Cutaneous Gene Expression by DNA Microarray in Murine Sclerodermatous Graft- Versus-Host Disease, a Model for Human Scleroderma Lixin Zhou, David Askew,

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Cutaneous Gene Expression by DNA Microarray in Murine Sclerodermatous Graft- Versus-Host Disease, a Model for Human Scleroderma Lixin Zhou, David Askew, Caiyun Wu, Anita C. Gilliam Journal of Investigative Dermatology Volume 127, Issue 2, Pages 281-292 (February 2007) DOI: 10.1038/sj.jid.5700517 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Mice on day 35 after BMT. (a) BALB/c mice transplanted with bone marrow and spleen cells from B10.D2 mice (Scl GVHD) show a chronic dermatitis on the ears, around the eyes, and on back and develop patchy alopecia (upper panel). BALB/c mice transplanted with cells from BALB/c mice (BMT control) do not show dermatitis or hair loss (lower panel). (b) Serial body weights (g) of mice by week after BMT. Normal mice (unirradiated littermate mice without BMT transplantation), lethally irradiated BALB/c mice transplanted with bone marrow and spleen cells from B10.D2 mice (Scl GVHD), BALB/c transplanted with cells from BALB/c mice (control mice) were compared. Five mice were in each group at the beginning of the experiments. As mice were killed at each time point, there was only one mouse in each group at day 120 after BMT. Journal of Investigative Dermatology 2007 127, 281-292DOI: (10.1038/sj.jid.5700517) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Routine histology of back skin from mice with Scl GVHD and BMT control on days 7, 30, 60, and 120 after BMT. Each panel shows the skin of the representative mouse selected for gene array studies. There is transient decreased subcutaneous fat, an influx of mononuclear cells, and increased the skin thickening that peaks at 30–60 days and begins to decline after BMT in Scl GVHD mice (top two panels, 10 ×). Skin thickening is less apparent by day 120 in Scl GVHD mice; however, the dermis is denser in appearance (lower panel × 40). Bar=100μm. Journal of Investigative Dermatology 2007 127, 281-292DOI: (10.1038/sj.jid.5700517) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Cytokine expression by T cells isolated from skin at day 7 post-BMT. Single cell suspensions of skin cells were cultured on anti-CD3-coated plates in the presence of 1μg/ml brefeldin A to isolate T cells. Adherent cells were then stained with fluorescently labeled anti-CD4 antibody, washed, fixed, and permeabilized with saponin to allow intracytoplasmic staining by antibodies. After incubation with fluorescent anticytokine antibodies or isotype control antibodies, cells were washed and examined by flow cytometry analysis for intracellular cytokine protein expression. Grey plot: isotype control; thin line plot: BMT control mice; thick line plot: mice with Scl GVHD. The numbers in the right upper panel represent % positive cells detected with a particular anticytokine antibody as follows: BMT control (normal font) and Scl GVHD (bold font). Journal of Investigative Dermatology 2007 127, 281-292DOI: (10.1038/sj.jid.5700517) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions