Characterization of CD200-Receptor Expression in the Murine Epidermis

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Characterization of CD200-Receptor Expression in the Murine Epidermis Michael D. Rosenblum, Jeffrey E. Woodliff, Nathaniel A. Madsen, Laura J. McOlash, Melissa R. Keller, Robert L. Truitt Journal of Investigative Dermatology Volume 125, Issue 6, Pages 1130-1138 (December 2005) DOI: 10.1111/j.0022-202X.2005.23948.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 CD200-receptor (CD200R) is expressed in murine epidermis. CD200R-specific quantitative real-time RT-PCR (qRT-PCR) was performed on epidermal cell suspensions or bone marrow isolated from B6 mice. Results represent pooled data from two of three replicate experiments. *Denotes statistically significant values for each isoform (p-value<0.05) by Student's t test. Although detected, the relative level of CD200R4 mRNA was too low to show above the X-axis. Journal of Investigative Dermatology 2005 125, 1130-1138DOI: (10.1111/j.0022-202X.2005.23948.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Characterization of CD200-receptor (CD200R) expression in epidermal cell (EC) populations. CD200R1-specific flow cytometry and CD200R1–R4 specific quantitative real-time RT-PCR (qRT-PCR) was performed on bulk EC (not shown) and sorted EC, respectively. Tables denote qRT-PCR values (arbitrary units normalized to 18S RNA in respective populations) for CD200R isoforms (R1–R4) in sorted cells. The median value from three to four replicate experiments is shown along with the mean±SEM. *Indicates that there is a statistically significant difference (p<0.05; Mann–Whitney U test) in mRNA expression within a subpopulation as compared to CD200R1. Journal of Investigative Dermatology 2005 125, 1130-1138DOI: (10.1111/j.0022-202X.2005.23948.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Langerhans cells from CD200–/– mice are maintained at a heightened state of activation. Four-color flow cytometry was performed on epidermal cells (EC) isolated from wild-type (WT) B6 or CD200–/– mice. Electronic gates were set on major histocompatibility complex class II (MHCII)bright, 7-aminoactinomycin-D (7AAD)- cells. Bold-line histograms represent staining with specific antibody, and light-line histograms represent staining with isotype-matched control antibody. Numeric values represent percent of cells staining positive and geometric mean fluorescence intensity of positive cells. Boxes around marker names denote statistically significant differences between WT and CD200–/– (four replicate experiments; p-value<0.05 by Student's t test). Journal of Investigative Dermatology 2005 125, 1130-1138DOI: (10.1111/j.0022-202X.2005.23948.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Dendritic epidermal T cells preferentially increase CD200-receptor (CD200R)1 upon activation in vitro. (a) Epidermal cells were isolated from B6 mice and cultured on anti-CD3-coated plates in the presence of interleukin (IL)-2. At various times, cells were harvested and subjected to CD200R1-specific flow cytometry. Cells are gated on γδ-TCR+/7-aminoactinomycin-D (7AAD)-. Results are representative of three replicate experiments. (b) γδ-TCR+/7AAD- cells were purified by fluorescent-activated cell sorting and cultured on anti-CD3-coated plates in the presence of IL-2. At various times, cell were harvested and subjected to CD200R-specific quantitative real-time RT-PCR. Results represent pooled means from three replicate experiments. The level of CD200R3 mRNA was too low to show above the X-axis, and no CD200R4 mRNA was detected. Journal of Investigative Dermatology 2005 125, 1130-1138DOI: (10.1111/j.0022-202X.2005.23948.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 7-17 dendritic epidermal T cells increase CD200-receptor (CD200R) expression upon activation in vitro. (a) Resting 7-17 cells (>8 days post-Con A activation) were cultured on anti-CD3-coated plates in the presence of interleukin (IL)-2. At various times, cells were harvested and subjected to CD200R1-specific flow cytometry. Quadrants were set based on isotype control staining (not shown) and numeric values represent % cells staining positive/mean fluorescence intensity of entire population. (b) Resting 7-17 cells were cultured on anti-CD3-coated plates in the presence of IL-2. At various times, cells were harvested and subjected to CD200R-specific quantitative real-time RT-PCR. Results represent pooled data from three replicate experiments. *Denotes statistically significant difference from time 0 (p-value<0.05) by Student's t test. Journal of Investigative Dermatology 2005 125, 1130-1138DOI: (10.1111/j.0022-202X.2005.23948.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 CD200 signaling inhibits 7-17 dendritic epidermal T cells (DETC) proliferation. 7-17 DETC were cultured on uncoated plates (no anti-CD3 mAb) or plates coated with either optimal anti-CD3, sub-optimal anti-CD3+ bacterial alkaline phosphatase (BAP).FLAG, or sub-optimal anti-CD3+CD200.FLAG (625 ng per well) in the absence of interleukin (IL)-2. Proliferation was measured at 88 h by [3H]-thymidine incorporation. Results are representative of 3 replicate experiments. *Denotes statistically significant from BAP.FLAG control (p-value<0.05) by Student's t test. Journal of Investigative Dermatology 2005 125, 1130-1138DOI: (10.1111/j.0022-202X.2005.23948.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions