baf457  (r&d systems)

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: DCs were left to migrate toward the bottom well at 37°C for 4 h. Chemotactic stimuli in the lower well consisted of 100 ng/ml murine CCL21 (PeproTech), 100 ng/ml human CCL21 (PeproTech), 100 ng/ml human CCL21-ΔC (generated as described above), 100 ng/ml murine CXCL12 (PeproTech), 100 ng/ml murine CXCL12 and 20 nM murine plasminogen (Molecular Innovations), 100 ng/ml murine CXCL12 and 8 nM murine plasmin (Molecular Innovations), or AIM-V medium alone.

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: 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: DCs were left to migrate toward the bottom well at 37°C for 4 h. Chemotactic stimuli in the lower well consisted of 100 ng/ml murine CCL21 (PeproTech), 100 ng/ml human CCL21 (PeproTech), 100 ng/ml human CCL21-ΔC (generated as described above), 100 ng/ml murine CXCL12 (PeproTech), 100 ng/ml murine CXCL12 and 20 nM murine plasminogen (Molecular Innovations), 100 ng/ml murine CXCL12 and 8 nM murine plasmin (Molecular Innovations), or AIM-V medium alone.

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: DCs were left to migrate toward the bottom well at 37°C for 4 h. Chemotactic stimuli in the lower well consisted of 100 ng/ml murine CCL21 (PeproTech), 100 ng/ml human CCL21 (PeproTech), 100 ng/ml human CCL21-ΔC (generated as described above), 100 ng/ml murine CXCL12 (PeproTech), 100 ng/ml murine CXCL12 and 20 nM murine plasminogen (Molecular Innovations), 100 ng/ml murine CXCL12 and 8 nM murine plasmin (Molecular Innovations), or AIM-V medium alone.

Techniques: Labeling, Derivative Assay, Recombinant, Migration

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: Impact of plasmin and plasmin(ogen) on in vitro DC migration. In vitro experiments were performed with LPS-matured bone marrow–derived DCs and primary LN LECs. (A) DC displayed greater chemotaxis toward CCL21-ΔC as compared with full-length CCL21. (B) DCs were allowed to transmigrate for 4 h across primary LN LEC monolayers. DCs displayed a near-significant in transmigration (i.e., in two out of three experiments) toward CCL21-ΔC compared with CCL21 added to the lower well compartment. (C and D) The presence of plasminogen (Plg) or plasmin (Plm), which were added to the upper and lower wells of the Transwell plate, did not impact DC chemotaxis toward (C) CCL21-ΔC and (D) CXCL12. Each data point represents an independent experiment. (E and F) The presence of plasminogen or plasmin in the assay (upper and lower wells) did not impact DC transmigration across LEC monolayers toward (E) CCL21-ΔC or (F) CXCL12. Each data point represents an independent experiment. (G and H) Crawling assay: YFP-expressing DCs were added on top of LEC monolayers, and their migration was recorded by time-lapse microscopy. The presence of plasminogen or plasmin in the medium did not impact the (G) velocity and (H) chemotactic index of DC crawling. Of note: Inhibition of ROCK with Y27632 was performed as positive CTR . Statistics: two-way ANOVA using multiple comparisons, followed by a Tukey correction. (I) qPCR-results demonstrating that in vitro –cultured primary LN LECs no longer express CCL21 and also do not express CCL19. S18: housekeeping gene; gp38: podoplanin. Raw CT values are shown. n = 3 different biological replicates (isolations).

Article Snippet: DCs were left to migrate toward the bottom well at 37°C for 4 h. Chemotactic stimuli in the lower well consisted of 100 ng/ml murine CCL21 (PeproTech), 100 ng/ml human CCL21 (PeproTech), 100 ng/ml human CCL21-ΔC (generated as described above), 100 ng/ml murine CXCL12 (PeproTech), 100 ng/ml murine CXCL12 and 20 nM murine plasminogen (Molecular Innovations), 100 ng/ml murine CXCL12 and 8 nM murine plasmin (Molecular Innovations), or AIM-V medium alone.

Techniques: In Vitro, Migration, Derivative Assay, Chemotaxis Assay, Transmigration Assay, Expressing, Time-lapse Microscopy, Inhibition, Cell Culture

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: 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, uPA mut 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 uPA mut 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.

Article Snippet: DCs were left to migrate toward the bottom well at 37°C for 4 h. Chemotactic stimuli in the lower well consisted of 100 ng/ml murine CCL21 (PeproTech), 100 ng/ml human CCL21 (PeproTech), 100 ng/ml human CCL21-ΔC (generated as described above), 100 ng/ml murine CXCL12 (PeproTech), 100 ng/ml murine CXCL12 and 20 nM murine plasminogen (Molecular Innovations), 100 ng/ml murine CXCL12 and 8 nM murine plasmin (Molecular Innovations), or AIM-V medium alone.

Techniques: Activity Assay, Staining, Enzyme-linked Immunosorbent Assay, Blocking 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: Blockade of uPA results in reduced CCL21-ΔC levels in skin-dLNs. (A and B) Western blot–based comparison of CCL21 proteoforms in steady-state LNs and ear skin. (A) Representative western blots performed on tissue protein extracts and (B) and quantification of the full-length CCL21 (gray) and CCL21-ΔC (white) relative band percentages. Pooled data from n = 4 western blots from independent experiments. (C) Representative western blot of CCL21 performed on LN extracts, LN eluates, and serum. (D) ELISA-based quantification of full-length CCL21 and CCL21-ΔC present in LN extracts, LN eluates, or serum. Antibody clone MAB457 detects full-length CCL21 only, whereas clone AF457 detects both full-length CCL21 and CCL21-ΔC. Data from n = 3 mice are shown. n.d., not detected. (E) ELISA-based quantification of full-length CCL21 and CCL21-ΔC present in LN eluates from WT and uPA mut mice. Data from n = 4–5 mice are shown, Student’s t test. n.d., not detected. (F and G) Protein extracts were prepared from LNs of PBS-perfused WT and uPA mut mice and used for ELISA-based quantification of (F) plasminogen and (G) assessment of plasmin activity (colorimetric assay). Pooled data from n = 6 mice per group are shown as mean ± SEM. Student’s t test. (H and I) Analysis of CCL21 levels in LNs of WT and uPA mut mice. Freshly cut LN sections were immediately (i.e., without fixation/permeabilization) stained for B220 (B cell follicles), LYVE-1, and CCL21. Images were subjected to AI-based tissue segmentation for differentiating between the T cell zone and B cell follicles/SCS. (H) Representative images from the immunofluorescent staining performed on LNs of WT and uPA mut mice. Scale bar: 100 μm. (I) Quantification of CCL21 staining intensity observed in the T cell zone. Each dot represents data from a stained auricular LN of one mouse (average of 4–6 images per LN). Student’s t test. Source data are available for this figure: .

Article Snippet: DCs were left to migrate toward the bottom well at 37°C for 4 h. Chemotactic stimuli in the lower well consisted of 100 ng/ml murine CCL21 (PeproTech), 100 ng/ml human CCL21 (PeproTech), 100 ng/ml human CCL21-ΔC (generated as described above), 100 ng/ml murine CXCL12 (PeproTech), 100 ng/ml murine CXCL12 and 20 nM murine plasminogen (Molecular Innovations), 100 ng/ml murine CXCL12 and 8 nM murine plasmin (Molecular Innovations), or AIM-V medium alone.

