Inflammation enhances the ROCK dependence of intralymphatic DC migration. (A) Gating scheme used for the analysis of ICAM-1 expression in LECs and BECs: CHS-inflamed and control ears of WT mice were enzymatically digested, stained for CD45, CD31, and podoplanin and analyzed by FACS for ICAM-1 expression in LECs (CD45⁻CD31⁺podoplanin⁺) and BECs (CD45⁻CD31⁺podoplanin⁻). (B) FACS analysis revealed an inflammation-induced up-regulation of ICAM-1 in LECs and BECs. ΔMFIs are shown, defined as the difference in the median fluorescent intensity between ICAM-1 expression and the corresponding isotope control staining. Representative data from 1 of at least 3 similar experiments are shown. (C-D) YFP⁺ BM-DCs were injected into the control or CHS-inflamed ear skin of VE-cadherin-Cre × RFP mice. (C) Quantification of the effect of Y27632-treatment (Y27) on the speed of interstitial DC migration in resting versus CHS-inflamed ear skin. (D) Quantification of the effect of Y27632-treatment on the speed of intralymphatic DC migration in resting versus CHS-inflamed ear skin (same dosing scheme as in Figure 4C). Inflamed dataset is pooled from 9 (baseline), 4 (PBS), and 5 (Y27) different experiments. (E) FITC painting experiments were performed in the control of CHS-inflamed ears of mice treated with Y27632 of with PBS vehicle control. Y27632 treatment reduced DC migration, as demonstrated by the quantification of total numbers of I-A/I-E⁺CD11c⁺FITC⁺ cells. Data from 1 of 3 similar experiments are shown (*P < .05; **P < .01; ***P < .001).