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Skin-Infiltrating Monocytes/Macrophages Migrate to Draining Lymph Nodes and Produce IL-10 After Contact Sensitizer Exposure to UV-Irradiated Skin  Eiko.

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Presentation on theme: "Skin-Infiltrating Monocytes/Macrophages Migrate to Draining Lymph Nodes and Produce IL-10 After Contact Sensitizer Exposure to UV-Irradiated Skin  Eiko."— Presentation transcript:

1 Skin-Infiltrating Monocytes/Macrophages Migrate to Draining Lymph Nodes and Produce IL-10 After Contact Sensitizer Exposure to UV-Irradiated Skin  Eiko Toichi, Kurt Q. Lu, Alan R. Swick, Thomas S. McCormick, Kevin D. Cooper  Journal of Investigative Dermatology  Volume 128, Issue 11, Pages (November 2008) DOI: /jid Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 IL-12p40 mRNA expression is upregulated after DNFB application or UVB irradiation, but not after combined exposure. Mice were (a) painted with DNFB on normal skin (DNFB), (b) UV-irradiated (UV), or painted with DNFB 48hours following UV irradiation (UV+DNFB, a, b). Time course of IL-12p40 mRNA expression in the DLN was examined using real-time RT-PCR. IL-12p40 expression levels were normalized to housekeeping gene. The results represent the mean±SE of 4–5 mice. *P<0.05 and **P<0.01 compared with normal (0hour). #P<0.05 compared with 48hours after UV irradiation (immediately before DNFB application). ##P<0.01 compared with DNFB at the same time point. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 The cellular source responsible for the rise of IL-12p40 mRNA in the DLN from DNFB or UVB-exposed skin is CD11c+ cells. DLNs were removed (a) 48hours after DNFB application to normal skin or (b) 72hours after UVB irradiation. DLN cells were incubated with anti-Thy1.2, anti-B220, anti-CD11c, or anti-F4/80 Ab. Positively selected fractions were enriched for T cells (T), B cells (B), CD11c+ cells or F4/80+ cells, as indicated. Negatively selected fractions were depleted of T cells (−T), B cells (−B), CD11c+ cells (−CD11c), or F4/80+ cells (−F4/80), respectively. Total RNA was extracted from each fraction and the unselected fraction (Total), and IL-12p40 mRNA expression was quantified by real-time RT-PCR. IL-12p40 expression levels were normalized to housekeeping gene. The results represent the mean±SE of three independent experiments. **P<0.01 compared with -CD11c and total. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 DNFB application to UV-irradiated skin induces a rapid spike of IL-10 mRNA in the DLN. Time course of IL-10 mRNA expression in the DLN of three groups was examined as described in Figure 1. The results represent the mean±SE of 4-5 mice. (a) ##P<0.01 compared with DNFB at the same time point. (b) **P<0.01 compared with 48hours after UV irradiation (immediately before DNFB application). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 The cellular source of the IL-10 mRNA in the DLN following UV+DNFB exposure is cells expressing CD11c and F4/80. DLNs were removed 3hours after DNFB application to UV-irradiated skin. IL-10 mRNA expression of the cellular fractions was quantified as described in Figure 2. The results represent the mean±SE of three independent experiments. #P<0.05 compared with -CD11c. *P<0.05 compared with -F4/80 and total. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Infiltration of F4/80+ cells into the UV-irradiated skin. Mouse back skin of normal and 48hours after UVB irradiation was removed and stained with anti-F4/80 Ab. Numerous cells expressing the F4/80 (brown, arrow) are observed in the UV-irradiated skin, but very few in normal skin. Bar=50μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 DNFB application to UV-irradiated skin induces upregulation of MCP-1 mRNA and is expressed by cells expressing CD11c and F4/80. (a) Time course of MCP-1 mRNA expression in the DLN was examined as described in Figure 1. The results represent the mean±SE of 3–4 mice. *P<0.05, **P<0.01compared with 48hours after UVB irradiation (immediately before DNFB application). (b) MCP-1 mRNA expression of the cellular fractions of the DLN 3hours after DNFB application to UV-irradiated skin was quantified as described in Figure 2. The results represent the mean±SE of three independent experiments. **P<0.01 compared with -CD11c (for CD11c) or –F4/80 (for F4/80) and total. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 DNFB application to UV-irradiated skin results in the appearance of F4/80+ cells in the DLN. DLN of normal skin (Normal), 3hours after DNFB application (DNFB), 51hours after UVB irradiation (UV), and 3hours after DNFB application to skin exposed to UV 48hours before (total 51hours after UVB) (UV+DNFB) were obtained. DLN cells were stained with phycoerythrin anti-CD11c, FITC anti-F4/80, allophycocyanin anti-CD3, and allophycocyanin anti-CD19 Abs, and three-color flow cytometric analysis was performed. In 100,000 collected cells, CD3−CD19− cells (∼1,500) were first selected and then analyzed for CD11c and F4/80 expression. Two mice were used in each group. (a) Percentage of each quadrant is given. Data are representative of three independent experiments. (b) Total cell numbers of DLNs were counted using trypan blue, and the yield of CD11c+F4/80− (CD11c+) cells or F4/80+CD11c− (F4/80+) cells were calculated. Data represent the mean±SE of three independent experiments. *P<0.05 compared with three other groups. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

9 Figure 8 F4/80+ cells migrate from skin to the DLN after hapten application to UV-irradiated skin. DLNs were obtained from mice 6hours after FITC/DNFB application to normal skin (FITC/DNFB) and to skin exposed to UV 48hours earlier (UV+ FITC/DNFB). T cells and B cells were depleted with magnetic beads. Negatively enriched populations were stained with phycoerythrin anti-F4/80 and allophycocyanin anti-CD11c Abs and three-color flow cytometric analysis was performed. FITC+ cells (∼800) were first selected and then analyzed for CD11c and F4/80 expression. Three mice were used in each group. Percentage of each quadrant is given. Data are representative of two independent experiments. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

10 Figure 9 F4/80+ cells migrated from UV-irradiated skin to the DLN following hapten-application produce IL-10. DLNs were obtained from mice 6hours after FITC/DNFB application to UV-irradiated skin. FITC+F4/80+CD11c− cells or FITC+CD11c+F4/80− cells were sorted and 2 × 104 cells per well were incubated with or without lipopolysaccharide. IL-10 ELISPOT was performed. Data are representative of three independent experiments. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

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