Both Pimecrolimus and Corticosteroids Deplete Plasmacytoid Dendritic Cells in Patients with Atopic Dermatitis Wolfram Hoetzenecker, Simone Meindl, Anton.

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Both Pimecrolimus and Corticosteroids Deplete Plasmacytoid Dendritic Cells in Patients with Atopic Dermatitis Wolfram Hoetzenecker, Simone Meindl, Anton Stuetz, Georg Stingl, Adelheid Elbe-Bürger Journal of Investigative Dermatology Volume 126, Issue 9, Pages 2141-2144 (September 2006) DOI: 10.1038/sj.jid.5700368 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Topical treatment with pimecrolimus and BMV significantly depletes BDCA-2+ cells in AD skin lesions. Cryostat sections (5μm) were air-dried and fixed in ice-cold acetone for 10minutes. To prevent nonspecific binding of the anti-BDCA-2 mAb (Miltenyi Biotec Inc., Auburn, CA), it was mixed with 1% normal horse serum and 2% BSA and applied to sections (1hour, room temperature). Immunolocalization of the primary antibody was performed using a biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA), followed by an avidin-biotinylated-HRP third layer (Vector Laboratories). AEC+substrate (DakoCytomation, Glostrup, Denmark) were used as chromogen and sections were counterstained with Gill's hematoxylin (Santa Cruz Biotechnologies, Santa Cruz, CA). In all staining experiments, an IgG1 isotype mAb served as negative control. Representative pictures of immunostained sections were taken and positive cells are marked by arrows (upper panels). pDC numbers were evaluated in a blinded fashion (25–45 counted fields per section under × 25 object lenses; numbers of patients per group: untreated=22, vehicle control=6, pimecrolimus=8, BMV=8) by two independent investigators and results are expressed as mean±SEM positive cells/mm2 dermis. One-way analysis of variance was used to evaluate statistically significant differences between test and control groups. *P<0.05 versus control. Bars=200μm. Journal of Investigative Dermatology 2006 126, 2141-2144DOI: (10.1038/sj.jid.5700368) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 CS but not pimecrolimus strongly reduce pDC numbers in vitro. Buffy coats from peripheral blood of healthy adult volunteers were purchased from the local transfusion service (Rotes Kreuz, Vienna, Austria). Peripheral blood monocytes were isolated as interface cells after density gradient centrifugation (Lymphoprep; Axis-Shield, Rodelokka, Norway) and erythrocytes were removed with ammonium chloride (0.8% NH4Cl/0.1mm EDTA). pDC (mean purity: 93.88%±5.21 (n=4)) were isolated using a negative isolation kit according to the manufacturer's manual (Miltenyi Biotec Inc.). (a) Freshly isolated cells were stained for the indicated markers and 30,000 events/sample were acquired. pDC were cultured (5 × 106/ml) for the indicated time periods (day 2: n=3; day 7: n=1) in RPMI 1640 medium (GIBCO Life Technologies, Carlsbad, CA) supplemented with 10% FCS, 2mm l-glutamine, antibiotics, and 10ng/ml recombinant human IL-3 (Pepro Tech, Rocky Hill, NJ) in the presence of vehicle, pimecrolimus, or BMV (all 10−6mol/l). Fluorescence was measured with a FACSCalibur flow cytometer, and data were analyzed using Cell Quest software (both Becton Dickinson, San Jose, CA). (b) Mean fluorescence intensity (MFI) was determined for BDCA-2 expression and (c) dead cells were identified by 7-amino-actinomycin D uptake (Sigma Chemicals Co., St Louis, MO). Journal of Investigative Dermatology 2006 126, 2141-2144DOI: (10.1038/sj.jid.5700368) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions