Figure 4.
LECs activate plasminogen to plasmin, thereby generating CCL21-ΔC with enhanced chemotactic activity. (A and B) Quantification of (A) plasminogen and (B) plasmin activity in tissue protein extracts generated from CTR or CHS-inflamed ear skin. n = 6–7 mice per condition. (C) CTR experiment with CHS-inflamed ears documenting that the plasmin activity observed in C can be completely blocked in presence of the plasmin inhibitor C3. (D) Schematic depiction of the experimental hypothesis: Inflammation leads to enhanced extravasation of plasminogen. uPA bound to uPAR on CCL21-secreting LECs converts plasminogen to plasmin, thereby inducing CCL21 cleavage into CCL21-ΔC. (E–G)In vitro CCL21 cleavage experiment: (E) Schematic depiction of the experiment: immortalized LECs were incubated with recombinant CCL21 (100 nM) and plasminogen (20 nM) for 4 h or 24 h at 37°C in absence or presence of the plasmin inhibitor C3, mU1, or PIC. Supernatants were analyzed by western blot for CCL21. (F) Representative western blot of the cell culture supernatant at indicated time points and conditions and (G) quantification of the full-length CCL21 (gray) and CCL21-ΔC (white) relative band percentage. Pooled data from n = 4 independent experiments. Mean ± SEM, one-way ANOVA, and P values are relative to the “plg only” condition. (H and I) Cell culture supernatants generated as in E were evaluated in a 3D collagen migration assay. Recombinant human CCL21 and CCL21-ΔC were used as positive CTRs (H) Cell trajectory plots of migrating BMDCs’ migratory tracks in response to the stimuli applied on either side of the collagen channel. (I) Quantification of DC directionality, displacement, and velocity in response to the stimuli applied. Pooled data from n = 2 independent experiments with a total of n = 40–50 tracks analyzed per condition. Mean ± SEM, unpaired Student's t test for each comparison. (J–L) Analysis of the CCL21 cleavage activity of LECs isolated from uPA−/− mice or mice with defective uPA binding to uPAR (uPAmut) (J) Schematic illustration of the three genotypes investigated. (K and L) Representative western blot of the cell culture supernatants after (K) 4 h and (L) 24 h of incubation (top) and quantification of the full-length CCL21 (gray) and CCL21-ΔC (white) relative band percentage (bottom). Pooled data from n = 5 independent experiments. Mean ± SEM, one-way ANOVA, and Source data are available for this figure: SourceData F4. plg, plasminogen.

LECs activate plasminogen to plasmin, thereby generating CCL21-ΔC with enhanced chemotactic activity. (A and B) Quantification of (A) plasminogen and (B) plasmin activity in tissue protein extracts generated from CTR or CHS-inflamed ear skin. n = 6–7 mice per condition. (C) CTR experiment with CHS-inflamed ears documenting that the plasmin activity observed in C can be completely blocked in presence of the plasmin inhibitor C3. (D) Schematic depiction of the experimental hypothesis: Inflammation leads to enhanced extravasation of plasminogen. uPA bound to uPAR on CCL21-secreting LECs converts plasminogen to plasmin, thereby inducing CCL21 cleavage into CCL21-ΔC. (E–G)In vitro CCL21 cleavage experiment: (E) Schematic depiction of the experiment: immortalized LECs were incubated with recombinant CCL21 (100 nM) and plasminogen (20 nM) for 4 h or 24 h at 37°C in absence or presence of the plasmin inhibitor C3, mU1, or PIC. Supernatants were analyzed by western blot for CCL21. (F) Representative western blot of the cell culture supernatant at indicated time points and conditions and (G) quantification of the full-length CCL21 (gray) and CCL21-ΔC (white) relative band percentage. Pooled data from n = 4 independent experiments. Mean ± SEM, one-way ANOVA, and P values are relative to the “plg only” condition. (H and I) Cell culture supernatants generated as in E were evaluated in a 3D collagen migration assay. Recombinant human CCL21 and CCL21-ΔC were used as positive CTRs (H) Cell trajectory plots of migrating BMDCs’ migratory tracks in response to the stimuli applied on either side of the collagen channel. (I) Quantification of DC directionality, displacement, and velocity in response to the stimuli applied. Pooled data from n = 2 independent experiments with a total of n = 40–50 tracks analyzed per condition. Mean ± SEM, unpaired Student's t test for each comparison. (J–L) Analysis of the CCL21 cleavage activity of LECs isolated from uPA−/− mice or mice with defective uPA binding to uPAR (uPAmut) (J) Schematic illustration of the three genotypes investigated. (K and L) Representative western blot of the cell culture supernatants after (K) 4 h and (L) 24 h of incubation (top) and quantification of the full-length CCL21 (gray) and CCL21-ΔC (white) relative band percentage (bottom). Pooled data from n = 5 independent experiments. Mean ± SEM, one-way ANOVA, and Source data are available for this figure: SourceData F4. plg, plasminogen.

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