Increased Expression of Carbonic Anhydrase II (CA II) in Lesional Skin of Atopic Dermatitis: Regulation by Th2 Cytokines Marijke Kamsteeg, Patrick L.J.M.

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Increased Expression of Carbonic Anhydrase II (CA II) in Lesional Skin of Atopic Dermatitis: Regulation by Th2 Cytokines Marijke Kamsteeg, Patrick L.J.M. Zeeuwen, Gys J. de Jongh, Diana Rodijk-Olthuis, Manon E.J. Zeeuwen-Franssen, Piet E.J. van Erp, Joost Schalkwijk Journal of Investigative Dermatology Volume 127, Issue 7, Pages 1786-1789 (July 2007) DOI: 10.1038/sj.jid.5700752 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 CA II is upregulated in lesional skin of AD patients and is present in differentiated keratinocytes. (a) CA II mRNA levels in epidermal sheets were significantly higher (P<0.0002) in lesional skin from AD patients (n=6) than in normal skin (NS) (n=5), whereas it was significantly lower (P<0.005) in lesional skin from psoriasis patients (PS) (n=9). CA II mRNA levels were determined by qPCR as described previously (Franssen et al., 2005). (b) Western blot analysis of epidermal sheets of four individuals in each group showing similar findings for CA II protein levels as found at the mRNA level. (c) Quantification of CA II protein levels in the same material using ELISA showed that in AD, CA II protein was significantly higher than in NS (P<0.0005), whereas it was significantly lower in PS (P<0.05). (d) CA II is predominantly expressed in the suprabasal keratinocytes as shown here by immunostaining of lesional AD skin. Bar=100μm. Journal of Investigative Dermatology 2007 127, 1786-1789DOI: (10.1038/sj.jid.5700752) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 CA II expression is induced by Th2 cytokines but not by Th1 cytokines. (a) No significant effect of the diagnosis on CA II expression was found in cultured keratinocytes derived from seven healthy donors (NS) and non-lesional skin of six AD patients and seven PS. Keratinocytes were cultured in keratinocyte growth medium (KGM) and allowed to differentiate by growth factor depletion as described before (Pfundt et al., 1996). Independent of the diagnosis, 48hours stimulation by Th2 cytokines (50ng/ml IL-4 and 50ng/ml IL-13; Peprotech, London, UK) significantly increased CA II mRNA levels (P<0.0002) compared with untreated keratinocytes (KGM without growth factors), whereas 48hours of Th1 cytokine stimulation (30ng/ml IL-1α (Peprotech), 30ng/ml tumor necrosis factor-α (Peprotech) and 10U/ml IFN-γ (Hycult Biotechnology, Uden, The Netherlands)) had no effect on CA II expression levels. CA II mRNA levels were determined as in Figure 1. (b) A Western blot representative for all these keratinocyte cultures shows that CA II protein levels were also increased after 48hours of Th2 cytokine stimulation and not by 48hours of Th1 cytokine stimulation. Purified CA II (Sigma-Aldrich, St Louis, MO) was used as a positive control (+C). Journal of Investigative Dermatology 2007 127, 1786-1789DOI: (10.1038/sj.jid.5700752) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions