Staphylococcus aureus fibronectin-binding protein specifically binds IgE from patients with atopic dermatitis and requires antigen presentation for cellular.

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Presentation transcript:

Staphylococcus aureus fibronectin-binding protein specifically binds IgE from patients with atopic dermatitis and requires antigen presentation for cellular immune responses Kavita Reginald, PhD, Kerstin Westritschnig, MD, Birgit Linhart, PhD, Margarete Focke-Tejkl, PhD, Beatrice Jahn-Schmid, PhD, Julia Eckl-Dorna, MD, PhD, Annice Heratizadeh, MD, Angelika Stöcklinger, PhD, Nadja Balic, Susanne Spitzauer, MD, Verena Niederberger, MD, Thomas Werfel, MD, Josef Thalhamer, PhD, Stephan Weidinger, MD, Natalija Novak, MD, Markus Ollert, MD, Alexander M. Hirschl, MD, Rudolf Valenta, MD Journal of Allergy and Clinical Immunology Volume 128, Issue 1, Pages 82-91.e8 (July 2011) DOI: 10.1016/j.jaci.2011.02.034 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Sequence of S aureus FBP. The nucleotides and deduced amino acids of FBP (GenBank ID HM245235) are shown. ∗TAA stop codon. The S aureus FBP protein contains 2 domains, FbpA (yellow) and domain DUF814 (green). IgE-reactive fragments determined by mass spectrometry are underlined (fragment 1, solid line; fragment 2, broken line). Journal of Allergy and Clinical Immunology 2011 128, 82-91.e8DOI: (10.1016/j.jaci.2011.02.034) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Purification, IgE reactivity, and epitope mapping of recombinant FBP. A, Left panel shows an SDS-PAGE containing intact affinity-purified rFBP and rFBP fragments (fragments 1 and 2). IgE reactivity of the nitrocellulose-blotted rFBP preparations (right panel, sera from patients with AD, lanes 12-37; sera from a person without allergy, lane N). B, SDS-PAGE of purified rFBP fragments (left) and IgE reactivity (right, sera from patients with AD, lanes 6-95; sera from a person without allergy, lane N). Journal of Allergy and Clinical Immunology 2011 128, 82-91.e8DOI: (10.1016/j.jaci.2011.02.034) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Allergenic activity of rFBP. RBL cells expressing human FcεRI were loaded with serum IgE from patients with AD with FBP-specific IgE (right panel), or patients with AD and nonallergic (NA) individuals without FBP-specific IgE (left panel) and challenged with rFBP. Percentages of total ß-hexosaminidase releases are shown. Journal of Allergy and Clinical Immunology 2011 128, 82-91.e8DOI: (10.1016/j.jaci.2011.02.034) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Immune reactivity of rFBP. A, FBP-specific levels of IgG1, IgG2 and IgG4 (2 different scales for the y-axis) antibodies in patients with AD and nonallergic (NA) individuals. Open circles represent outliers, and filled circles represent extreme values. B, PBMCs from patients with AD or NA individuals were stimulated with FBP, Phl p 1, IL-2, or medium control (MC), and mean lymphoproliferative responses of triplicate measurements are shown. Horizontal lines denote the median value of each group. C, T-cell lines from 5 individuals with allergy were stimulated in duplicates with FBP, SEA, SEB, SEE, or MC, and the lymphoproliferative responses are shown as counts per minute (CPM). Journal of Allergy and Clinical Immunology 2011 128, 82-91.e8DOI: (10.1016/j.jaci.2011.02.034) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Cytokine releases induced from PBMCs of patients with AD and nonallergic (NA) individuals by FBP. PBMCs were stimulated with FBP, Phl p 1, or medium control (MC), and the amounts of released cytokines are presented. Statistically significant differences between groups are marked. ∗P < .05. Journal of Allergy and Clinical Immunology 2011 128, 82-91.e8DOI: (10.1016/j.jaci.2011.02.034) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 FBP induces an allergic immune response in Balb/c mice. A, FBP-specific IgE levels were measured in a group of FBP-sensitized mice at different times. Open circles represent outliers. B, β-Hexosaminidase release from RBL cells that had been sensitized with preimmune (Pre) or immune (Imm) sera and were stimulated with FBP. RBL cells without sera exposure were stimulated with FBP or medium alone. Levels of FBP-specific IgG1 (C) and IgG2a (D) were measured as for IgE in A. Journal of Allergy and Clinical Immunology 2011 128, 82-91.e8DOI: (10.1016/j.jaci.2011.02.034) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 FBP-specific lymphoproliferation and cytokine responses. A, Lymphoproliferative responses toward different agonists as determined by 3H-thymidine incorporation are shown as stimulation indices of triplicate measurements. Open circles represent outliers, whereas filled circles represent extreme values. B, FBP-specific cytokine responses. Statistically significant differences between groups are marked. ∗P < .05. MC, Medium control; SI, stimulation index. Journal of Allergy and Clinical Immunology 2011 128, 82-91.e8DOI: (10.1016/j.jaci.2011.02.034) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

FBP-specific T-cell proliferation using viable or fixed APCs FBP-specific T-cell proliferation using viable or fixed APCs. Purified T cells from 2 patients with AD were cocultured with either viable or fixed APCs and stimulated with rFBP or SEA. The PBMCs of each patient served as controls. Cell proliferation responses were measured by 3H thymidine incorporation and are displayed as counts per minute (cpm). MC, Medium control. Journal of Allergy and Clinical Immunology 2011 128, 82-91.e8DOI: (10.1016/j.jaci.2011.02.034) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions