Figure 8. Skin IMCs release CD4+ T cell chemokine CCL4. (A) Chemotaxis of CD4+ T cells isolated from control mice to supernatant from IMCs. IMCs were either stimulated or not with LPS. T cells were stimulated or not with CD3/CD28 antibody. CD4+ T cells were placed in the top chamber and supernatants (30% vol/vol) in the bottom chamber. For control, LPS was added to the medium in the bottom chamber. Mean and SD of cumulative results of six independent experiments are shown. ***, P < 0.001. (B) Quantitative RT-PCR analysis of the expression of ccl3, ccl4, ccl5, and ccl22 in skin of WT or S100A9Tg mice treated with TPA or acetone. Differences in expression of ccl3 and ccl4 were significant. Mean and SD are shown. *, P < 0.01. (C) Amount of CCL22 (left) and CCL3 (right) protein measured by ELISA in lysates of various organs from TPA-treated WT and S100A9Tg mice. Mean and SD from four experiments with three mice per group are shown. Each measurement was performed in duplicate and normalized for total protein. Cumulative results from three experiments are shown. (D) Amount of CCL4 protein measured by ELISA in lysates of various organs from TPA-treated WT and S100A9Tg mice. Mean and SD from four experiments with three mice per group are shown. Each measurement was performed in duplicate normalized for total protein. **, P < 0.01. (E) Amount of CCL4 determined by ELISA in supernatants of IMCs isolated from BM of WT or S100A9Tg mice treated with TPA for 4 wk. Cells were activated with LPS overnight. Measurements were performed twice in duplicates. Mean and SD are shown. (F) Expression of ccl4 in Gr-1+CD11b+ IMCs isolated from BM of naive mice treated for 24 h with the indicated cytokines. The range of most commonly used concentrations was tested. Concentrations shown: 250 ng/ml IFN-γ, 50 ng/ml TNF, 50 ng/ml IL-1β, and 100 ng/ml LPS. Ccl4 expression was measured by quantitative PCR and normalized to β-actin. Mean and SD are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001 from control (n = 3). (G) Expression of ccl4 in Gr-1+ and Gr-1− cells isolated from skin of WT or S100A9Tg mice treated for 5 wk with TPA (n = 3). Mean and SD are shown.
Figure 8.

Skin IMCs release CD4+ T cell chemokine CCL4. (A) Chemotaxis of CD4+ T cells isolated from control mice to supernatant from IMCs. IMCs were either stimulated or not with LPS. T cells were stimulated or not with CD3/CD28 antibody. CD4+ T cells were placed in the top chamber and supernatants (30% vol/vol) in the bottom chamber. For control, LPS was added to the medium in the bottom chamber. Mean and SD of cumulative results of six independent experiments are shown. ***, P < 0.001. (B) Quantitative RT-PCR analysis of the expression of ccl3, ccl4, ccl5, and ccl22 in skin of WT or S100A9Tg mice treated with TPA or acetone. Differences in expression of ccl3 and ccl4 were significant. Mean and SD are shown. *, P < 0.01. (C) Amount of CCL22 (left) and CCL3 (right) protein measured by ELISA in lysates of various organs from TPA-treated WT and S100A9Tg mice. Mean and SD from four experiments with three mice per group are shown. Each measurement was performed in duplicate and normalized for total protein. Cumulative results from three experiments are shown. (D) Amount of CCL4 protein measured by ELISA in lysates of various organs from TPA-treated WT and S100A9Tg mice. Mean and SD from four experiments with three mice per group are shown. Each measurement was performed in duplicate normalized for total protein. **, P < 0.01. (E) Amount of CCL4 determined by ELISA in supernatants of IMCs isolated from BM of WT or S100A9Tg mice treated with TPA for 4 wk. Cells were activated with LPS overnight. Measurements were performed twice in duplicates. Mean and SD are shown. (F) Expression of ccl4 in Gr-1+CD11b+ IMCs isolated from BM of naive mice treated for 24 h with the indicated cytokines. The range of most commonly used concentrations was tested. Concentrations shown: 250 ng/ml IFN-γ, 50 ng/ml TNF, 50 ng/ml IL-1β, and 100 ng/ml LPS. Ccl4 expression was measured by quantitative PCR and normalized to β-actin. Mean and SD are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001 from control (n = 3). (G) Expression of ccl4 in Gr-1+ and Gr-1− cells isolated from skin of WT or S100A9Tg mice treated for 5 wk with TPA (n = 3). Mean and SD are shown.

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