Intermedin: A Skin Peptide that Is Downregulated in Atopic Dermatitis Friederike Kindt, Silke Wiegand, Christoph Löser, Martin Nilles, Volker Niemeier, Sheau Yu Teddy Hsu, Martin Steinhoff, Wolfgang Kummer, Uwe Gieler, Rainer Viktor Haberberger  Journal of Investigative Dermatology  Volume 127, Issue 3, Pages 605-613 (March 2007) DOI: 10.1038/sj.jid.5700576 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 IMD mRNA and peptide in human skin, human keratinocytes, and HaCaT cells. (a) RT-PCR analysis of the IMD expression in human skin and in HaCaT cells. Primers specific for IMD and the house keeping gene 18S rRNA were used to amplify cDNA prepared from skin of healthy control persons (Contr. skin), from non-lesional (AD non-les.) and lesional (AD les.) skin biopsies of AD patients and from HaCaT cells. Omission of the template leads to absence of amplification products. M=100bp Marker. (b) RT-PCR analysis of the IMD expression in HaCaT cells and cultured human keratinocytes. Primers specific for IMD and the house keeping gene 18S rRNA were used to amplify cDNA prepared from HaCaT cells and cultured human keratinocytes. Aliquots from total RNA after DNA digestion before RT were used as a control (Control). Journal of Investigative Dermatology 2007 127, 605-613DOI: (10.1038/sj.jid.5700576) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Expression of IMD in human skin studied via confocal laser scanning microscopy (CLSM). IMD immunoreactivity is present in (a) human skin and (c) HaCaT cells, and (e) primary human keratinocytes. The specificity of the immunolabelling is demonstrated by the absence of immunoreactivity in the preabsorption controls (Pre b, d, f). Bar=50μm. Journal of Investigative Dermatology 2007 127, 605-613DOI: (10.1038/sj.jid.5700576) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 The localization of IMD in human skin in CD31-negative and in α-SMA-immunoreactive cells studied via CLSM. IMD immunoreactivity is present in cells that run together with CD31-positve endothelial cells (a, b, bar=20μm). (c) The signet ring-like IMD-positive cells are localized close to but not identical with CD31-positive capillary endothelial cells (d, bar=8μm). The localization of IMD in human skin studied via CLSM. IMD-immunoreactive cells and α-SMA-immunoreactive cells were found in the dermis (e, f, bar=40μm). The IMD immunoreactivity was present in α-SMA-positive cells (g, h, bar=8μm). Journal of Investigative Dermatology 2007 127, 605-613DOI: (10.1038/sj.jid.5700576) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Localization of IMD in the dermis of human skin studied via CLSM. IMD immunoreactivity was found in the deeper dermis in the smooth muscle of arterioles (large arrow in a, b) and in cells close to endothelial cells that exhibited only minor or virtually no α-SMA staining (small arrow in a, b). Subpopulations of Nf68-positive axons in nerve fiber bundles also expressed IMD immunoreactivity (small arrow in c, d). Smooth muscle cells of arrector pili muscles were also positive for IMD (e, TO-PRO-positive nuclei are shown in blue). Single nerve fibers were found in close apposition to dermal arterioles (small arrow in f). An IMD-immunoreactive cell layer (red) surrounded the acini of eccrine glands (small arrow in g) close to α-SMA-positive myoepithelial cells (green, g). The elongated TO-PRO-positive nuclei (blue) of these IMD-positive cells (red) were found in close apposition to the acini (small arrow h). Bar=20μm. Journal of Investigative Dermatology 2007 127, 605-613DOI: (10.1038/sj.jid.5700576) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Quantitative RT-PCR analysis of IMD expression in AD skin. Quantitative RT-PCR analysis revealed that the expression level of IMD is reduced in AD. The data were normalized by subtracting the CT levels between the IMD and 18S rRNA. The ΔCT values were subtracted from 50 showing higher values with higher expression. The expression of IMD in healthy skin was significantly higher compared to the IMD expression in lesional and non-lesional skin of AD patients. ***P<0.001. Journal of Investigative Dermatology 2007 127, 605-613DOI: (10.1038/sj.jid.5700576) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Localization of IMD in the skin of healthy controls and in lesional and non-lesional skin areas of AD patients studied via CLSM, nuclear counterstain with TO-PRO. The thickness of the epidermis was increased in the lesional (P32K) and non-lesional area (P32G) of AD patients. Lesional areas of “mixed type” AD patients (P32K) showed in some cases stronger IMD immunoreactivity compared to healthy control skin (K32) and non-lesional areas (P32G). The IMD immunoreactivity of pericytes (arrowheads) at the epidermis (Ep) dermis border was, compared to healthy control skin (K30) decreased in the lesional (P31K) and non-lesional area (P31G) of AD patients. Bar=20μm. Journal of Investigative Dermatology 2007 127, 605-613DOI: (10.1038/sj.jid.5700576) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions