Normal dendritic cell mobilization to lymph nodes under conditions of severe lymphatic hypoplasia

Abstract

To address the requirement for lymphatic capillaries in dendritic cell (DC) mobilization from skin to lymph nodes (LNs), we used mice bearing one inactivated allele of vascular endothelial growth factor receptor 3 (VEGFR3) where skin lymphatic capillaries are reported absent. Unexpectedly, DC mobilization from the back skin to draining LNs was similar in magnitude, and kinetics to control mice and humoral immunity appeared intact. By contrast, DC migration from body extremities, including ear and forepaws, was ablated. An evaluation in different regions of skin revealed rare patches of lymphatic capillaries only in body trunk areas where migration was intact. That is, whereas the ear skin was totally devoid of lymphatic capillaries, residual capillaries in the back skin were present though retained only at ∼10% normal density. This reduction in density markedly reduced the clearance of soluble tracers, indicating that normal cell migration was spared under conditions when lymphatic transport function was poor. Residual lymphatic capillaries expressed slightly higher levels of CCL21 and migration of skin DCs to LNs remained dependent on CCR7 in Chy mice. DC migration from the ear could be rescued by the introduction of a limited number of lymphatic capillaries through skin transplantation. Thus, the development of lymphatic capillaries in the skin of body extremities was more severely impacted by a mutant copy of VEGFR3 than trunk skin, but lymphatic transport function was markedly reduced throughout the skin, demonstrating that even under conditions when a marked loss in lymphatic capillary density reduces lymph transport, DC migration from skin to LNs remains normal.

Fig. 1. Normal proportions of lymph-trafficking DCs in some lymph nodes of Chy mice

(A) Total cellularity was measured in the auricular, brachial and inguinal LNs of WT and Chy mice. (B) LN preparations from WT and Chy mice were analyzed for CD11c and MHC-II expression and the proportion of CD11c⁺MHC-II^hi cells are shown. n=6–7 mice. (C) Immunofluorescence images of LNs stained for CD207 (green), CD3 (red), B220 (pink) and LYVE-1 (blue). Bar=12µm. (D) Representative dot plots (D) and proportion (E) of CD207 expression by CD11c⁺ cells in skin-draining LNs. (F) CD8α expression among CD11c⁺CD207⁺ cells in brachial LNs of Chy and WT mice. (G) Proportion of CD11c⁺MHC-II^hi DCs from pooled brachial, inguinal and axillary LNs from WT and K14-VEGFR3-Ig mice. Open circles, WT mice; closed circles, Chy or K14-VEGFR3-Ig mice. Each circle shows one mouse. n=3–7 mice. *p<0.05; **p<0.01; ***p<0.001; ns=not significant. Results are from at least 3 independent experiments.

Fig. 2. No kinetic delay in DC migration to lymph nodes in Chy mice

(A) Representative dot plots of FITC⁺CD11c⁺ cells in skin-draining LNs of WT and Chy mice 18 h after FITC application to the dorsal ear and upper and lower back skin. (B) Percentage of CD11c⁺FITC⁺ cells in LNs of WT and Chy mice. n=5–7. (C) Percentage of CD11c⁺FITC⁺ cells in brachial LNs of WT and Chy mice 12 h after FITC application to upper back skin. (D) Representative dot plots of MHC-II⁺ bead⁺ cells in skin-draining LNs of WT and Chy mice 3 days after i.d. injection of 1µm green fluorescent beads in the ear and back skin. (E) Percentage of green bead⁺ cells in LNs of WT and Chy mice. Open circles, WT mice; closed circles, Chy mice. n=4–7. (F) Spleen CD11b⁺ cells from CD45.1 WT and CD45.2 WT (left) or CD45.2 CCR7^−/− (middle, right) mice were labelled with CMRA and mixed at 1:1 ratio, then injected into the upper back skin dermis of WT/Chy CD45.2⁺ mice. At 18 h, CD45.1 and CD45.2 expression was examined in brachial LNs on total CMRA⁺ cells (F). (G) Results are shown as the mean percentage ± SD of CMRA⁺ cells that were CD45.1⁺ vs. CD45.2⁺ in WT and Chy brachial LNs. (H) WT (red) and CCR7^−/− (green) BM-DCs were pulsed overnight with 1µm beads, stimulated with 100ng/mL LPS for 5 h, and injected subcutaneously in the back skin over the inguinal LN. At 16h, inguinal skin and LNs were collected and the migration of bead-labelled cells towards CCL21⁺ (white) lymphatic capillary-rich dermis was examined (H). (I) Images of the draining inguinal LN in both WT and Chy mice 16 h after injection of bead⁺ DCs. Bar=80µm. n=4. **p<0.01; ***p<0.001; ns=not significant. Results derive from 2–4 independent experiments.

