Up-regulation of chemokines in CHS-inflamed LECs. (A) The analysis of normalized array intensity levels revealed a strong up-regulation of chemokines in LECs isolated from inflamed skin. On the y-axis, the average signal intensities recorded in all replicates are shown. In the case of genes that were represented in the arrays by multiple probe sets, the average signal intensity of all probes is displayed. (B) The inflammation-induced up-regulation of selected chemokines was confirmed by quantitative PCR analysis performed on amplified cDNA derived from ex vivo isolated LECs. Data represent pooled values from 4 sample pairs. (C) The up-regulation of CXCL9 protein was analyzed by FACS. Histogram plots showing CXCL9 expression (intracellular staining) in LECs (gated on CD45⁻CD31⁺podoplanin⁺ cells) derived from CHS-inflamed or control ear single-cell suspensions. The number in the top right of each image represents the ΔMFI, defined as the difference in the median fluorescent intensity between the candidate and the corresponding isotope control staining. Black line represents CXCL9 staining; and gray, tinted line, isotype control staining. (D) Summary of the ΔMFIs of CXCL9 measured in all experiments. Data points (control [CTR] − inflamed [INFL]) from the same experiment are connected by a line. (E) The expression of CXCL1 and CCL2 (red) in CHS-inflamed ears was validated at the protein level by whole-mount immunofluorescence. Whole mounts were costained for LYVE-1 (green). White arrows indicate regions of chemokine expression and colocalization with lymphatic vessels. Scale bar represents 100 μm.