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Miriam Wittmann, MD, Jana Zeitvogel, Dong Wang, MD, Thomas Werfel, MD 

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Presentation on theme: "Miriam Wittmann, MD, Jana Zeitvogel, Dong Wang, MD, Thomas Werfel, MD "— Presentation transcript:

1 IL-27 is expressed in chronic human eczematous skin lesions and stimulates human keratinocytes 
Miriam Wittmann, MD, Jana Zeitvogel, Dong Wang, MD, Thomas Werfel, MD  Journal of Allergy and Clinical Immunology  Volume 124, Issue 1, Pages (July 2009) DOI: /j.jaci Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 IL-27 subunits are expressed in eczematous skin lesions. Skin biopsy specimens (solid circles, atopic dermatitis; open circles, allergic contact dermatitis) were analyzed for expression of IL-27 subunits. A, Relative expression levels normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as determined by means of quantitative RT-PCR, are shown. Medians are indicated. B, Immunohistologic staining for EBI3 is depicted along with appropriate isotype staining. A typical example of acute (n = 5) and chronic (n = 7) eczema is shown (original magnification ×200). Journal of Allergy and Clinical Immunology  , 81-89DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 WSX-1 expression and STAT1/STAT3 activation by keratinocytes. A, WSX-1 was analyzed by means of flow cytometry after stimulation with IFN-γ (20 ng/mL) for 24 hours. The percentage of cells positive for WSX-1 is depicted. A histogram of a representative staining (non stimulated vs IFN-γ stimulated) is depicted along with the isotype control. B, Phospho-STAT (pSTAT) 1/3 and total STAT1/3 levels were determined by means of Western blot analysis 15 minutes after stimulation with IL-27. One representative experiment of 3 total experiments from different donors is shown. C, Activation of STAT1, as determined by means of intracellular staining of phospho-STAT1, is depicted as the mean fluorescence intensity (MFI) of non stimulated versus cells stimulated for 15 minutes with IL-27 (25 ng/mL). In addition, a histogram of a representative staining along with isotype control is shown. ns, Non stimulated. Journal of Allergy and Clinical Immunology  , 81-89DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 CXCL10 production and chemotactic activity of IL-27–stimulated keratinocytes. A, Keratinocytes were stimulated for 24 hours with different doses of IL-27, IL-6, or IL-12, and the supernatants were analyzed by means of ELISA for CXCL10 (n = 15). B, Supernatants of IL-27–stimulated keratinocytes were used in chemotaxis assays. The number of migrated CD4 T cells is depicted. ns, Non stimulated. Journal of Allergy and Clinical Immunology  , 81-89DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 IL-27 induces MHC class I, IL-18Rα, and CD54 surface expression. A, Keratinocytes were stimulated with IL-27 (25 ng/mL, n = 13) and analyzed the next day for MHC class I (MHCI) surface expression. The mean fluorescence intensity (MFI) is depicted. B, For analysis of CD54 and IL-18Rα, cell-surface expression with 50 ng/mL IL-27 was used. Data were normalized by setting the MFI of nonstimulated cells to 100% for each independent experiment (n = 8). Statistical analysis was performed with original data by using the paired t test. In addition, histograms of representative stainings along with isotype controls are shown. ns, Non stimulated. Journal of Allergy and Clinical Immunology  , 81-89DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 IL-27 acts as a priming signal on keratinocytes. A, Keratinocytes were stimulated with IL-27 (25 ng/mL) or TNF-α (10 ng/mL) alone or with a combination of both signals with a 2-hour time gap. Supernatants were analyzed for CXCL10 after 24 hours of stimulation (n = 10). B and C, Keratinocytes were stimulated with IL-6 (25 ng/mL) or IL-12 (25 ng/mL, n = 9; Fig 5, B) or IL-23 (25 ng/mL, n = 4; Fig 5, C) as a priming signal, as described for IL-27. ns, Non stimulated. Journal of Allergy and Clinical Immunology  , 81-89DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Protection from activation-induced cell death by IL-12 and IL-27. Apoptosis was induced in keratinocytes by using TRAIL (50 ng/mL) and TNF-α (10 ng/mL), as determined by means of H2A.X staining. IL-27 and IL-12 were used at 10 ng/mL. The mean fluorescence intensity for H2A.X was determined, and the values of the proapoptotic stimuli TRAIL plus TNF-α were set to 100% for each independent experiment. Values relative to this positive control are depicted. Statistical analysis was performed with raw data by using the Wilcoxon signed-rank test (n = 10). In addition, a histogram of a representative staining along with isotype control is shown. Journal of Allergy and Clinical Immunology  , 81-89DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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