Figure 1.
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.

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.

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