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A Chronic Contact Eczema Impedes Migration of Antigen-Presenting Cells in Alopecia Areata  Pooja Gupta, Pia Freyschmidt-Paul, Mario Vitacolonna, Sabine.

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Presentation on theme: "A Chronic Contact Eczema Impedes Migration of Antigen-Presenting Cells in Alopecia Areata  Pooja Gupta, Pia Freyschmidt-Paul, Mario Vitacolonna, Sabine."— Presentation transcript:

1 A Chronic Contact Eczema Impedes Migration of Antigen-Presenting Cells in Alopecia Areata 
Pooja Gupta, Pia Freyschmidt-Paul, Mario Vitacolonna, Sabine Kiessling, Susanne Hummel, Dagmar Hildebrand, Rachid Marhaba, Margot Zöller  Journal of Investigative Dermatology  Volume 126, Issue 7, Pages (July 2006) DOI: /sj.jid Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Allergen treatment has no impact on the high recovery of LNC and SkIL of AA mice. The number of draining LNC and SkIL (mean±SD) is shown for control, AA-, SADBE-treated, and AA/SADBE mice. Values are derived from eight experiments, each with a pool of leukocytes from three mice. Significant differences in comparison to control mice are indicated by an asterisk. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 The impact of allergen treatment on the distribution of leukocyte subpopulations in draining lymph nodes and skin. (a, b, and g) The main leukocyte subpopulations, (c and d) monocytic subpopulations, and (e and f) common leukocyte subpopulations were evaluated in draining LNC and SkIL of control, AA-, SADBE-treated, and AA/SADBE mice by (a–f) flow cytometry and (g) immunohistology of 5μm sections of shock-frozen skin, which were counterstained by hematoxilin (bar=50μm). (a–f) The percentage (mean±SD of five experiments) of stained cells is shown. Significant differences to LNC and SkIL of control mice are indicated by an asterisk. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Cytokine expression in LNC and SkIL of untreated and allergen-treated AA mice. (a and b) Cytokine expression was evaluated by intracellular staining and flow cytometric analysis in fixed and permeabilized draining LNC and SkIL of control, AA, SADBE, and AA/SADBE mice. The percentage (mean±SD of five experiments) of stained cells is shown. Significant differences to LNC and SkIL of control mice are indicated by an asterisk. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Chemokine profile of LNC, SkIL and the dermis of untreated and allergen-treated AA mice. (a and b) LNC and SkIL of control, AA, SADBE, and AA/SADBE mice were fixed and permeabilized and stained with antibodies for the indicated chemokines and chemokine receptors for flow cytometric analysis. The percentage (mean±SD of five experiments) of stained cells is shown. Significant differences to LNC and SkIL of control mice are indicated by an asterisk. (c) Sections (5μm) of shock-frozen skin of control, AA-, SADBE-treated, and AA/SADBE mice were stained with the indicated antibodies. For staining with cytokine-specific antibodies, sections were fixed and permeabilized. Sections were counterstained with hematoxilin. Representative examples are shown. Bar=50μm. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 High migratory activity of LNC of AA mice. LNC (5 × 104) from control, AA, SADBE, and AA/SADBE mice were seeded in the upper well of a Boyden chamber, where the lower well contained either RPMI with 0.1% bovine serum albumin or 40ng OPN or 4ng CXCL10. After 4hours incubation, cells in the lower chamber were counted. The percentage (mean±SD of triplicates from five experiments) of migrating cells is presented. Significant differences in comparison to LNC from control mice are indicated by an asterisk. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Unimpaired lymph node and skin homing of LNC from allergen-treated as compared to untreated AA mice. LNC from control, AA- and SADBE-treated mice were stained with CFSE and were i.v. injected into control, AA-, SADBE-treated, and AA/SADBE mice, that were killed after 3 days. (a and b) The percentage of dye-labelled LNC and SkIL was evaluated by flow cytometry. (a) A representative example and (b) mean values±SD of three experiments each with pools of cells from three mice are shown. Significant differences in the percentage of labelled cells from control versus AA- and SADBE-treated mice are indicated by an asterisk. Significant differences in dependence on the AA/SADBE host (as compared to the untreated AA or SADBE-treated host) are indicated by s. (c–e) LNC and SkIL were stained with the indicated antibodies and counterstained with allophycocyanin-labelled secondary antibodies. (c) An example for the recovery of dye-labelled CD4+ cells in draining lymph nodes and SkIL of control, AA-, SADBE-treated, and AA/SADBE mice is presented. (d and e) The percentage of marker-positive SkIL as well as of dye-labelled SkIL was evaluated. The percentage (mean±SD of three experiments with pooled cells of three mice per group) of double fluorescent (dye labelled, marker positive) SkIL in comparison to marker-positive SkIL (=100%) is shown. Significant differences (Wilcoxon rank-sum test) are indicated by an asterisk (control versus AA- and SADBE-treated mice) or by s (the AA/SADBE versus the control or the AA- or the SADBE-treated host). Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 6 Unimpaired lymph node and skin homing of LNC from allergen-treated as compared to untreated AA mice. LNC from control, AA- and SADBE-treated mice were stained with CFSE and were i.v. injected into control, AA-, SADBE-treated, and AA/SADBE mice, that were killed after 3 days. (a and b) The percentage of dye-labelled LNC and SkIL was evaluated by flow cytometry. (a) A representative example and (b) mean values±SD of three experiments each with pools of cells from three mice are shown. Significant differences in the percentage of labelled cells from control versus AA- and SADBE-treated mice are indicated by an asterisk. Significant differences in dependence on the AA/SADBE host (as compared to the untreated AA or SADBE-treated host) are indicated by s. (c–e) LNC and SkIL were stained with the indicated antibodies and counterstained with allophycocyanin-labelled secondary antibodies. (c) An example for the recovery of dye-labelled CD4+ cells in draining lymph nodes and SkIL of control, AA-, SADBE-treated, and AA/SADBE mice is presented. (d and e) The percentage of marker-positive SkIL as well as of dye-labelled SkIL was evaluated. The percentage (mean±SD of three experiments with pooled cells of three mice per group) of double fluorescent (dye labelled, marker positive) SkIL in comparison to marker-positive SkIL (=100%) is shown. Significant differences (Wilcoxon rank-sum test) are indicated by an asterisk (control versus AA- and SADBE-treated mice) or by s (the AA/SADBE versus the control or the AA- or the SADBE-treated host). Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

9 Figure 7 Leukocyte migration from the skin towards the draining lymph node in untreated and allergen-treated AA mice. FITC (250μg/mouse) was dissolved in ethanol and was gently rubbered into the shaved skin. Mice were sacrificed after 3 days. (a and b) The percentage of fluorescent cells was evaluated in SkIL and draining lymph nodes. (a) A representative example and (b) the percentage of fluorescent cells (mean±SD of three experiments with pools of cells of three mice per group) is shown. Significant differences in the percentage of stained draining LNC between control, AA-, SADBE-treated, and AA/SADBE mice are indicated by an asterisk. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

10 Figure 8 Impaired migration of FITC-labelled leukocyte subpopulations from the skin to draining lymph nodes of allergen-treated AA mice. Mice were treated as described in Figure 7. (a and e) Draining LNC and SkIL of control, AA-, SADBE-treated, and AA/SADBE mice were stained with antibodies for the indicated markers, where cells had been fixed and permeabilized in advance for chemokine staining. Cells were counterstained with the allophycocyanin-labelled secondary antibodies. Representative examples are shown. (b, c, f, and g) The percentage of FITC-labelled, marker expressing LNC and SkIL was evaluated as described in Figure 6, i.e. the percentage of FITC-labelled, marker-positive cells in comparison to marker-positive cells (=100%) is presented. In (d and h) the relative contribution of FITC-labelled, marker-positive cells in the draining lymph node and the skin are presented as the ratio of marker-positive, FITC-labelled LNC: marker-positive, FITC-labelled SkIL (mean±SD of three experiments with pools of cells from three mice per group). Significant differences in the percentage of double-stained draining LNC and SkIL (b, c, f, and g) between control, AA-, SADBE-treated, and AA/SADBE mice (Student's t-test), and (d and h) significance of differences in the ratio of double-labelled LNC versus SkIL (Wilcoxon rank-sum test) are indicated by an asterisk. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

11 Figure 8 Impaired migration of FITC-labelled leukocyte subpopulations from the skin to draining lymph nodes of allergen-treated AA mice. Mice were treated as described in Figure 7. (a and e) Draining LNC and SkIL of control, AA-, SADBE-treated, and AA/SADBE mice were stained with antibodies for the indicated markers, where cells had been fixed and permeabilized in advance for chemokine staining. Cells were counterstained with the allophycocyanin-labelled secondary antibodies. Representative examples are shown. (b, c, f, and g) The percentage of FITC-labelled, marker expressing LNC and SkIL was evaluated as described in Figure 6, i.e. the percentage of FITC-labelled, marker-positive cells in comparison to marker-positive cells (=100%) is presented. In (d and h) the relative contribution of FITC-labelled, marker-positive cells in the draining lymph node and the skin are presented as the ratio of marker-positive, FITC-labelled LNC: marker-positive, FITC-labelled SkIL (mean±SD of three experiments with pools of cells from three mice per group). Significant differences in the percentage of double-stained draining LNC and SkIL (b, c, f, and g) between control, AA-, SADBE-treated, and AA/SADBE mice (Student's t-test), and (d and h) significance of differences in the ratio of double-labelled LNC versus SkIL (Wilcoxon rank-sum test) are indicated by an asterisk. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

12 Figure 9 Reduced DC migration from the skin towards the draining lymph node in allergen-treated mice. CD11c+ and CD11b+ LNC were separated from AA- and SADBE-treated mice as described in Materials and Methods. Cells were mixed at a 1:1 ratio, were labelled with CFSE and were s.c. injected at eight different sites under the dorsal skin of AA-, SADBE-treated, and AA/SADBE mice. Mice were killed after 3 days. Draining LNC and SkIL were stained with antibodies for the indicated markers and allophycocyanin-labelled secondary antibodies. The percentage of dye-labelled, (a) marker-positive LNC and (b) SkIL in comparison to marker positive cells was evaluated as described in Figure 6. In (c) the ratio of double-labelled LNC versus SkIL is presented. Significant differences (mean±SD from three separate experiments with pools of cells from two to three mice per group) between AA- and SADBE-treated mice are indicated by an asterisk; significant differences between the AA/SADBE host, that received CD11c+ and CD11b+ cells from AA- or SADBE-treated mice, as compared to the AA- or SADBE-treated host are indicated by s. Significant differences in the ratio of double-labelled LNC versus (c) was calculated by the Wilcoxon rank-sum test. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

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