Journal: The Journal of Cell Biology

Article Title: uPA-mediated remodeling of CCL21 gradients regulates lymphatic migration of dendritic cells

doi: 10.1083/jcb.202412190

Figure Lengend Snippet: A soluble proteoform of CCL21 (CCL21-ΔC) with chemotactic activity is present in murine skin and increased in CHS-inflamed skin. (A) Representative western blot of CCL21 performed on steady-state (CTR) and CHS-inflamed (CHS) murine ear skin protein extracts. Recombinant CCL21 was loaded as a CTR. (B) Western blot analysis of recombinant human full-length CCL21 and CCL21-ΔC protein. One out of two experiments is shown. (C) Quantification of the full-length CCL21 (gray) and CCL21-ΔC (white) relative band percentages. Pooled data from n = 4 independent biological replicates. (D) ELISA-based quantification of total CCL21 in tissue protein extracts, performed with antibody clone AF457, which detects full-length CCL21 and CCL21-ΔC. Pooled data from n = 5 mice/condition. Statistics: unpaired Student’s t test. (E–J) Skin elution assay and analyses were performed on the supernatants. (E) Schematic depiction of the assay and representative western blot analysis. (F) Image-based quantification of the CCL21-ΔC band intensity from western blots as in E. A.U., arbitrary units, as produced by the western blot imager (G) ELISA-based quantification of total CCL21, performed with antibody clone AF457. Pooled data from n = 6–7 mice/condition, with one CHS-inflamed and a contralateral CTR ear, are shown in E and F. Data from the same mouse are connected by a line. The mean is shown in red, paired Student’s t test. (H–J) Transwell chemotaxis assays were performed on elution assay supernatants (see E) with 1:1 mixtures of LPS-matured labelled WT and CCR7 −/− DCs in presence/absence of a CCL21-blocking antibody. Flow cytometry–based quantification of the total numbers of transmigrated (H) WT DCs and (I) CCR7 −/− DCs, as well as of (J) the ratio of transmigrated WT to CCR7 −/− DCs. Data points from 3–7 experiments per condition are shown. Mixed effects statistical analysis. (K) Ratio of WT: CCR7 −/− DCs measured in seven paired experiments performed for CTR and CHS-inflamed condition. Statistical analysis: paired Student's t test. (L and M) Western blot analysis of protein extracts of (L) steady-state human skin (CTR) and (M) donor-matched steady-state (CTR) and inflamed (INF) human skin from a psoriasis patient. Data from one out of two experiments in L and one experiment in M are shown. Recombinant human full-length CCL21 was loaded as a CTR. Source data are available for this figure: .

Article Snippet: Murine recombinant CCL21 (#250-13; PeproTech) and recombinant murine plasmin (Molecular Innovations) at molar ratios of CCL21 to plasmin of 1:0.02–1:0.16 (starting with 100 nM CCL21 and 2 nM plasmin) were incubated in PBS or serum-free ProCHO medium (Lonza) for 4 h at 37°C.

Techniques: Activity Assay, Western Blot, Recombinant, Enzyme-linked Immunosorbent Assay, Produced, Chemotaxis Assay, Blocking Assay, Flow Cytometry

Journal: The Journal of Cell Biology

Article Title: uPA-mediated remodeling of CCL21 gradients regulates lymphatic migration of dendritic cells

doi: 10.1083/jcb.202412190

Figure Lengend Snippet: In vitro assays demonstrating CCL21 cleavage by plasmin. (A and B) Western blot analysis of (A) human and (B) murine CCL21 cleavage after incubation with recombinant plasmin with a fixed molar ratio of 1:0.08 for increasing times at 37°C, as indicated in the figure. (C) Dose titration of the plasmin inhibitor C3 to a fixed molar ratio of murine CCL21:plasmin (1:0.08) and incubation for up to 4 h, as indicated in the figure. Representative western blots of n = 2 (A) or n = 3 (B and C) experiments are shown. (D) Fluorometric plasmin activation assay, with indicated plasmin (12 μM) and inhibitor concentrations. The % increase from T 0 (0 min) is depicted. PIC: broad spectrum protease inhibitor. Representative results of n = 3 independent experiments are shown. Source data are available for this figure: .

Article Snippet: Murine recombinant CCL21 (#250-13; PeproTech) and recombinant murine plasmin (Molecular Innovations) at molar ratios of CCL21 to plasmin of 1:0.02–1:0.16 (starting with 100 nM CCL21 and 2 nM plasmin) were incubated in PBS or serum-free ProCHO medium (Lonza) for 4 h at 37°C.

Techniques: In Vitro, Western Blot, Incubation, Recombinant, Titration, Activation Assay, Protease Inhibitor

Journal: The Journal of Cell Biology

Article Title: uPA-mediated remodeling of CCL21 gradients regulates lymphatic migration of dendritic cells

doi: 10.1083/jcb.202412190

Figure Lengend Snippet: 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 (uPA mut ) (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: . plg, plasminogen.

Article Snippet: Murine recombinant CCL21 (#250-13; PeproTech) and recombinant murine plasmin (Molecular Innovations) at molar ratios of CCL21 to plasmin of 1:0.02–1:0.16 (starting with 100 nM CCL21 and 2 nM plasmin) were incubated in PBS or serum-free ProCHO medium (Lonza) for 4 h at 37°C.

Techniques: Activity Assay, Generated, In Vitro, Incubation, Recombinant, Western Blot, Cell Culture, Migration, Comparison, Isolation, Binding Assay

Journal: The Journal of Cell Biology

Article Title: uPA-mediated remodeling of CCL21 gradients regulates lymphatic migration of dendritic cells

doi: 10.1083/jcb.202412190

Figure Lengend Snippet: Video showing fluorescently labeled bone marrow–derived DCs (green) moving in 3D collagen toward recombinant human CCL21 (up) and CCL21+plg (down) provided in reservoirs on left and right, respectively, of the chamber. WT DCs display enhanced migration toward CCL21-ΔC provided on the left. Video specifications: 5-min intervals; 5 frames/s (1500-fold accelerated). The original length of the recording: 200 min. Video length: 8 s. plg, plasminogen.

Article Snippet: Murine recombinant CCL21 (#250-13; PeproTech) and recombinant murine plasmin (Molecular Innovations) at molar ratios of CCL21 to plasmin of 1:0.02–1:0.16 (starting with 100 nM CCL21 and 2 nM plasmin) were incubated in PBS or serum-free ProCHO medium (Lonza) for 4 h at 37°C.

Techniques: Labeling, Derivative Assay, Recombinant, Migration