Techniques: Western Blot, Comparison, Enzyme-linked Immunosorbent Assay, Activity Assay, Colorimetric Assay, Staining

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: Summary diagram. Summary of the main findings and the overall model. Top: Summary of events happening at the level of LECs: continuous low-level extravasation of plasminogen from blood vessels leads to uPA/uPAR-mediated activation of plasmin, which in turn cleaves immobilized CCL21 into soluble CCL21-ΔC (WT steady-state—left). When uPA-mediated activation of plasminogen is compromised (uPA mut ), less CCL21 gets cleaved, shifting the balance toward more immobilized CCL21 accumulating on/around LECs (uPA mut steady-state—middle). Under inflammatory conditions, with higher extravasation of plasminogen and higher expression of uPA and uPAR by LECs, more plasmin is activated, resulting in more CCL21 cleavage (WT inflammation—right). Bottom: The bottom part of the figure illustrates how these changes affect the balance between immobilized CCL21 and soluble CCL21-ΔC around afferent lymphatics and in the dLN. Additionally, the impact on distinct CCR7-dependent steps (1–3) in lymphatic migration of DCs are indicated. Question marks (?) indicate steps that were not specifically investigated in this study and thus represent speculations based on indirect findings and/or the literature.

Article Snippet: DCs were left to migrate toward the bottom well at 37°C for 4 h. Chemotactic stimuli in the lower well consisted of 100 ng/ml murine CCL21 (PeproTech), 100 ng/ml human CCL21 (PeproTech), 100 ng/ml human CCL21-ΔC (generated as described above), 100 ng/ml murine CXCL12 (PeproTech), 100 ng/ml murine CXCL12 and 20 nM murine plasminogen (Molecular Innovations), 100 ng/ml murine CXCL12 and 8 nM murine plasmin (Molecular Innovations), or AIM-V medium alone.

Techniques: Activation Assay, Expressing, Migration

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: CHS-induced dermal inflammation impairs DC entry into lymphatics in in vitro crawl-in assays but enhances their in vivo migration. (A) Current model of the different steps in DC migration from skin to dLNs and their documented dependence on CCL21 gradients. DCs approach dermal lymphatics migrating along a perilymphatic CCL21 gradient (1). Upon entry into lymphatic capillaries (i), DCs actively migrate in a semidirected manner, following the immobilized CCL21 gradient (2) deposited in the capillary lumen (ii). Once lymph flow picks up due to LV contractions in collectors, DCs are passively transported (iii) to the LN SCS. Egress from the SCS into the LN parenchyma (iv) occurs along another CCL21 gradient (3) (B and C) Crawl-in assay: Mice were sensitized with 2% oxazolone on the belly on day 0 and challenged on day 5 by applying 1% oxazolone to the skin of one ear. Crawl-in assays with both ears, i.e., the CTR and the CHS-inflamed ear, were performed 1 day later by adding fluorescently labelled LPS-matured WT and CCR7 −/− bone marrow–derived DCs (1:1 ratio) onto the dermal ear skin to allow DCs to migrate into lymphatics for 4 h. (B) Representative images of WT and CCR7 −/− bone marrow–derived DCs and the vasculature (stained for CD31) at the end of the experiment. Scale bar: 100 μm. (C) Quantification of the ratio of WT (left) and CCR7 −/− (right) bone marrow–derived DCs located inside vs. outside of lymphatics. n = 4 experiments performed with 1 mouse each. Paired Student’s t test. (D–H) In vivo DC migration assay: 1:1 mixture of LPS-matured and CCR7 −/− DCs, labelled in two fluorescent colors, were transferred into either CHS-inflamed or uninflamed CTR footpads. DC numbers in draining popliteal LNs were quantified by flow cytometry 16–18 h later. (D) Popliteal LN weight. (E) Popliteal LN cellularity. (F) Gating scheme used for the identification of transferred DCs. (G) Quantification of DC numbers in popliteal LNs. Pooled data from six experiments are shown (total n = 25 CTR and n = 25 CHS). (H) Ratio of migrated WT:CCR7 −/− DCs per experiment. Statistics: unpaired (D, E, and F) and paired (H—since in same animal) Student’s t test.

Article Snippet: In some cases, 1 μg/ml of CCL21 blocking antibody (polyclonal, AF457; R&D Systems) was added to the lower wells.

Techniques: In Vitro, In Vivo, Migration, Derivative Assay, Staining, 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: The immobilized perilymphatic CCL21 gradient is diminished during CHS-induced skin inflammation. (A–E) Analysis of the immobilized perilymphatic CCL21 gradient stained in fresh (unfixed) CTR and CHS-inflamed ear skin (24 h after challenge). (A) Representative images showing the CCL21 and LYVE-1 signal at 20× magnification (scale bar: 50 μm). (B) Based on the LYVE-1 signal, a mask was generated to analyze the CCL21 intensity in relation to distance from the nearest LV. Scale bar: 100 μm. (C–E) Quantification of the CCL21 staining intensity as a function of the distance from the nearest LYVE-1 + LV. CCL21 staining intensity was measured at (D) 0 μm or (E) 30 μm from the LV. n = 6 mice per condition (4–6 images analyzed per mouse). The dotted horizontal line in D and E indicates the level of background (isotype) staining. (F–H) Analysis of intracellular CCL21 deposits, revealed by staining in PFA-fixed CTR and CHS-inflamed ear skin. (F) Representative images. Scale bar: 50 μm. Quantification of G the intracellular CCL21 staining intensity and (H) the number of CCL21 deposits present within lymphatics. Pooled data from n = 4 mice/condition with 3–6 images/ear skin are shown. Data from the same experiment (i.e., same mouse) in G and H are connected by a line and analyzed by paired Student’s t test.

Article Snippet: In some cases, 1 μg/ml of CCL21 blocking antibody (polyclonal, AF457; R&D Systems) was added to the lower wells.

Techniques: Staining, Generated

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: In some cases, 1 μg/ml of CCL21 blocking antibody (polyclonal, AF457; R&D Systems) was added to the lower wells.

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: In some cases, 1 μg/ml of CCL21 blocking antibody (polyclonal, AF457; R&D Systems) was added to the lower wells.

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: In some cases, 1 μg/ml of CCL21 blocking antibody (polyclonal, AF457; R&D Systems) was added to the lower wells.

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: Expression of components of the plasmin activation pathway and CCL21 cleavage activity of cultured cells. (A and B) Flow cytometry–based analysis of uPA, uPAR, and plasminogen expression in conditionally immortalized LECs and primary LN LECs. (A) Representative histogram plots and corresponding (B) summary of the delta mean fluorescent intensity (ΔMFI; specific-isotype staining) values measured in 4–5 different experiments. (C) CCL21 cleavage assay performed in presence or absence of LECs and plasminogen (plg), revealing the dependence of CCL21 cleavage on both factors (i.e., LECs and plg). One representative out of two similar experiments is shown. (D and E) CCL21 cleavage assay performed with (D) bone marrow–derived DCs and (E) primary keratinocytes, revealing their ability to cleave CCL21 in presence of plg. One representative out of two similar experiments is shown in D and E. (F) qRT-PCR–based analysis of mRNA from LN LECs isolated from WT, uPA mut , and uPA −/− mice. (G) Absolute CT values and (H) relative expression levels. Data from four LN LEC isolations are shown. One-way ANOVA. (H and I) Impact of heparitinase treatment on the CCL21 gradient in uPA mut mice. (H) Representative images showing LYVE-1 and the immobilized perilymphatic CCL21 gradient in the steady-state ear skin of uPA mut mice upon in vitro treatment with heparitinase (HEP) or in untreated CTRs. Scale bar: 50 μm. (I) Quantification of the CCL21 staining intensity as a function of the distance from the nearest LYVE-1 + LV. n = 3 mice per condition, two-way ANOVA. Source data are available for this figure: .

Article Snippet: In some cases, 1 μg/ml of CCL21 blocking antibody (polyclonal, AF457; R&D Systems) was added to the lower wells.

Techniques: Expressing, Activation Assay, Activity Assay, Cell Culture, Flow Cytometry, Staining, Cleavage Assay, Derivative Assay, Quantitative RT-PCR, Isolation, In Vitro

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: In some cases, 1 μg/ml of CCL21 blocking antibody (polyclonal, AF457; R&D Systems) was added to the lower wells.

Techniques: Labeling, Derivative Assay, Recombinant, Migration

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: Impact of plasmin and plasmin(ogen) on in vitro DC migration. In vitro experiments were performed with LPS-matured bone marrow–derived DCs and primary LN LECs. (A) DC displayed greater chemotaxis toward CCL21-ΔC as compared with full-length CCL21. (B) DCs were allowed to transmigrate for 4 h across primary LN LEC monolayers. DCs displayed a near-significant in transmigration (i.e., in two out of three experiments) toward CCL21-ΔC compared with CCL21 added to the lower well compartment. (C and D) The presence of plasminogen (Plg) or plasmin (Plm), which were added to the upper and lower wells of the Transwell plate, did not impact DC chemotaxis toward (C) CCL21-ΔC and (D) CXCL12. Each data point represents an independent experiment. (E and F) The presence of plasminogen or plasmin in the assay (upper and lower wells) did not impact DC transmigration across LEC monolayers toward (E) CCL21-ΔC or (F) CXCL12. Each data point represents an independent experiment. (G and H) Crawling assay: YFP-expressing DCs were added on top of LEC monolayers, and their migration was recorded by time-lapse microscopy. The presence of plasminogen or plasmin in the medium did not impact the (G) velocity and (H) chemotactic index of DC crawling. Of note: Inhibition of ROCK with Y27632 was performed as positive CTR . Statistics: two-way ANOVA using multiple comparisons, followed by a Tukey correction. (I) qPCR-results demonstrating that in vitro –cultured primary LN LECs no longer express CCL21 and also do not express CCL19. S18: housekeeping gene; gp38: podoplanin. Raw CT values are shown. n = 3 different biological replicates (isolations).

Article Snippet: In some cases, 1 μg/ml of CCL21 blocking antibody (polyclonal, AF457; R&D Systems) was added to the lower wells.

Techniques: In Vitro, Migration, Derivative Assay, Chemotaxis Assay, Transmigration Assay, Expressing, Time-lapse Microscopy, Inhibition, Cell Culture

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: The immobilized perilymphatic CCL21 gradient is diminished during TPA-induced skin inflammation. The skin of one ear of each mouse was inflamed by topical application of the irritant TPA. Experiments with both ears, i.e., the uninflamed (CTR and the TPA-inflamed ear, were performed 1 day later. (A–D) Analysis of the extracellular, immobilized perilymphatic CCL21 gradient stained in fresh (unfixed) CTR and TPA-inflamed ear skin. (A) Representative images showing the CCL21 and LYVE-1 signal at 20× magnification (scale bar: 50 μm). (B) Quantification of the CCL21 staining intensity as a function of the distance from the nearest LYVE-1 + LV. CCL21 staining intensity was measured at (C) 0 μm or (D) 30 μm from the LV. n = 3 mice per condition (4–6 images analyzed per mouse). The dotted horizontal line in C and D indicates the level of background (isotype) staining. (E) Elution assay: CTR and TPA-inflamed ear skin were placed in medium overnight, and the amount of CCL21 protein eluted into the medium was determined by ELISA. (F and G) Quantification of (F) plasminogen and (G) plasmin activity in tissue protein extracts generated from CTR or TPA-inflamed ear skin. n = 6–7 mice per condition. All graphs: unpaired Student’s t test.

Article Snippet: In some cases, 1 μg/ml of CCL21 blocking antibody (polyclonal, AF457; R&D Systems) was added to the lower wells.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Generated

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: 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, uPA mut 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 uPA mut 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.

Article Snippet: In some cases, 1 μg/ml of CCL21 blocking antibody (polyclonal, AF457; R&D Systems) was added to the lower wells.

Techniques: Activity Assay, Staining, Enzyme-linked Immunosorbent Assay, Blocking 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: Blockade of uPA results in reduced CCL21-ΔC levels in skin-dLNs. (A and B) Western blot–based comparison of CCL21 proteoforms in steady-state LNs and ear skin. (A) Representative western blots performed on tissue protein extracts and (B) and quantification of the full-length CCL21 (gray) and CCL21-ΔC (white) relative band percentages. Pooled data from n = 4 western blots from independent experiments. (C) Representative western blot of CCL21 performed on LN extracts, LN eluates, and serum. (D) ELISA-based quantification of full-length CCL21 and CCL21-ΔC present in LN extracts, LN eluates, or serum. Antibody clone MAB457 detects full-length CCL21 only, whereas clone AF457 detects both full-length CCL21 and CCL21-ΔC. Data from n = 3 mice are shown. n.d., not detected. (E) ELISA-based quantification of full-length CCL21 and CCL21-ΔC present in LN eluates from WT and uPA mut mice. Data from n = 4–5 mice are shown, Student’s t test. n.d., not detected. (F and G) Protein extracts were prepared from LNs of PBS-perfused WT and uPA mut mice and used for ELISA-based quantification of (F) plasminogen and (G) assessment of plasmin activity (colorimetric assay). Pooled data from n = 6 mice per group are shown as mean ± SEM. Student’s t test. (H and I) Analysis of CCL21 levels in LNs of WT and uPA mut mice. Freshly cut LN sections were immediately (i.e., without fixation/permeabilization) stained for B220 (B cell follicles), LYVE-1, and CCL21. Images were subjected to AI-based tissue segmentation for differentiating between the T cell zone and B cell follicles/SCS. (H) Representative images from the immunofluorescent staining performed on LNs of WT and uPA mut mice. Scale bar: 100 μm. (I) Quantification of CCL21 staining intensity observed in the T cell zone. Each dot represents data from a stained auricular LN of one mouse (average of 4–6 images per LN). Student’s t test. Source data are available for this figure: .

Article Snippet: In some cases, 1 μg/ml of CCL21 blocking antibody (polyclonal, AF457; R&D Systems) was added to the lower wells.

Techniques: Western Blot, Comparison, Enzyme-linked Immunosorbent Assay, Activity Assay, Colorimetric Assay, Staining

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: Supplemental data to FITC painting experiments and analysis of LNs draining CHS-inflamed skin. (A) Schematic depiction of the experiment: FITC was applied to the ear skin in WT and uPA mut mice, and ear skin or ear-draining auricular LNs were collected for analysis after 24 h. (B and C) Analysis of dermal DC numbers. Ear skin was enzymatically digested, and single-cell suspensions were generated for flow cytometry–based analysis. (D and E) Gatings used to identify migratory DCs (CD11c + MHCII hi ), and, amongst those, FITC + DCs in single-cell suspensions generated from (D) enzymatically digested LNs and (E) undigested LNs. (F–I) Analysis of immobilized CCL21 and plasmin(ogen) in CHS-dLNs. A CHS response was induced in the ear skin of WT mice, and ear-draining auricular LNs were collected 24 h later. (F and G) Analysis of CCL21 levels in LNs draining CTR or CHS-inflamed ear skin. Freshly cut LN sections were immediately (i.e., without fixation/permeabilization) stained for B220 (B cell follicles), LYVE-1, and CCL21. (F) Representative images of the immunofluorescent staining and of the AI-based tissue segmentation used for differentiating between the T cell zone and B cell follicles/SCS. Scale bar: 100 μm. (G) Quantification of CCL21 staining intensity of observed in the T cell zone. Each dot represents data from a stained auricular LN of one mouse (average of 4–6 images per LN). Student’s t test. (H) ELISA-based quantification of (H) plasminogen and (I) plasmin activity in LN protein extracts, generated after perfusing the mice with PBS. Pooled data from n = 6 mice per group are shown in H and I, Student’s t test. (J) Representative gating strategy used for the quantification of FITC + CD11c + cells in LN sections from WT and uPA mut FITC-painted auricular LNs in . CD11c + cells were identified based on CD11c-AF647 positivity. From the CD11c + population, FITC + cells were identified and subsequently quantified. Marker-negative cells (red color) served as negative control to check background staining and to set the fluorescence thresholds. Please note that the data points of the WT CTR group in C are identical to those shown in , as these extracts were prepared and measured simultaneously.

Article Snippet: In some cases, 1 μg/ml of CCL21 blocking antibody (polyclonal, AF457; R&D Systems) was added to the lower wells.

Techniques: Generated, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Marker, Negative Control, Fluorescence

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: Summary diagram. Summary of the main findings and the overall model. Top: Summary of events happening at the level of LECs: continuous low-level extravasation of plasminogen from blood vessels leads to uPA/uPAR-mediated activation of plasmin, which in turn cleaves immobilized CCL21 into soluble CCL21-ΔC (WT steady-state—left). When uPA-mediated activation of plasminogen is compromised (uPA mut ), less CCL21 gets cleaved, shifting the balance toward more immobilized CCL21 accumulating on/around LECs (uPA mut steady-state—middle). Under inflammatory conditions, with higher extravasation of plasminogen and higher expression of uPA and uPAR by LECs, more plasmin is activated, resulting in more CCL21 cleavage (WT inflammation—right). Bottom: The bottom part of the figure illustrates how these changes affect the balance between immobilized CCL21 and soluble CCL21-ΔC around afferent lymphatics and in the dLN. Additionally, the impact on distinct CCR7-dependent steps (1–3) in lymphatic migration of DCs are indicated. Question marks (?) indicate steps that were not specifically investigated in this study and thus represent speculations based on indirect findings and/or the literature.

Article Snippet: In some cases, 1 μg/ml of CCL21 blocking antibody (polyclonal, AF457; R&D Systems) was added to the lower wells.

Techniques: Activation Assay, Expressing, Migration

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: CHS-induced dermal inflammation impairs DC entry into lymphatics in in vitro crawl-in assays but enhances their in vivo migration. (A) Current model of the different steps in DC migration from skin to dLNs and their documented dependence on CCL21 gradients. DCs approach dermal lymphatics migrating along a perilymphatic CCL21 gradient (1). Upon entry into lymphatic capillaries (i), DCs actively migrate in a semidirected manner, following the immobilized CCL21 gradient (2) deposited in the capillary lumen (ii). Once lymph flow picks up due to LV contractions in collectors, DCs are passively transported (iii) to the LN SCS. Egress from the SCS into the LN parenchyma (iv) occurs along another CCL21 gradient (3) (B and C) Crawl-in assay: Mice were sensitized with 2% oxazolone on the belly on day 0 and challenged on day 5 by applying 1% oxazolone to the skin of one ear. Crawl-in assays with both ears, i.e., the CTR and the CHS-inflamed ear, were performed 1 day later by adding fluorescently labelled LPS-matured WT and CCR7 −/− bone marrow–derived DCs (1:1 ratio) onto the dermal ear skin to allow DCs to migrate into lymphatics for 4 h. (B) Representative images of WT and CCR7 −/− bone marrow–derived DCs and the vasculature (stained for CD31) at the end of the experiment. Scale bar: 100 μm. (C) Quantification of the ratio of WT (left) and CCR7 −/− (right) bone marrow–derived DCs located inside vs. outside of lymphatics. n = 4 experiments performed with 1 mouse each. Paired Student’s t test. (D–H) In vivo DC migration assay: 1:1 mixture of LPS-matured and CCR7 −/− DCs, labelled in two fluorescent colors, were transferred into either CHS-inflamed or uninflamed CTR footpads. DC numbers in draining popliteal LNs were quantified by flow cytometry 16–18 h later. (D) Popliteal LN weight. (E) Popliteal LN cellularity. (F) Gating scheme used for the identification of transferred DCs. (G) Quantification of DC numbers in popliteal LNs. Pooled data from six experiments are shown (total n = 25 CTR and n = 25 CHS). (H) Ratio of migrated WT:CCR7 −/− DCs per experiment. Statistics: unpaired (D, E, and F) and paired (H—since in same animal) Student’s t test.

Article Snippet: Inguinal LNs of adult (6–10 wk) WT and uPA mut mice were embedded in OCT. Unfixed, freshly cut 10-μm sections were blocked with 5% donkey serum (D9663; Sigma-Aldrich) in PBS and stained immediately with goat anti-mouse CCL21 (AF457; R&D Systems, 13 μg/ml), rabbit anti-mouse LYVE-1 (polyclonal, AngioBio), and rat anti-mouse B220/CD45R (BD Pharmingen) in blocking solution for 1 h at RT, as previously described ( ).

Techniques: In Vitro, In Vivo, Migration, Derivative Assay, Staining, 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: The immobilized perilymphatic CCL21 gradient is diminished during CHS-induced skin inflammation. (A–E) Analysis of the immobilized perilymphatic CCL21 gradient stained in fresh (unfixed) CTR and CHS-inflamed ear skin (24 h after challenge). (A) Representative images showing the CCL21 and LYVE-1 signal at 20× magnification (scale bar: 50 μm). (B) Based on the LYVE-1 signal, a mask was generated to analyze the CCL21 intensity in relation to distance from the nearest LV. Scale bar: 100 μm. (C–E) Quantification of the CCL21 staining intensity as a function of the distance from the nearest LYVE-1 + LV. CCL21 staining intensity was measured at (D) 0 μm or (E) 30 μm from the LV. n = 6 mice per condition (4–6 images analyzed per mouse). The dotted horizontal line in D and E indicates the level of background (isotype) staining. (F–H) Analysis of intracellular CCL21 deposits, revealed by staining in PFA-fixed CTR and CHS-inflamed ear skin. (F) Representative images. Scale bar: 50 μm. Quantification of G the intracellular CCL21 staining intensity and (H) the number of CCL21 deposits present within lymphatics. Pooled data from n = 4 mice/condition with 3–6 images/ear skin are shown. Data from the same experiment (i.e., same mouse) in G and H are connected by a line and analyzed by paired Student’s t test.

Article Snippet: Inguinal LNs of adult (6–10 wk) WT and uPA mut mice were embedded in OCT. Unfixed, freshly cut 10-μm sections were blocked with 5% donkey serum (D9663; Sigma-Aldrich) in PBS and stained immediately with goat anti-mouse CCL21 (AF457; R&D Systems, 13 μg/ml), rabbit anti-mouse LYVE-1 (polyclonal, AngioBio), and rat anti-mouse B220/CD45R (BD Pharmingen) in blocking solution for 1 h at RT, as previously described ( ).

