Histamine Upregulates Keratinocyte MMP-9 Production via the Histamine H1 Receptor Maria Gschwandtner, Rahul Purwar, Miriam Wittmann, Wolfgang Bäumer,

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Histamine Upregulates Keratinocyte MMP-9 Production via the Histamine H1 Receptor Maria Gschwandtner, Rahul Purwar, Miriam Wittmann, Wolfgang Bäumer, Manfred Kietzmann, Thomas Werfel, Ralf Gutzmer Journal of Investigative Dermatology Volume 128, Issue 12, Pages 2783-2791 (December 2008) DOI: 10.1038/jid.2008.153 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Histamine and the H1R agonist β-histine induce MMP-9 in keratinocytes. (a) Histamine (10μM) and β-histine (10μM) induce MMP-9 production in keratinocyte supernatants after 48hours of stimulation. (b) Additive effect of TGF-β1 (1ngml−1) and histamine in the upregulation of MMP-9. Mean and SEM of (a) 11 and (b) 7 independent experiments are shown. Journal of Investigative Dermatology 2008 128, 2783-2791DOI: (10.1038/jid.2008.153) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Dose and time dependency of MMP-9 induction. Dose–response curve of MMP-9 production for (a) histamine and (b) β-histine. Basal level of MMP-9 production in nonstimulated controls was (a) 910pgml−1 and (b) 827pgml−1. Time course of MMP-9 induction by histamine (10μM); mRNA levels determined by (c) quantitative RT-PCR and protein levels measured in (d) ELISA are plotted. Mean and SEM of (a, b) 3, (c) 7, and (d) 6 independent experiments are shown. Journal of Investigative Dermatology 2008 128, 2783-2791DOI: (10.1038/jid.2008.153) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Specific H1R antagonists clemastine and levocetirizine diminish induction of MMP-9. (a) Upregulation of MMP-9 by histamine and H1R agonist β-histine. (b) Upregulation is blocked by 30minutes preincubation with H1R-specific antagonist clemastine hydrogen fumarate (10μM). (c) Dose–response of β-histine-stimulated cells to clemastine. (d) H1R antagonist levocetirizine, but not H2R antagonist ranitidine and H3R and H4R antagonist thioperamide, can block histamine-induced MMP-9 production. Mean and SEM of (a, b) 10, (c) 2, and (d) 6 independent experiments are depicted. Journal of Investigative Dermatology 2008 128, 2783-2791DOI: (10.1038/jid.2008.153) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Histamine induces MMP-9 as determined by gelatin zymography. (a) Zymogram of healthy skin homogenates shows increase in MMP-9 levels by histamine (2) as compared to nonstimulated control (1). Endogenous histamine released from mast cells after compound 48/80 (0.3mM) treatment leads to MMP-9 upregulation (3) that can be blocked by 30minutes pre-incubation with 10μM H1R-specific antagonist clemastine (4). (b) Zymogram of keratinocyte supernatants: nonstimulated (1), stimulated with histamine (2), TGF-β1 (3), or histamine plus TGF-β1 (4). One representative analysis of (a) 2 and (b) 3 independent experiments is shown. Journal of Investigative Dermatology 2008 128, 2783-2791DOI: (10.1038/jid.2008.153) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Increase in MMP-9 and destruction of type IV collagen at the basement membrane after histamine stimulation. Immunohistological staining shows increased amount of (a–c) MMP-9 and (d–f) decreased amount of type IV collagen at the basement membrane after histamine stimulation. (a,d) Nontreated skin, (b, e) skin treated with 10μM histamine, (c, f) isotype staining. (g) MMP inhibitor GM6001 blocks histamine induced destruction of type IV collagen, but not its (h) negative control. One representative analysis of (a–c) 8 and (d–h) 3 independent experiments is depicted. Journal of Investigative Dermatology 2008 128, 2783-2791DOI: (10.1038/jid.2008.153) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Induction of T cell migration across an artificial basement membrane. (a) Prestimulation of keratinocytes with 10μM histamine upregulates the migration of Jurkat T cells across an artificial basement membrane in a coculture system. Increased migration is blocked by (b) MMP inhibitor GM6001 but not its (c) negative control. A total of 10000 beads were counted and the cells/bead ratio of (a) 9 and (b, c) 7 independent experiments was calculated. Journal of Investigative Dermatology 2008 128, 2783-2791DOI: (10.1038/jid.2008.153) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions