Plasmacytoid Dendritic Cells: A New Cutaneous Dendritic Cell Subset with Distinct Role in Inflammatory Skin Diseases Andreas Wollenberg, Sandra Günther,

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Plasmacytoid Dendritic Cells: A New Cutaneous Dendritic Cell Subset with Distinct Role in Inflammatory Skin Diseases Andreas Wollenberg, Sandra Günther, Martina Moderer, Stefanie Wetzel Journal of Investigative Dermatology Volume 119, Issue 5, Pages 1096-1102 (November 2002) DOI: 10.1046/j.1523-1747.2002.19515.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Identification of DC subsets in human skin. (A) Detection of myeloid DC and PDC in PBMC. PBMC isolated from peripheral blood of healthy volunteers were stained with HLA-DR (PerCP), lineage markers (FITC), CD123 (PE), and CD11c (APC). A morphologic gate (FSC vs. SSC) was set to exclude cell multiples from acquisition (not in figure). HLA-DR+ and lineage– cells were examined for the expression of CD11c (myeloid DC) and of CD123 (PDC) by flow cytometry. The FSC and SSC characteristics of PDC are indicated. (B) Detection of Langerhans cells and IDEC in normal and inflammatory skin and comparison of CD11b and CD11c expression. Epidermal single cell suspensions of biopsies of normal skin and of skin lesions of a patient with AD were prepared and stained with CD1a (PE) in combination with either CD11b (FITC) or CD11c (FITC) (no additional staining with lineage markers and HLA-DR). Langerhans cells both in normal and inflamed skin are identified by high expression of CD1a and the lack of CD11b. IDEC identified by coexpression of CD1a and CD11b are detected in inflamed skin but not in normal skin. For both CD11b and CD11c, expression on IDEC is higher than on Langerhans cells. (C) Simultaneous detection of PDC, IDEC, and Langerhans cells in inflamed skin: Epidermal single cell suspensions of biopsies of skin lesions of a patient with psoriasis were prepared and stained with HLA-DR (PerCP), lineage markers (FITC), CD11c (APC), and either CD123 (PE) or CD1a (PE). HLA-DR+ and lineage– cells were gated and examined for the expression of CD123 (PDC) and CD11c (bright: IDEC; intermediate/low: Langerhans cells). IDEC show a lower CD1a staining than Langerhans cells. The identity of PDC is confirmed by FSC and SSC characteristics (cf. A). Journal of Investigative Dermatology 2002 119, 1096-1102DOI: (10.1046/j.1523-1747.2002.19515.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Comparison of BDCA-2 staining and CD123-based identification of PDC. Epidermal single cell suspensions of biopsies of lesional skin were prepared. For the detection of PDC, a four-color staining protocol was used to identify DC subsets: HLA-DR (PerCP), lineage markers (FITC), CD11c (APC), and CD123 (PE) (indicated as CD123+). Alternatively, HLA-DR (PerCP) staining was combined with the PDC-specific anti-BDCA-2 antibody (indicated as BDCA-2). (A) HLA-DR+ cells were gated and examined for the expression of BDCA-2. (B) The mean frequency of PDC in skin biopsies from patients with AD (n=3), psoriasis (n=6), CD (n=4), and LE (n=2) is depicted (mean±SEM). The results with the two different staining protocols (CD123 or BDCA-2) are compared, showing no statistical difference between both methods. Journal of Investigative Dermatology 2002 119, 1096-1102DOI: (10.1046/j.1523-1747.2002.19515.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 PDC, Langerhans cells, and IDEC in normal skin and in lesional skin of patients with AD, psoriasis vulgaris, CD, and LE. Epidermal single cell suspensions of biopsies of normal skin and of lesional skin of patients with AD, psoriasis vulgaris, CD, and LE were prepared. (A) DC subsets (PDC, IDEC, and Langerhans cells) were quantified by four-color-staining with HLA-DR (PerCP), lineage markers (FITC), CD11c (APC), and CD123 (PE) as indicated. (B) The identity of PDC is confirmed by FSC and SSC characteristics as compared with PBMC (see Figure 1a). (C) The mean frequencies of PDC, IDEC, and Langerhans cells (±SEM) are compared between normal skin (n=3), AD (n=9), psoriasis vulgaris (n=6, patients not identical with the six patients in Figure 2), and CD (n=5). Note different scale of the x axis. Statistical significance was determined by Mann–Whitney U test (*p<0.05). Journal of Investigative Dermatology 2002 119, 1096-1102DOI: (10.1046/j.1523-1747.2002.19515.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunohistologic analysis of PDC in skin. Cryosections of human tonsils (A) were stained with the PDC-specific MoAb BDCA-2 as a positive control. Positive cells are located in the area of high endothelial venules and in the perifollicular T cell areas of the tonsil. Next, cryosections of lesional psoriasis skin (B,C) were stained with the PDC-specific MoAb BDCA-2, demonstrating BDCA-2 positive cells with a membranous staining pattern in the basal layer of the epidermis (B) as well as in the papillary dermis (B,C) of psoriatic skin lesions. Skin samples from three individual patients gave similar results. Journal of Investigative Dermatology 2002 119, 1096-1102DOI: (10.1046/j.1523-1747.2002.19515.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions