Dorothea Dijkstra, Holger Stark, Paul L. Chazot, Fiona C

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Human Inflammatory Dendritic Epidermal Cells Express a Functional Histamine H4 Receptor Dorothea Dijkstra, Holger Stark, Paul L. Chazot, Fiona C. Shenton, Rob Leurs, Thomas Werfel, Ralf Gutzmer Journal of Investigative Dermatology Volume 128, Issue 7, Pages 1696-1703 (July 2008) DOI: 10.1038/sj.jid.5701250 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of the H4R by IDECs in skin sections. Skin sections (1μm) of formalin-fixed and paraffin-embedded lesional AD skin biopsies were stained with either FITC–CD36 or FITC–CD206 (to identify IDECs) and counterstained for the expression of the H4R (Texas Red). One representative experiment of five is shown. Bar=20μm. Journal of Investigative Dermatology 2008 128, 1696-1703DOI: (10.1038/sj.jid.5701250) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Expression of the H4R by IDECs in epidermal cell suspensions performed on freshly obtained shave biopsies from lesional AD skin. IDECs were stained for CD206 (FITC) and H4R (peridinin–chlorophyll–protein complex), CD206-positive cells were gated and analyzed for H4R expression. (a) One representative experiment is shown. GM, geometric mean of fluorescence. (b) Nine experiments are summarized. Statistical analysis was performed by Wilcoxon's signed-rank test; **P<0.005. Journal of Investigative Dermatology 2008 128, 1696-1703DOI: (10.1038/sj.jid.5701250) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Expression and regulation of the H4R on Mo-IDECs. (a) Staining of H4R (thick line), isotype (gray filled thin line), and blocked H4R antibody (thin line). (b) Regulation of H4R expression by IFN-γ (20ngml−1), IL-4 (20ngml−1), TNF-α (0.5ngml−1), poly I:C (TLR3 ligand, 2μgml−1), or combinations of these (mean±SEM of five independent experiments is shown). Statistical analysis was performed by ANOVA; **P<0.005. (c) Upregulation by IFN-γ, IFN-γ+TNF-α and IFN-γ+TNF-α and IFN-γ+TNF-α+poly I:C (thick lines) as compared with non-stimulated cells (thin lines) in one representative experiment. Journal of Investigative Dermatology 2008 128, 1696-1703DOI: (10.1038/sj.jid.5701250) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Histamine downregulates CCL2 protein via H4R. Mo-IDECs were stimulated with HA or agonists of H1R (β-histine; β his), H2R (dimaprit; Dim), H3R (methimepip; MP), and H4R (Clob and 4-MH), for 48hours at 10μM or at the indicated concentrations. JNJ7777120 (JNJ) was used as an H4R-neutral antagonist. CCL2 was measured by ELISA, normalized to non-stimulated cells: 3,261±1,902ngml−1 (mean±SEM). (a) HA downregulated CCL2 solely via H4R. (b) Dose–response curves of CCL2 downregulation by HA, Clob, and 4-MH. Mean±SEM of five different experiments are shown. Statistical analysis was performed by ANOVA; *P<0.05, **P<0.005. Journal of Investigative Dermatology 2008 128, 1696-1703DOI: (10.1038/sj.jid.5701250) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 H4R agonist stimulation downregulates chemotaxis induced by supernatants from Mo-IDECs. Supernatants from Mo-IDECs stimulated for 48hours with various H4R ligands were used as chemoattractants. Values normalized to chemotaxis induced by supernatant from non-stimulated Mo-IDECs (NS). HA, Clob, and 4-MH (10μM), CCL2 neutralizing antibody (a-CCL2). Mean±SEM of four experiments are shown. Statistical analysis was performed by ANOVA; *P<0.05, **P<0.005, ***P<0.0005. Journal of Investigative Dermatology 2008 128, 1696-1703DOI: (10.1038/sj.jid.5701250) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Histamine downregulates IL-12p40 via H4R. Mo-IDECs were incubated with the same ligands as described in Figure 4 for 48hours. After 24hours of incubation poly I:C (2μgml−1) was added to the culture as second stimulus. Values are normalized to IL-12p40 levels produced by IDEC stimulated only with poly I:C (2,950±2,340pgml−1, mean±SEM). Statistical analysis was performed by ANOVA; **P<0.005. Journal of Investigative Dermatology 2008 128, 1696-1703DOI: (10.1038/sj.jid.5701250) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions