Blockade of uPA or plasmin activity alters the perilymphatic CCL21 gradient. (A) Representative images showing LYVE-1 and the immobilized perilymphatic CCL21 gradient in the steady-state ear skin of WT, uPAmut and uPA−/−, ACKR4−/−, and CCR7−/− mice. Scale bar: 50 μm. (B) Quantification of the CCL21 staining intensity as a function of the distance from the nearest LYVE-1+ LV. n = 4–6 mice per condition, two-way ANOVA (C and D) Quantification of the CCL21 staining intensity at (C) 0 μm or 30 μm from the LV. (E) Ratio of the CCL21 signal intensity measured at 60 vs. 0 μm from the LV. n = 4–6 mice per condition; one-way ANOVA. (F) ELISA-based quantification of total CCL21 in culture supernatants from ear skin of WT and uPAmut mice, performed with antibody clone AF457 which detects full-length CCL21 and CCL21-ΔC. Pooled data from n = 4 mice/condition. Statistics: unpaired Student's t test. (G–J) Impact of 24 h of combined treatment with the plasmin-selective inhibitor C3 and uPA-blocking antibody mU1 on the perilymphatic CCL21 gradient. (G) Representative images showing the CCL21 and LYVE-1 signal in the ear skin of mice. Scale bar: 50 μm. (H) Quantification of the CCL21 staining intensity as a function of the distance from the nearest LYVE-1+ LV. Two-way ANOVA, n = 3 mice. (I and J) Quantification of the CCL21 staining intensity at (I) 0 μm or (J) 30 μm from the LV. n = 3 mice per condition. Paired Student’s t test.