Techniques: Staining, Generated

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: Inguinal LNs of adult (6–10 wk) WT and uPA mut mice were embedded in OCT. Unfixed, freshly cut 10-μm sections were blocked with 5% donkey serum (D9663; Sigma-Aldrich) in PBS and stained immediately with goat anti-mouse CCL21 (AF457; R&D Systems, 13 μg/ml), rabbit anti-mouse LYVE-1 (polyclonal, AngioBio), and rat anti-mouse B220/CD45R (BD Pharmingen) in blocking solution for 1 h at RT, as previously described ( ).

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: Inguinal LNs of adult (6–10 wk) WT and uPA mut mice were embedded in OCT. Unfixed, freshly cut 10-μm sections were blocked with 5% donkey serum (D9663; Sigma-Aldrich) in PBS and stained immediately with goat anti-mouse CCL21 (AF457; R&D Systems, 13 μg/ml), rabbit anti-mouse LYVE-1 (polyclonal, AngioBio), and rat anti-mouse B220/CD45R (BD Pharmingen) in blocking solution for 1 h at RT, as previously described ( ).

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: Inguinal LNs of adult (6–10 wk) WT and uPA mut mice were embedded in OCT. Unfixed, freshly cut 10-μm sections were blocked with 5% donkey serum (D9663; Sigma-Aldrich) in PBS and stained immediately with goat anti-mouse CCL21 (AF457; R&D Systems, 13 μg/ml), rabbit anti-mouse LYVE-1 (polyclonal, AngioBio), and rat anti-mouse B220/CD45R (BD Pharmingen) in blocking solution for 1 h at RT, as previously described ( ).

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: Expression of components of the plasmin activation pathway and CCL21 cleavage activity of cultured cells. (A and B) Flow cytometry–based analysis of uPA, uPAR, and plasminogen expression in conditionally immortalized LECs and primary LN LECs. (A) Representative histogram plots and corresponding (B) summary of the delta mean fluorescent intensity (ΔMFI; specific-isotype staining) values measured in 4–5 different experiments. (C) CCL21 cleavage assay performed in presence or absence of LECs and plasminogen (plg), revealing the dependence of CCL21 cleavage on both factors (i.e., LECs and plg). One representative out of two similar experiments is shown. (D and E) CCL21 cleavage assay performed with (D) bone marrow–derived DCs and (E) primary keratinocytes, revealing their ability to cleave CCL21 in presence of plg. One representative out of two similar experiments is shown in D and E. (F) qRT-PCR–based analysis of mRNA from LN LECs isolated from WT, uPA mut , and uPA −/− mice. (G) Absolute CT values and (H) relative expression levels. Data from four LN LEC isolations are shown. One-way ANOVA. (H and I) Impact of heparitinase treatment on the CCL21 gradient in uPA mut mice. (H) Representative images showing LYVE-1 and the immobilized perilymphatic CCL21 gradient in the steady-state ear skin of uPA mut mice upon in vitro treatment with heparitinase (HEP) or in untreated CTRs. Scale bar: 50 μm. (I) Quantification of the CCL21 staining intensity as a function of the distance from the nearest LYVE-1 + LV. n = 3 mice per condition, two-way ANOVA. Source data are available for this figure: .

Article Snippet: Inguinal LNs of adult (6–10 wk) WT and uPA mut mice were embedded in OCT. Unfixed, freshly cut 10-μm sections were blocked with 5% donkey serum (D9663; Sigma-Aldrich) in PBS and stained immediately with goat anti-mouse CCL21 (AF457; R&D Systems, 13 μg/ml), rabbit anti-mouse LYVE-1 (polyclonal, AngioBio), and rat anti-mouse B220/CD45R (BD Pharmingen) in blocking solution for 1 h at RT, as previously described ( ).

Techniques: Expressing, Activation Assay, Activity Assay, Cell Culture, Flow Cytometry, Staining, Cleavage Assay, Derivative Assay, Quantitative RT-PCR, Isolation, In Vitro

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: Inguinal LNs of adult (6–10 wk) WT and uPA mut mice were embedded in OCT. Unfixed, freshly cut 10-μm sections were blocked with 5% donkey serum (D9663; Sigma-Aldrich) in PBS and stained immediately with goat anti-mouse CCL21 (AF457; R&D Systems, 13 μg/ml), rabbit anti-mouse LYVE-1 (polyclonal, AngioBio), and rat anti-mouse B220/CD45R (BD Pharmingen) in blocking solution for 1 h at RT, as previously described ( ).

Techniques: Labeling, Derivative Assay, Recombinant, Migration

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: Impact of plasmin and plasmin(ogen) on in vitro DC migration. In vitro experiments were performed with LPS-matured bone marrow–derived DCs and primary LN LECs. (A) DC displayed greater chemotaxis toward CCL21-ΔC as compared with full-length CCL21. (B) DCs were allowed to transmigrate for 4 h across primary LN LEC monolayers. DCs displayed a near-significant in transmigration (i.e., in two out of three experiments) toward CCL21-ΔC compared with CCL21 added to the lower well compartment. (C and D) The presence of plasminogen (Plg) or plasmin (Plm), which were added to the upper and lower wells of the Transwell plate, did not impact DC chemotaxis toward (C) CCL21-ΔC and (D) CXCL12. Each data point represents an independent experiment. (E and F) The presence of plasminogen or plasmin in the assay (upper and lower wells) did not impact DC transmigration across LEC monolayers toward (E) CCL21-ΔC or (F) CXCL12. Each data point represents an independent experiment. (G and H) Crawling assay: YFP-expressing DCs were added on top of LEC monolayers, and their migration was recorded by time-lapse microscopy. The presence of plasminogen or plasmin in the medium did not impact the (G) velocity and (H) chemotactic index of DC crawling. Of note: Inhibition of ROCK with Y27632 was performed as positive CTR . Statistics: two-way ANOVA using multiple comparisons, followed by a Tukey correction. (I) qPCR-results demonstrating that in vitro –cultured primary LN LECs no longer express CCL21 and also do not express CCL19. S18: housekeeping gene; gp38: podoplanin. Raw CT values are shown. n = 3 different biological replicates (isolations).

Article Snippet: Inguinal LNs of adult (6–10 wk) WT and uPA mut mice were embedded in OCT. Unfixed, freshly cut 10-μm sections were blocked with 5% donkey serum (D9663; Sigma-Aldrich) in PBS and stained immediately with goat anti-mouse CCL21 (AF457; R&D Systems, 13 μg/ml), rabbit anti-mouse LYVE-1 (polyclonal, AngioBio), and rat anti-mouse B220/CD45R (BD Pharmingen) in blocking solution for 1 h at RT, as previously described ( ).

Techniques: In Vitro, Migration, Derivative Assay, Chemotaxis Assay, Transmigration Assay, Expressing, Time-lapse Microscopy, Inhibition, Cell Culture

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: The immobilized perilymphatic CCL21 gradient is diminished during TPA-induced skin inflammation. The skin of one ear of each mouse was inflamed by topical application of the irritant TPA. Experiments with both ears, i.e., the uninflamed (CTR and the TPA-inflamed ear, were performed 1 day later. (A–D) Analysis of the extracellular, immobilized perilymphatic CCL21 gradient stained in fresh (unfixed) CTR and TPA-inflamed ear skin. (A) Representative images showing the CCL21 and LYVE-1 signal at 20× magnification (scale bar: 50 μm). (B) Quantification of the CCL21 staining intensity as a function of the distance from the nearest LYVE-1 + LV. CCL21 staining intensity was measured at (C) 0 μm or (D) 30 μm from the LV. n = 3 mice per condition (4–6 images analyzed per mouse). The dotted horizontal line in C and D indicates the level of background (isotype) staining. (E) Elution assay: CTR and TPA-inflamed ear skin were placed in medium overnight, and the amount of CCL21 protein eluted into the medium was determined by ELISA. (F and G) Quantification of (F) plasminogen and (G) plasmin activity in tissue protein extracts generated from CTR or TPA-inflamed ear skin. n = 6–7 mice per condition. All graphs: unpaired Student’s t test.

Article Snippet: Inguinal LNs of adult (6–10 wk) WT and uPA mut mice were embedded in OCT. Unfixed, freshly cut 10-μm sections were blocked with 5% donkey serum (D9663; Sigma-Aldrich) in PBS and stained immediately with goat anti-mouse CCL21 (AF457; R&D Systems, 13 μg/ml), rabbit anti-mouse LYVE-1 (polyclonal, AngioBio), and rat anti-mouse B220/CD45R (BD Pharmingen) in blocking solution for 1 h at RT, as previously described ( ).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Generated

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: 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, uPA mut 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 uPA mut 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.

Article Snippet: Inguinal LNs of adult (6–10 wk) WT and uPA mut mice were embedded in OCT. Unfixed, freshly cut 10-μm sections were blocked with 5% donkey serum (D9663; Sigma-Aldrich) in PBS and stained immediately with goat anti-mouse CCL21 (AF457; R&D Systems, 13 μg/ml), rabbit anti-mouse LYVE-1 (polyclonal, AngioBio), and rat anti-mouse B220/CD45R (BD Pharmingen) in blocking solution for 1 h at RT, as previously described ( ).

Techniques: Activity Assay, Staining, Enzyme-linked Immunosorbent Assay, Blocking 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: Blockade of uPA results in reduced CCL21-ΔC levels in skin-dLNs. (A and B) Western blot–based comparison of CCL21 proteoforms in steady-state LNs and ear skin. (A) Representative western blots performed on tissue protein extracts and (B) and quantification of the full-length CCL21 (gray) and CCL21-ΔC (white) relative band percentages. Pooled data from n = 4 western blots from independent experiments. (C) Representative western blot of CCL21 performed on LN extracts, LN eluates, and serum. (D) ELISA-based quantification of full-length CCL21 and CCL21-ΔC present in LN extracts, LN eluates, or serum. Antibody clone MAB457 detects full-length CCL21 only, whereas clone AF457 detects both full-length CCL21 and CCL21-ΔC. Data from n = 3 mice are shown. n.d., not detected. (E) ELISA-based quantification of full-length CCL21 and CCL21-ΔC present in LN eluates from WT and uPA mut mice. Data from n = 4–5 mice are shown, Student’s t test. n.d., not detected. (F and G) Protein extracts were prepared from LNs of PBS-perfused WT and uPA mut mice and used for ELISA-based quantification of (F) plasminogen and (G) assessment of plasmin activity (colorimetric assay). Pooled data from n = 6 mice per group are shown as mean ± SEM. Student’s t test. (H and I) Analysis of CCL21 levels in LNs of WT and uPA mut mice. Freshly cut LN sections were immediately (i.e., without fixation/permeabilization) stained for B220 (B cell follicles), LYVE-1, and CCL21. Images were subjected to AI-based tissue segmentation for differentiating between the T cell zone and B cell follicles/SCS. (H) Representative images from the immunofluorescent staining performed on LNs of WT and uPA mut mice. Scale bar: 100 μm. (I) Quantification of CCL21 staining intensity observed in the T cell zone. Each dot represents data from a stained auricular LN of one mouse (average of 4–6 images per LN). Student’s t test. Source data are available for this figure: .

Article Snippet: Inguinal LNs of adult (6–10 wk) WT and uPA mut mice were embedded in OCT. Unfixed, freshly cut 10-μm sections were blocked with 5% donkey serum (D9663; Sigma-Aldrich) in PBS and stained immediately with goat anti-mouse CCL21 (AF457; R&D Systems, 13 μg/ml), rabbit anti-mouse LYVE-1 (polyclonal, AngioBio), and rat anti-mouse B220/CD45R (BD Pharmingen) in blocking solution for 1 h at RT, as previously described ( ).

Techniques: Western Blot, Comparison, Enzyme-linked Immunosorbent Assay, Activity Assay, Colorimetric Assay, Staining

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: Supplemental data to FITC painting experiments and analysis of LNs draining CHS-inflamed skin. (A) Schematic depiction of the experiment: FITC was applied to the ear skin in WT and uPA mut mice, and ear skin or ear-draining auricular LNs were collected for analysis after 24 h. (B and C) Analysis of dermal DC numbers. Ear skin was enzymatically digested, and single-cell suspensions were generated for flow cytometry–based analysis. (D and E) Gatings used to identify migratory DCs (CD11c + MHCII hi ), and, amongst those, FITC + DCs in single-cell suspensions generated from (D) enzymatically digested LNs and (E) undigested LNs. (F–I) Analysis of immobilized CCL21 and plasmin(ogen) in CHS-dLNs. A CHS response was induced in the ear skin of WT mice, and ear-draining auricular LNs were collected 24 h later. (F and G) Analysis of CCL21 levels in LNs draining CTR or CHS-inflamed ear skin. Freshly cut LN sections were immediately (i.e., without fixation/permeabilization) stained for B220 (B cell follicles), LYVE-1, and CCL21. (F) Representative images of the immunofluorescent staining and of the AI-based tissue segmentation used for differentiating between the T cell zone and B cell follicles/SCS. Scale bar: 100 μm. (G) Quantification of CCL21 staining intensity of observed in the T cell zone. Each dot represents data from a stained auricular LN of one mouse (average of 4–6 images per LN). Student’s t test. (H) ELISA-based quantification of (H) plasminogen and (I) plasmin activity in LN protein extracts, generated after perfusing the mice with PBS. Pooled data from n = 6 mice per group are shown in H and I, Student’s t test. (J) Representative gating strategy used for the quantification of FITC + CD11c + cells in LN sections from WT and uPA mut FITC-painted auricular LNs in . CD11c + cells were identified based on CD11c-AF647 positivity. From the CD11c + population, FITC + cells were identified and subsequently quantified. Marker-negative cells (red color) served as negative control to check background staining and to set the fluorescence thresholds. Please note that the data points of the WT CTR group in C are identical to those shown in , as these extracts were prepared and measured simultaneously.

Article Snippet: Inguinal LNs of adult (6–10 wk) WT and uPA mut mice were embedded in OCT. Unfixed, freshly cut 10-μm sections were blocked with 5% donkey serum (D9663; Sigma-Aldrich) in PBS and stained immediately with goat anti-mouse CCL21 (AF457; R&D Systems, 13 μg/ml), rabbit anti-mouse LYVE-1 (polyclonal, AngioBio), and rat anti-mouse B220/CD45R (BD Pharmingen) in blocking solution for 1 h at RT, as previously described ( ).

Techniques: Generated, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Marker, Negative Control, Fluorescence

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: Summary diagram. Summary of the main findings and the overall model. Top: Summary of events happening at the level of LECs: continuous low-level extravasation of plasminogen from blood vessels leads to uPA/uPAR-mediated activation of plasmin, which in turn cleaves immobilized CCL21 into soluble CCL21-ΔC (WT steady-state—left). When uPA-mediated activation of plasminogen is compromised (uPA mut ), less CCL21 gets cleaved, shifting the balance toward more immobilized CCL21 accumulating on/around LECs (uPA mut steady-state—middle). Under inflammatory conditions, with higher extravasation of plasminogen and higher expression of uPA and uPAR by LECs, more plasmin is activated, resulting in more CCL21 cleavage (WT inflammation—right). Bottom: The bottom part of the figure illustrates how these changes affect the balance between immobilized CCL21 and soluble CCL21-ΔC around afferent lymphatics and in the dLN. Additionally, the impact on distinct CCR7-dependent steps (1–3) in lymphatic migration of DCs are indicated. Question marks (?) indicate steps that were not specifically investigated in this study and thus represent speculations based on indirect findings and/or the literature.

Article Snippet: Inguinal LNs of adult (6–10 wk) WT and uPA mut mice were embedded in OCT. Unfixed, freshly cut 10-μm sections were blocked with 5% donkey serum (D9663; Sigma-Aldrich) in PBS and stained immediately with goat anti-mouse CCL21 (AF457; R&D Systems, 13 μg/ml), rabbit anti-mouse LYVE-1 (polyclonal, AngioBio), and rat anti-mouse B220/CD45R (BD Pharmingen) in blocking solution for 1 h at RT, as previously described ( ).

Techniques: Activation Assay, Expressing, Migration

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: CHS-induced dermal inflammation impairs DC entry into lymphatics in in vitro crawl-in assays but enhances their in vivo migration. (A) Current model of the different steps in DC migration from skin to dLNs and their documented dependence on CCL21 gradients. DCs approach dermal lymphatics migrating along a perilymphatic CCL21 gradient (1). Upon entry into lymphatic capillaries (i), DCs actively migrate in a semidirected manner, following the immobilized CCL21 gradient (2) deposited in the capillary lumen (ii). Once lymph flow picks up due to LV contractions in collectors, DCs are passively transported (iii) to the LN SCS. Egress from the SCS into the LN parenchyma (iv) occurs along another CCL21 gradient (3) (B and C) Crawl-in assay: Mice were sensitized with 2% oxazolone on the belly on day 0 and challenged on day 5 by applying 1% oxazolone to the skin of one ear. Crawl-in assays with both ears, i.e., the CTR and the CHS-inflamed ear, were performed 1 day later by adding fluorescently labelled LPS-matured WT and CCR7 −/− bone marrow–derived DCs (1:1 ratio) onto the dermal ear skin to allow DCs to migrate into lymphatics for 4 h. (B) Representative images of WT and CCR7 −/− bone marrow–derived DCs and the vasculature (stained for CD31) at the end of the experiment. Scale bar: 100 μm. (C) Quantification of the ratio of WT (left) and CCR7 −/− (right) bone marrow–derived DCs located inside vs. outside of lymphatics. n = 4 experiments performed with 1 mouse each. Paired Student’s t test. (D–H) In vivo DC migration assay: 1:1 mixture of LPS-matured and CCR7 −/− DCs, labelled in two fluorescent colors, were transferred into either CHS-inflamed or uninflamed CTR footpads. DC numbers in draining popliteal LNs were quantified by flow cytometry 16–18 h later. (D) Popliteal LN weight. (E) Popliteal LN cellularity. (F) Gating scheme used for the identification of transferred DCs. (G) Quantification of DC numbers in popliteal LNs. Pooled data from six experiments are shown (total n = 25 CTR and n = 25 CHS). (H) Ratio of migrated WT:CCR7 −/− DCs per experiment. Statistics: unpaired (D, E, and F) and paired (H—since in same animal) Student’s t test.

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, In Vivo, Migration, Derivative Assay, Staining, 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: The immobilized perilymphatic CCL21 gradient is diminished during CHS-induced skin inflammation. (A–E) Analysis of the immobilized perilymphatic CCL21 gradient stained in fresh (unfixed) CTR and CHS-inflamed ear skin (24 h after challenge). (A) Representative images showing the CCL21 and LYVE-1 signal at 20× magnification (scale bar: 50 μm). (B) Based on the LYVE-1 signal, a mask was generated to analyze the CCL21 intensity in relation to distance from the nearest LV. Scale bar: 100 μm. (C–E) Quantification of the CCL21 staining intensity as a function of the distance from the nearest LYVE-1 + LV. CCL21 staining intensity was measured at (D) 0 μm or (E) 30 μm from the LV. n = 6 mice per condition (4–6 images analyzed per mouse). The dotted horizontal line in D and E indicates the level of background (isotype) staining. (F–H) Analysis of intracellular CCL21 deposits, revealed by staining in PFA-fixed CTR and CHS-inflamed ear skin. (F) Representative images. Scale bar: 50 μm. Quantification of G the intracellular CCL21 staining intensity and (H) the number of CCL21 deposits present within lymphatics. Pooled data from n = 4 mice/condition with 3–6 images/ear skin are shown. Data from the same experiment (i.e., same mouse) in G and H are connected by a line and analyzed by paired Student’s t test.

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: Staining, Generated

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: Expression of components of the plasmin activation pathway and CCL21 cleavage activity of cultured cells. (A and B) Flow cytometry–based analysis of uPA, uPAR, and plasminogen expression in conditionally immortalized LECs and primary LN LECs. (A) Representative histogram plots and corresponding (B) summary of the delta mean fluorescent intensity (ΔMFI; specific-isotype staining) values measured in 4–5 different experiments. (C) CCL21 cleavage assay performed in presence or absence of LECs and plasminogen (plg), revealing the dependence of CCL21 cleavage on both factors (i.e., LECs and plg). One representative out of two similar experiments is shown. (D and E) CCL21 cleavage assay performed with (D) bone marrow–derived DCs and (E) primary keratinocytes, revealing their ability to cleave CCL21 in presence of plg. One representative out of two similar experiments is shown in D and E. (F) qRT-PCR–based analysis of mRNA from LN LECs isolated from WT, uPA mut , and uPA −/− mice. (G) Absolute CT values and (H) relative expression levels. Data from four LN LEC isolations are shown. One-way ANOVA. (H and I) Impact of heparitinase treatment on the CCL21 gradient in uPA mut mice. (H) Representative images showing LYVE-1 and the immobilized perilymphatic CCL21 gradient in the steady-state ear skin of uPA mut mice upon in vitro treatment with heparitinase (HEP) or in untreated CTRs. Scale bar: 50 μm. (I) Quantification of the CCL21 staining intensity as a function of the distance from the nearest LYVE-1 + LV. n = 3 mice per condition, two-way ANOVA. 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: Expressing, Activation Assay, Activity Assay, Cell Culture, Flow Cytometry, Staining, Cleavage Assay, Derivative Assay, Quantitative RT-PCR, Isolation, In Vitro

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

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: Impact of plasmin and plasmin(ogen) on in vitro DC migration. In vitro experiments were performed with LPS-matured bone marrow–derived DCs and primary LN LECs. (A) DC displayed greater chemotaxis toward CCL21-ΔC as compared with full-length CCL21. (B) DCs were allowed to transmigrate for 4 h across primary LN LEC monolayers. DCs displayed a near-significant in transmigration (i.e., in two out of three experiments) toward CCL21-ΔC compared with CCL21 added to the lower well compartment. (C and D) The presence of plasminogen (Plg) or plasmin (Plm), which were added to the upper and lower wells of the Transwell plate, did not impact DC chemotaxis toward (C) CCL21-ΔC and (D) CXCL12. Each data point represents an independent experiment. (E and F) The presence of plasminogen or plasmin in the assay (upper and lower wells) did not impact DC transmigration across LEC monolayers toward (E) CCL21-ΔC or (F) CXCL12. Each data point represents an independent experiment. (G and H) Crawling assay: YFP-expressing DCs were added on top of LEC monolayers, and their migration was recorded by time-lapse microscopy. The presence of plasminogen or plasmin in the medium did not impact the (G) velocity and (H) chemotactic index of DC crawling. Of note: Inhibition of ROCK with Y27632 was performed as positive CTR . Statistics: two-way ANOVA using multiple comparisons, followed by a Tukey correction. (I) qPCR-results demonstrating that in vitro –cultured primary LN LECs no longer express CCL21 and also do not express CCL19. S18: housekeeping gene; gp38: podoplanin. Raw CT values are shown. n = 3 different biological replicates (isolations).

