors). *P < .05. D, Effect of diminution of MrgX2 on histamine release and PGD2 production from MCs in response to SP. Levels of histamine release and PGD2 production of nontransduced, nontargeted shRNA-transduced and MrgX2 shRNA-transduced skin-derived cultured MCs are shown as white, gray, and black bars, respectively. Three independent experiments were performed with 3 independent donors. Data are shown as means ± SEMs. *P < .05 and **P < .01, respectively. E, Histamine release from skin-derived cultured MCs stimulated with SP in the absence or presence of CP Data are representative of similar results obtained from 2 independent experiments with 2 different donors. DMSO, Dimethyl sulfoxide. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Eosinophil infiltration and colocalization of MCs and eosinophils in the urticarial region of patients with CU. Representative images of the colocalization of MCs and eosinophils in skin tissues from a patient with CU are shown (A). Skin specimens from patients with CU (Fig 4, A) and NC subjects (C) were immunostained with anti-tryptase mAb (green), anti-ECP antibody (red), and 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI; blue). Bar = 10 μm. As controls, skin specimens from patients with CU (B) and NC subjects (D) were stained with mouse IgG1 or rabbit IgG. A comparison of the numbers of eosinophils in the skin tissues from NC subjects (n = 13) and patients with CU (n = 9) is shown (E). Data are expressed as medians and interquartile ranges. ***P < .001. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 MBP and EPO induced histamine release through MrgX2. A, SP induced an intracellular Ca2+ increase through MrgX2. CHO cells transfected with MrgX2 or vector alone (Mock) were incubated with 10−9 to 10−4 mol/L SP, and intracellular Ca2+ levels were measured. Data are shown as the relative fluorescent unit (RFU; percentage of intracellular Ca2+ increase by 1 μmol/L of the calcium ionophore A23187). B, MBP and EPO induced intracellular Ca2+ increase through MrgX2. CHO cells transfected with MrgX2 or vector alone (Mock) were incubated with 10, 30, and 100 μg/mL eosinophil granule proteins, and intracellular Ca2+ levels were measured. Data were shown as the relative fluorescent unit (RFU; percentage of intracellular Ca2+ increase by 1 μmol/L of the calcium ionophore A23187). C, Effect of diminution of MrgX2 on histamine release from skin-derived cultured MCs in response to eosinophil granule proteins. Levels of histamine release of nontransduced, nontargeted shRNA-transduced, and MrgX2 shRNA-transduced skin-derived cultured MCs are shown as white, gray, and black bars, respectively. Three independent experiments were performed with 3 independent donors. Data are shown as means ± SEMs. *P < .05. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E1 Comparison of numbers of MCs (A), numbers of MrgX2+ MCs (B), and percentages of MrgX2+ MCs among all MCs (C) in skin tissues between patients with CU less than 50 years of age and those older than 50 years. Triangles, squares, and circles indicate skin biopsy sites at the arms, legs, and trunks, respectively. Data are expressed as medians and interquartile ranges. N.S., Not significant. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E2 Comparison of numbers of MCs (A), numbers of MrgX2+ MCs (B), and percentage of MrgX2+ MCs among all MCs (C) in skin tissues between NC subjects less than 35 years of age and those older than 35 years. Triangles, squares, and circles indicate skin biopsy sites at the arms, legs, and trunks, respectively. Data are expressed as medians and interquartile ranges. N.S., Not significant. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E3 A, Immunochemical staining of skin specimens with anti-PGP 9.5 mAb (green, a), anti-MrgX2 antibody (red, b), and 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI; blue, c). White arrows indicate PGP 9.5-MrgX2 double-positive cells. Bar = 30 μm. B, As controls, skin specimens from patients with CU were stained with mouse IgG1 and rabbit IgG. Bar = 50 μm. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E4 Comparisons of numbers of MCs among anatomic sites for biopsy specimens. A, Comparison of numbers of MCs in skin tissues from the arms, legs, or trunks of NC subjects. B, Comparison of numbers of MCs in skin tissues from the trunks of NC subjects and patients with CU. N.S., Not significant. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E5 Comparisons of MC phenotypes among anatomic sites for biopsy specimens from NC subjects. A-C, Representative images of tryptase-positive cells and chymase-positive cells in the skin tissues of arm (Fig E5, A), leg (Fig E5, B), and trunk (Fig E5, C) biopsy specimens from NC subjects are shown. Immunochemical staining of skin specimens with anti-tryptase mAb (green, a), anti-chymase mAb (red, b), and 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI; blue, c) is shown. White arrows indicate tryptase-chymase double-positive cells. Bar = 50 μm. D, As negative controls, skin specimens from NC subjects were stained with mouse IgG1. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E6 Comparisons of MC phenotypes among anatomic sites for biopsy specimens from patients with CU. A-C, Representative images of tryptase-positive cells and chymase-positive cells in skin tissues of arm (Fig E6, A), leg (Fig E6, B), and trunk (Fig E6, C) biopsy specimens from patients with CU are shown. Immunochemical staining of skin specimens with anti-tryptase mAb (green, a), anti-chymase mAb (red, b), and 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI; blue, c) is shown. White arrows indicate tryptase-chymase double-positive cells. Bar = 50 μm. D, As negative controls, skin specimens from patients with CU were stained with mouse IgG1. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 Fig E7 Effect of drugs used for treatment (A) and neurotransmitters and cytokines (B) on MrgX2 expression levels on human skin MCs and effect of drugs used for treatment on SP-induced histamine release from skin MCs (C). Human skin MCs were incubated with olopatadine hydrochloride (H1RA; 10 μmol/L), famotidine (H2RA; 10 μmol/L), and dexamethasone (10 μmol/L; Fig E7, A) or histamine dihydrochloride (10 μmol/L), SP (3 μmol/L), rhIL-33 (30 ng/mL), rhTSLP (10 ng/mL), or both rhL-33 (30 ng/mL) and rhTSLP (10 ng/mL; Fig E7, B) for 24 hours before flow cytometric analysis of MrgX2 expression levels on human skin MCs. Right upper values indicate mean fluorescence intensity ratios. Fig E7, C, Skin MCs were pretreated with olopatadine hydrochloride (H1RA; 10 μmol/L) or famotidine (H2RA; 10 μmol/L) for 30 minutes or dexamethasone (10 μmol/L) for 16 hours before being challenged by a suboptimal concentration (0.3 μmol/L) of SP (n = 3 donors). DEX, Dexamethasone; DMSO, dimethyl sulfoxide; H1RA, histamine H1 receptor antagonist; H2RA, histamine H2 receptor antagonist; N.S., not significant. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

14 Fig E8 Cell-surface and intracellular expression of MrgX2 on human PB-derived cultured MCs. A, MrgX2 mRNA expression levels of PB-derived MCs and LAD2 cells. The expression level of MrgX2 mRNA in LAD2 cells was designated as 1. Data are shown as means ± SEMs of 3 different donors of PB-derived cultured MCs (PBMC) and 3 different experiments (LAD2 cells). B, Cell-surface and intracellular expression levels of MrgX2 on PB-derived cultured MCs. Solid lines indicate histograms of nonpermeabilized MCs (left panel) and permeabilized MCs (right panel) treated with anti-MrgX2 mAb. Isotype immunoglobulin-treated MCs are shown as gray filled histograms. Right upper values indicate mean fluorescence intensity ratios. Data are representative of similar results obtained from 2 independent experiments. C, Permeabilized PB-derived cultured MCs were immunostained with anti-MrgX2 antibody (red, a). As controls, cells stained with rabbit IgG are shown (c). As controls, nonpermeabilized MCs were immunostained with anti-MrgX2 antibody (b) or rabbit IgG (d). Cells were stained with 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI; blue). Bar = 10 μm. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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