The IgE-Reactive Autoantigen Hom s 2 Induces Damage of Respiratory Epithelial Cells and Keratinocytes via Induction of IFN-γ Irene Mittermann, Renate.

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The IgE-Reactive Autoantigen Hom s 2 Induces Damage of Respiratory Epithelial Cells and Keratinocytes via Induction of IFN-γ Irene Mittermann, Renate Reininger, Maya Zimmermann, Katharina Gangl, Jürgen Reisinger, Karl J. Aichberger, Elli K. Greisenegger, Verena Niederberger, Joachim Seipelt, Barbara Bohle, Tamara Kopp, Cezmi A. Akdis, Susanne Spitzauer, Peter Valent, Rudolf Valenta Journal of Investigative Dermatology Volume 128, Issue 6, Pages 1451-1459 (July 2008) DOI: 10.1038/sj.jid.5701195 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Coomassie blue-stained SDS-PAGE containing purified rHom s 2 and a molecular weight marker (lane M). Molecular weights are indicated at the left side in kDa. Journal of Investigative Dermatology 2008 128, 1451-1459DOI: (10.1038/sj.jid.5701195) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Mapping of IgE and IgG epitopes using rHom s 2 fragments. (a) Schematic diagram of β-galactosidase (1) and Hom s 2-β–galactosidase fusion proteins, containing the N-terminal (2) or C-terminal (3) part of Hom s 2. (b) Coomassie-stained SDS-PAGE of E. coli extracts containing β-galactosidase (lane 1), the N-terminal (lane 2), or the C-terminal (lane 3) Hom s 2–β-galactosidase fusion proteins. Nitrocellulose membranes containing the blotted extracts were reacted with serum IgE from two Hom s 2-sensitized patients (AD1, AD2), with a rabbit antiserum raised against purified rHom s 2 (rabbit α-Hom s 2) or with the corresponding rabbit preimmune serum (preimmune-Ig). Molecular weights (kDa) and the position of the β-galactosidase fusion proteins (asterisks) are indicated. Journal of Investigative Dermatology 2008 128, 1451-1459DOI: (10.1038/sj.jid.5701195) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Basophil histamine release experiments. Basophils from patients (a) AD1 and (b) AD2 containing rHom s 2-specific IgE antibodies were exposed to increasing concentrations of rHom s 2, anti-IgE, or the grass pollen allergen, rPhl p 1 (x axis). The percentages of total histamine release are displayed on the y axis. Journal of Investigative Dermatology 2008 128, 1451-1459DOI: (10.1038/sj.jid.5701195) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Cellular, cytokine, and humoral immune responses to rHom s 2 and rPhl p 1. Box plots showing (a) lymphoproliferative responses (y axis: mean counts per minute (c.p.m.)) of AD patients, RC patients, non-atopic individuals (NA), or CA patients to rHom s 2 (gray boxes), rPhl p 1 (dotted boxes), or medium control (MC) (white boxes). (b) INF-γ, (c) IL-10, and (d) IL-5 levels (y axis: pgml−1) as determined in the cultures. Statistically significant differences are indicated (*P<0.05 or **P<0.01). (e) IgG1, (f), IgG2, and (g) IgG4 subclass reactivity to Hom s 2 and to the grass pollen allergen Phl p 1. Optical density (OD) values corresponding to the amount of bound antibodies are displayed. The cutoff levels (median ODs for HSA) of the ELISAs are displayed as horizontal lines. Open dots represent outliers and triangles represent extreme values. HSA, human serum albumin. Journal of Investigative Dermatology 2008 128, 1451-1459DOI: (10.1038/sj.jid.5701195) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Culture supernatants from Hom s 2-stimulated PBMCs decrease transepithelial resistance in respiratory epithelial cells. (a) INF-γ levels (y axis: pgml−1) determined in supernatants of PBMC cultures stimulated with rHom s 2 or rPhl p 1 from AD and non-atopic (NA) individuals. Statistically significant differences are indicated (*P<0.05). Change of transepithelial resistance (y axis: % change of resistance) over time (x axis) in epithelial cell layers exposed to culture supernatants from (b) Hom s 2- or (c) Phl p 1-stimulated PBMCs is shown. Journal of Investigative Dermatology 2008 128, 1451-1459DOI: (10.1038/sj.jid.5701195) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Culture supernatants from Hom 2-stimulated PBMCs induce IFN-γ-dependent (a) disintegration of respiratory epithelial cell layers and (b) apoptosis in keratinocytes. (a) Respiratory epithelial cells or (b) HaCat keratinocytes were incubated with medium (1) or supernatants of Hom s 2-stimulated PBMCs with an isotype antibody control (2) or with a neutralizing anti-IFN-γ mAb (3). (a) The percentage of the change of transepithelial resistance and (b) the percentages of annexin-V- and/or 7AAD-negative viable keratinocytes are shown. Journal of Investigative Dermatology 2008 128, 1451-1459DOI: (10.1038/sj.jid.5701195) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions