David A. Groneberg, MDab, Pia Welker, PhDcd, Tanja C. Fischer, MDc, Q

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Down-regulation of vasoactive intestinal polypeptide receptor expression in atopic dermatitis David A. Groneberg, MDab, Pia Welker, PhDcd, Tanja C. Fischer, MDc, Q.Thai Dinh, MDe, Andreas Grützkau, PhDc, Christian Peiser, MD, PhDa, Ulrich Wahn, MDa, Beate M. Henz, MDc, Axel Fischer, MDa Journal of Allergy and Clinical Immunology Volume 111, Issue 5, Pages 1099-1105 (May 2003) DOI: 10.1067/mai.2003.1477 Copyright © 2003 Mosby, Inc. Terms and Conditions

Fig. 1 Mast cell numbers and VPAC2 expression in AD. Sections of normal (A) and AD (B) human skin were evaluated for mast cell numbers, and toluidine blue staining showed an increased number of toluidine blue mast cells (arrows , 90 ± 8 per mm2) in AD tissues (C) . VPAC2 staining was found in mast cells (arrows in D ), and signals for the ligand VIP were present in cutaneous nerve fibers (arrows in E ). VPAC2 mRNA in situ hybridization confirmed staining of mast cells (arrows in F ). No specific staining was found when the sense probes were used (D) . Scale bar represents 120 μm (E = 60 μm). Journal of Allergy and Clinical Immunology 2003 111, 1099-1105DOI: (10.1067/mai.2003.1477) Copyright © 2003 Mosby, Inc. Terms and Conditions

Fig. 2 RT-PCR and gene array studies. Amplification products of the VPAC2 gene were found in extracts of mammary gland skin (mamma) and foreskin (preputium) mast cell preparations and HMC-1 cells. There was no signal in KU-812 cells (A) . The control gene was present in every preparation (reduced glyceraldehyde-phosphate dehydrogenase). HMC-1 cells were then examined for VPAC2 gene expression under basal and stimulated conditions and showed high basal expression and a significant downregulation of VPAC2 under stimulation in contrast to other receptors such as VPAC1, GABA-A, or the ligand VIP (B) . Journal of Allergy and Clinical Immunology 2003 111, 1099-1105DOI: (10.1067/mai.2003.1477) Copyright © 2003 Mosby, Inc. Terms and Conditions

Fig. 3 Mast cell VPCAC2 protein levels in situ. Staining intensity of VPAC2 protein was analyzed in mast cells of normal subjects (A and B ) and AD patients (C and D ) by using image quantification methods, constant staining and microscope settings, and pseudo-color imaging (B and D ). Staining intensity that was measured in single mast cells (MC) was decreased in AD if compared to normal individuals (E) . Scale bar represents 100 μm. Journal of Allergy and Clinical Immunology 2003 111, 1099-1105DOI: (10.1067/mai.2003.1477) Copyright © 2003 Mosby, Inc. Terms and Conditions