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, Migration, Derivative Assay, Chemotaxis Assay, Transmigration Assay, Expressing, Time-lapse Microscopy, Inhibition, Cell Culture

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: The immobilized perilymphatic CCL21 gradient is diminished during TPA-induced skin inflammation. The skin of one ear of each mouse was inflamed by topical application of the irritant TPA. Experiments with both ears, i.e., the uninflamed (CTR and the TPA-inflamed ear, were performed 1 day later. (A–D) Analysis of the extracellular, immobilized perilymphatic CCL21 gradient stained in fresh (unfixed) CTR and TPA-inflamed ear skin. (A) Representative images showing the CCL21 and LYVE-1 signal at 20× magnification (scale bar: 50 μm). (B) Quantification of the CCL21 staining intensity as a function of the distance from the nearest LYVE-1 + LV. CCL21 staining intensity was measured at (C) 0 μm or (D) 30 μm from the LV. n = 3 mice per condition (4–6 images analyzed per mouse). The dotted horizontal line in C and D indicates the level of background (isotype) staining. (E) Elution assay: CTR and TPA-inflamed ear skin were placed in medium overnight, and the amount of CCL21 protein eluted into the medium was determined by ELISA. (F and G) Quantification of (F) plasminogen and (G) plasmin activity in tissue protein extracts generated from CTR or TPA-inflamed ear skin. n = 6–7 mice per condition. All graphs: unpaired Student’s t test.

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: Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Generated

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: 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, uPA mut 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 uPA mut 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.

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, Staining, Enzyme-linked Immunosorbent Assay, Blocking 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: Blockade of uPA results in reduced CCL21-ΔC levels in skin-dLNs. (A and B) Western blot–based comparison of CCL21 proteoforms in steady-state LNs and ear skin. (A) Representative western blots performed on tissue protein extracts and (B) and quantification of the full-length CCL21 (gray) and CCL21-ΔC (white) relative band percentages. Pooled data from n = 4 western blots from independent experiments. (C) Representative western blot of CCL21 performed on LN extracts, LN eluates, and serum. (D) ELISA-based quantification of full-length CCL21 and CCL21-ΔC present in LN extracts, LN eluates, or serum. Antibody clone MAB457 detects full-length CCL21 only, whereas clone AF457 detects both full-length CCL21 and CCL21-ΔC. Data from n = 3 mice are shown. n.d., not detected. (E) ELISA-based quantification of full-length CCL21 and CCL21-ΔC present in LN eluates from WT and uPA mut mice. Data from n = 4–5 mice are shown, Student’s t test. n.d., not detected. (F and G) Protein extracts were prepared from LNs of PBS-perfused WT and uPA mut mice and used for ELISA-based quantification of (F) plasminogen and (G) assessment of plasmin activity (colorimetric assay). Pooled data from n = 6 mice per group are shown as mean ± SEM. Student’s t test. (H and I) Analysis of CCL21 levels in LNs of WT and uPA mut mice. Freshly cut LN sections were immediately (i.e., without fixation/permeabilization) stained for B220 (B cell follicles), LYVE-1, and CCL21. Images were subjected to AI-based tissue segmentation for differentiating between the T cell zone and B cell follicles/SCS. (H) Representative images from the immunofluorescent staining performed on LNs of WT and uPA mut mice. Scale bar: 100 μm. (I) Quantification of CCL21 staining intensity observed in the T cell zone. Each dot represents data from a stained auricular LN of one mouse (average of 4–6 images per LN). Student’s t test. 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: Western Blot, Comparison, Enzyme-linked Immunosorbent Assay, Activity Assay, Colorimetric Assay, Staining

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: Supplemental data to FITC painting experiments and analysis of LNs draining CHS-inflamed skin. (A) Schematic depiction of the experiment: FITC was applied to the ear skin in WT and uPA mut mice, and ear skin or ear-draining auricular LNs were collected for analysis after 24 h. (B and C) Analysis of dermal DC numbers. Ear skin was enzymatically digested, and single-cell suspensions were generated for flow cytometry–based analysis. (D and E) Gatings used to identify migratory DCs (CD11c + MHCII hi ), and, amongst those, FITC + DCs in single-cell suspensions generated from (D) enzymatically digested LNs and (E) undigested LNs. (F–I) Analysis of immobilized CCL21 and plasmin(ogen) in CHS-dLNs. A CHS response was induced in the ear skin of WT mice, and ear-draining auricular LNs were collected 24 h later. (F and G) Analysis of CCL21 levels in LNs draining CTR or CHS-inflamed ear skin. Freshly cut LN sections were immediately (i.e., without fixation/permeabilization) stained for B220 (B cell follicles), LYVE-1, and CCL21. (F) Representative images of the immunofluorescent staining and of the AI-based tissue segmentation used for differentiating between the T cell zone and B cell follicles/SCS. Scale bar: 100 μm. (G) Quantification of CCL21 staining intensity of observed in the T cell zone. Each dot represents data from a stained auricular LN of one mouse (average of 4–6 images per LN). Student’s t test. (H) ELISA-based quantification of (H) plasminogen and (I) plasmin activity in LN protein extracts, generated after perfusing the mice with PBS. Pooled data from n = 6 mice per group are shown in H and I, Student’s t test. (J) Representative gating strategy used for the quantification of FITC + CD11c + cells in LN sections from WT and uPA mut FITC-painted auricular LNs in . CD11c + cells were identified based on CD11c-AF647 positivity. From the CD11c + population, FITC + cells were identified and subsequently quantified. Marker-negative cells (red color) served as negative control to check background staining and to set the fluorescence thresholds. Please note that the data points of the WT CTR group in C are identical to those shown in , as these extracts were prepared and measured simultaneously.

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: Generated, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Marker, Negative Control, Fluorescence

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: Summary diagram. Summary of the main findings and the overall model. Top: Summary of events happening at the level of LECs: continuous low-level extravasation of plasminogen from blood vessels leads to uPA/uPAR-mediated activation of plasmin, which in turn cleaves immobilized CCL21 into soluble CCL21-ΔC (WT steady-state—left). When uPA-mediated activation of plasminogen is compromised (uPA mut ), less CCL21 gets cleaved, shifting the balance toward more immobilized CCL21 accumulating on/around LECs (uPA mut steady-state—middle). Under inflammatory conditions, with higher extravasation of plasminogen and higher expression of uPA and uPAR by LECs, more plasmin is activated, resulting in more CCL21 cleavage (WT inflammation—right). Bottom: The bottom part of the figure illustrates how these changes affect the balance between immobilized CCL21 and soluble CCL21-ΔC around afferent lymphatics and in the dLN. Additionally, the impact on distinct CCR7-dependent steps (1–3) in lymphatic migration of DCs are indicated. Question marks (?) indicate steps that were not specifically investigated in this study and thus represent speculations based on indirect findings and/or the literature.

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: Activation Assay, Expressing, Migration

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