Isolation of LECs from uninflamed and CHS-inflamed mouse ears. (A) A CHS response toward oxazolone was induced in the ears of WT mice. Twenty-four hours after induction, CHS-challenged ears were markedly red and swollen. (B) IFN-γ and TNF-α protein levels were significantly increased in CHS-inflamed ears (n = 3 mice per group). **P < .01. ***P < .001. (C) Whole-mount immunofluorescence of inflamed and control ears detected large numbers of MHCII^high cells (red) around (middle panel) and within (right panel, orthogonal slice produced by confocal microscopy) LYVE-1-positive lymphatic vessels (green) in inflamed mouse ears. Scale bars represent 100 μm (left and middle) and 20 μm (right). (D) Ear tissues were digested enzymatically and stained for CD45, CD31, and podoplanin to differentiate between leukocytes (CD45⁺CD31⁻), BECs (CD45⁻CD31⁺podoplanin⁻), and LECs (CD45⁻CD31⁺podoplanin⁺). LECs were isolated from ear single-cell suspensions by FACS sorting. (E) Podoplanin was significantly down-regulated in inflamed LECs (INFL) compared with control LECs (CTR), as demonstrated by analysis of the median fluorescent intensity (MFI) of podoplanin expression found on cells in the LEC gate. *P < .05 (paired Student t test). (F) RNA was extracted from sorted LECs. cDNA was synthesized and subjected to one round of linear amplification. LEC preparations isolated from control ears or inflamed ears that were processed in parallel were treated as pairs. The induction of ICAM-1 and VCAM-1 in pairs of control and inflamed samples was analyzed by quantitative PCR.