Chronic Mouse Model of TMA-Induced Contact Hypersensitivity Claudia Schneider, Wolf-Dietrich F. Döcke, Thomas M. Zollner, Lars Röse  Journal of Investigative Dermatology  Volume 129, Issue 4, Pages 899-907 (April 2009) DOI: 10.1038/jid.2008.307 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Inflammation parameters in acute, subacute, and chronic TMA-induced mouse CHS models. Acute (A), subacute (SA), and chronic (C) TMA-induced CHS models were executed as described in the Materials and Methods. (a) Ear thickness (n=11), (b) activity of myeloperoxidase (n=5), (c) serum IgE levels (n=10), and (d) weight of auricular lymph nodes (n=11) are shown as mean±SD. Percentage values for inhibition of inflammation parameters at the end of experiment are given at 24hours either after single prednisolone application (day 5) for acute model, after three applications (days 5–7) for subacute model, or after six daily doses of 30mgkg−1 prednisolone (days 9–14) in chronic model. **P<0.01, ***P<0.001 TMA group compared with solvent group; +P<0.05, ++P<0.01, +++P<0.001, treatment group compared with TMA group; #P<0.05, ###P<0.001, comparison of TMA groups among the three models. Journal of Investigative Dermatology 2009 129, 899-907DOI: (10.1038/jid.2008.307) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Cutaneous cell infiltration in acute, subacute, and chronic TMA-induced mouse CHS models. Skin from acute solvent control as well as acute, subacute, and chronic TMA-induced ear inflammation models was stained by HE (a), toluidine blue (b) and antibodies against MHCII (I-Ad; c), CD4 (d), and CD8 (e). In the acute TMA-induced CHS model, only a weak infiltration by eosinophils, mast cells, MHCII-, CD4-, and CD8-positive cells is seen. Stronger cell infiltration is visible in subacute model. Strongest immune cell infiltration as well as acanthosis is seen in the chronic TMA-induced CHS model. Although CD8+ cells are predominantly found in the epidermis, CD4+ T-helper cells are mainly located in the thickened dermis of the dorsum of the ears. For eosinophils and mast cells in chronic model also see the inserts in (a) and (b), respectively. Scale bar=100μm. Journal of Investigative Dermatology 2009 129, 899-907DOI: (10.1038/jid.2008.307) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Cutaneous cytokine profile in acute, subacute, and chronic TMA-induced mouse CHS models. Cytokines in ear homogenates from acute (A), subacute (SA), and chronic (C) TMA-induced CHS models are given as mean±SD (n=5). Expression of type 1 cytokines (IFNγ, IL-12) and Th2 cytokines (IL-4, protein IL-13) (a) as well as of the inflammatory cytokines, TNFα, and IL-1β (b) is given on protein and/or mRNA level. Percentages exhibit the percent inhibition of cytokine expression in the treatment group compared to the increase of inflammation from solvent to the respective TMA group (100%). *P<0.05, **P<0.01, TMA group compared with solvent group; +P<0.05, ++P<0.01, treatment group compared with TMA group; #P<0.05, ##P<0.01, comparison of TMA groups among the three models. Journal of Investigative Dermatology 2009 129, 899-907DOI: (10.1038/jid.2008.307) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Cytokine profile and T-cell characterization in lymph nodes from acute, subacute, and chronic mouse CHS models. (a) Weight of auricular lymph nodes (n=10), derived from acute, subacute, and chronic models, is given as mean±SD. (b) The mRNA expression of TNFα, IL-1β, Langerin, and CD3δ is given as fold expression of HPRT. (c) IFNγ and IL-4 expression and the expression of the Th1- and Th2-typifying transcription factors, T-bet and GATA-3, are shown as fold expression of CD3δ (n=5, mean±SD). (d) Ratio of CD3+CD4+ and CD3+CD4− lymph node cells is given as mean±SD (n=5). CD4+CD3+ and CD4−CD3+ cells were further characterized by flowcytometry for the surface expression of CD69 (CLEC2C), CD25 (IL-2R-α), MHCII (I-Ad), CD49d (Itga4), CD62L (L-selectin), and CD45RB (n=5, geometric mean fluorescence intensity±SD expressed as percentage of mean expression in acute TMA model). #P<0.05, ##P<0.01, ###P<0.001, comparison of TMA groups among the three models. Journal of Investigative Dermatology 2009 129, 899-907DOI: (10.1038/jid.2008.307) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions