Differential Effects of Corticosteroids and Pimecrolimus on the Developing Skin Immune System in Humans and Mice  Simone Meindl, Christine Vaculik, Josef G. Meingassner, Gero Kramer, Johnnie Akgün, Marion Prior, Anton Stuetz, Georg Stingl, Adelheid Elbe-Bürger  Journal of Investigative Dermatology  Volume 129, Issue 9, Pages 2184-2192 (September 2009) DOI: 10.1038/jid.2009.50 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Emergence kinetics and function of LCs in the developing human foreskin. (a and b) Epidermal sheets and (c) EC suspensions from human foreskin of the indicated age groups (infants: ≤2 years; children: 3–7 years; adults: ≥18 years) show an increase of LCs numbers in the developing human epidermis analyzed by immunofluorescence and flow cytometry, respectively (n=numbers of individuals/age group). (a and b) LCs were stained with a purified anti-CD1a mAb, visualized with an Alexa Fluor 546-conjugated second step reagent and analyzed. Significant differences were observed between infant and adult LC densities (**P<0.01), whereas those between children and adults were not significant (NS; P<0.20); Bars=20μm. (c) EC suspensions were labeled with a PE-conjugated anti-CD1a mAb directly after isolation and analyzed. A total of 20,000 events per sample were acquired. Bars represent the mean percentage of CD1a+ cells±SD. Significant differences were observed between the percentages of infant and adult LCs (***P<0.001), and those between children and adults (***P<0.001). (d) EC suspensions from foreskins (infants and individuals ≥10 years old) were incubated for 20minutes with 1mgml−1 FITC-dextran. Then cells were stained with a PE-labeled anti-CD1a mAb or isotype control before acquisition by flow cytometry. The surface binding of the reagent at 4°C was measured and subtracted from 37°C values. Data are presented as the percentage of internalized FITC-dextran. The percentage of FITC-dextran uptake by LCs from individuals older than 10 years was taken as 100%. Data shown are means (geometric mean fluorescence intensity)±SD of double-positive cells (n=6) **P<0.002. Journal of Investigative Dermatology 2009 129, 2184-2192DOI: (10.1038/jid.2009.50) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 BMV but not PIM affects the viability of unfractionated infant ECs in vitro. EC suspensions isolated from infants (foreskin) and adults (breast skin) were cultured (1 × 106 cells per ml) in the presence of vehicle (=100%), PIM (□), and BMV (▪) (10−6moll−1). After 48hours, 50,000 cells were acquired by flow cytometry and viability of (a) ECs and (b) CD1a+ LCs was determined. Viability of the vehicle group was taken as 100% (mean percentage±SD; (n=4)) **P<0.002. (c) Almost confluent NHEKs (normal human epidermal keratinocytes) were exposed to vehicle, PIM, and BMV (10−6moll−1) for 24 and 48hours and then analyzed by flow cytometry. A total of 50,000 cells were acquired and viability of the vehicle group was taken as 100% (mean percentage±SD (n=3)). (d) Sections from infant and adult human skin biopsy samples were exposed to anti-GR-Alexa Fluor 488/PE-anti-CD207/allophycocyanin-anti-CD45 labeling and analyzed with a CLSM. Bars=50μm. Journal of Investigative Dermatology 2009 129, 2184-2192DOI: (10.1038/jid.2009.50) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 BMV affects the maturation process of infant LCs in vitro. (a) ECs (1 × 106 cells per ml) isolated from infant foreskin were cultured for 48hours in the presence of vehicle, PIM, and BMV (10−6moll−1). Cells were harvested, stained, and 500 CD45+CD1a+ cells per sample were gated and analyzed. Symbols show mean fluorescence intensity±SD (five independent experiments with different donors) of the indicated markers cultured with PIM and BMV in relation to vehicle group (100%) ***P<0.001. (b) Different concentrations of BMV (10−6–10−9moll−1) showed a concentration-dependent inhibition of CD86 expression (n=4). (c) In some experiments, ECs were incubated with dexamethasone (DEX) (10−6moll−1), and CD86 expression was determined by flow cytometry (n=3). Journal of Investigative Dermatology 2009 129, 2184-2192DOI: (10.1038/jid.2009.50) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Neither BMV nor PIM significantly influence the release of cytokines in infant LCs and ECs in vitro. (a) EC suspensions isolated from infant foreskin were highly enriched for CD1c+ LCs by using MACS and subsequently one part of the cells was analyzed by flow cytometry for CD207 and HLA-DR co-expression. Supernatants of (b and c) highly enriched LCs or (c and d) LC-depleted ECs were harvested 24hours after culture in the presence or absence of indicated compounds (10−6moll−1) and assayed by the Luminex technology. In panel c, IL-8 production of highly enriched LCs was compared with that of LC-depleted ECs. Bars shown are mean values±SD (n=4). Journal of Investigative Dermatology 2009 129, 2184-2192DOI: (10.1038/jid.2009.50) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Epicutaneous treatment of newborn mice with corticosteroids but not PIM induces apoptosis in LC precursors in situ. (a) Newborn BALB/c mice were treated and analyzed as indicated in the scheme. Sixty hours after cessation of treatment, (b and c) EC suspensions and (d–f) epidermal sheets were prepared, stained, and analyzed as described in Materials and Methods. For flow cytometry, a total of 50,000 events per sample were acquired. (b) A representative experiment of three independent experiments is shown and (c) data analyzed are significant (*P<0.05, **P<0.01, ***P<0.001). One part of epidermal sheets from treated mice was stained with an anti-MHC class II mAb. (d) MHC class II+ cells are depicted (bar=20μm) and (e) their enumeration is shown. The other part was subjected either to a TUNEL assay or to caspase 3 staining (f) and counterstained with an anti-MHC class II mAb. Data shown are representative of two independent experiments. Journal of Investigative Dermatology 2009 129, 2184-2192DOI: (10.1038/jid.2009.50) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions