Figure 5.
Rapid DC migration from inflamed skin to dLNs is integrin β1 dependent.(A–C) Fluorescently labeled WT DCs were injected into INF or CTR mouse ear skin and footpads. DC numbers in dLNs were quantified by FACS at different time points after transfer. (A) Gating scheme. Numbers in graphs show percentage of parent. (B and C) FACS-based quantification of DCs in ear-draining auricular LNs (B) and footpad-draining popliteal LNs (C). Each dot represents the value from one dLN. (D) Representative whole-mounts prepared 5 h after DC injection. Scale bar, 50 µm. (E–K) 1:1 ratios of differently labeled (F–I) WT and Tln−/− DCs (F–I) or WT and Itgb1−/− DCs (J and K) were injected into CTR and INF mouse ear skin or footpads and the DC ratio quantified in the dLNs at different time points after transfer. (E) Representative FACS plots of an ear-draining auricular LN 17 h after DC transfer. (F and G) Analysis of Tln1−/−:WT DC ratios in auricular LNs (F) and popliteal LNs (G). (H and I) Impact of anti–VCAM-1 (H) or anti–integrin α4 (I) treatment on the ratio Tln1−/−:WT DCs recovered from LNs draining inflamed ears or footpads at 14–17 h after DC transfer. (J and K) Analysis of Itgb1−/−:WT DC ratios in footpad-draining popliteal LNs (J) and ear-draining auricular LNs (K). Each dot in F, G, J, and K represents the DC ratio quantified in one dLN of three or four experiments. Each dot in H and I represents the average ratio obtained in 7 or 10 experiments (with 1–3 mice per condition/experiment). Ratios from the same experiment are connected with a line. (L) 1:1 ratios of fluorescent WT and Itgb1−/− DCs were injected into INF mouse footpads and DC ratios quantified in popliteal dLNs 17 h after transfer. Left: Representative picture of a popliteal LN sections at the time of analysis. Middle: Zoom-ins of the subcapsular sinus (SCS) and parenchyma (PAR) areas. All scale bars, 100 µm. Right: Quantification of the Itgb1−/−:WT DC ratios in the respective area. Each dot represents the ratio in 1 LN (analysis of 5–10 images/LN) of three experiments (two LNs per experiment). Statistical significances either compare with the normalized input ratio of 1 or between the groups. (B, C, F, G, and H–L) Unpaired one-way ANOVA followed by Tukey post hoc test (B, C, F, G, J, and K), paired Student’s t test (H and I), or unpaired Student’s t test (L). *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. FSC-A, forward scatter area; SSC-A, side scatter area.

Rapid DC migration from inflamed skin to dLNs is integrin β1 dependent. (A–C) Fluorescently labeled WT DCs were injected into INF or CTR mouse ear skin and footpads. DC numbers in dLNs were quantified by FACS at different time points after transfer. (A) Gating scheme. Numbers in graphs show percentage of parent. (B and C) FACS-based quantification of DCs in ear-draining auricular LNs (B) and footpad-draining popliteal LNs (C). Each dot represents the value from one dLN. (D) Representative whole-mounts prepared 5 h after DC injection. Scale bar, 50 µm. (E–K) 1:1 ratios of differently labeled (F–I) WT and Tln−/− DCs (F–I) or WT and Itgb1−/− DCs (J and K) were injected into CTR and INF mouse ear skin or footpads and the DC ratio quantified in the dLNs at different time points after transfer. (E) Representative FACS plots of an ear-draining auricular LN 17 h after DC transfer. (F and G) Analysis of Tln1−/−:WT DC ratios in auricular LNs (F) and popliteal LNs (G). (H and I) Impact of anti–VCAM-1 (H) or anti–integrin α4 (I) treatment on the ratio Tln1−/−:WT DCs recovered from LNs draining inflamed ears or footpads at 14–17 h after DC transfer. (J and K) Analysis of Itgb1−/−:WT DC ratios in footpad-draining popliteal LNs (J) and ear-draining auricular LNs (K). Each dot in F, G, J, and K represents the DC ratio quantified in one dLN of three or four experiments. Each dot in H and I represents the average ratio obtained in 7 or 10 experiments (with 1–3 mice per condition/experiment). Ratios from the same experiment are connected with a line. (L) 1:1 ratios of fluorescent WT and Itgb1−/− DCs were injected into INF mouse footpads and DC ratios quantified in popliteal dLNs 17 h after transfer. Left: Representative picture of a popliteal LN sections at the time of analysis. Middle: Zoom-ins of the subcapsular sinus (SCS) and parenchyma (PAR) areas. All scale bars, 100 µm. Right: Quantification of the Itgb1−/−:WT DC ratios in the respective area. Each dot represents the ratio in 1 LN (analysis of 5–10 images/LN) of three experiments (two LNs per experiment). Statistical significances either compare with the normalized input ratio of 1 or between the groups. (B, C, F, G, and H–L) Unpaired one-way ANOVA followed by Tukey post hoc test (B, C, F, G, J, and K), paired Student’s t test (H and I), or unpaired Student’s t test (L). *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. FSC-A, forward scatter area; SSC-A, side scatter area.

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