Fig. 3. Regionalized lymphatic defects dictate DC mobilization

(A) Representative dot plots of cells in the draining auricular LN 3 days after injection of red beads into the cheek skin dermis and green beads into the ear dermis. (B) Percentage of red bead⁺ cells (cheek skin) and green bead⁺ cells (ear) in the auricular LN. (C) Representative dot plots of red bead⁺ and green bead⁺ cells in the draining brachial LN 3 days after injection of red beads into the upper back skin dermis and green beads into the front footpad dermis. (D) Percentage of red bead⁺ cells (back skin) and green bead⁺ cells (footpad) in the brachial LN. Open circles, WT mice; closed circles, Chy mice. n=10–11; ***p<0.001; ns=not significant. Results depict 3 independent experiments.

Fig. 4. Rare lymphatic capillaries exist in the back skin dermis of Chy mice

(A) Whole mount staining of ear dermis with LYVE-1 (green) and smooth muscle actin (SMA; red). Bar=60µm. (B) 10µm cross-sections of back skin from WT and Chy mice stained for LYVE-1 (green) and SMA (red). Bar=60µm. (C) Podoplanin (green), LYVE-1 (white) and SMA (red) staining of en face sections of back skin from WT (left) and Chy (middle and right) mice. Bar=150µm. (D) CCL21 staining of lymphatic capillaries in back skin sections from WT and Chy mice. Bar=150µm. (E) Quantification of lymphatic capillary density. (F) Local density of lymphatic capillaries examining only the areas of Chy back skin that contained vessels. Results are from 8 mice/group. (G) Normalized mean fluorescence intensity of CCL21 staining. Measurements were taken from 3 mice/group. (H–I) Whole mount images of the ear dermis (H) and en face images of back skin dermis (I) of WT and Chy mice show podoplanin (green), LYVE-1 (white) and SMA (red) staining. Bar=32µm (H) and 38 (I) µm. Note that pre-collectors are podoplanin⁺LYVE-1⁻ and exhibit partial SMA coverage. *p<0.05; ***p<0.001.

Fig. 5. Molecular transport from back skin lymphatic vessels of Chy mice is impaired

(A–B) Images show staining with LYVE-1 (green) and VE-Cadherin (red) on lymphatic capillaries in the back skin dermis of WT and Chy mice. Bar=24µm. (B) Enlargement of inset in (A) showing that the structural pattern is highlighted more clearly after isosurface rendering of the image. Unit=5.2µm (left) and 5.08µm (right). (C) Images show staining of back skin lymphatic capillaries with collagen IV (white) and CCL21 (red) in WT and Chy mice. Bar=24µm. (D) Enlargement of inset in (C). Yellow arrowheads indicate lymphatic portals. Bar=11.1µm (left) and 12µm (right). (E–F) Sections of auricular (E) and brachial (F) LNs 5 min. after injection of 50µg TRITC-dextran (red) into the ear and back i.d. Counterstained with DAPI (blue). Bar=60 µm. (G) Lymphatic transport measured in the ear and back skin. Each symbol represents pooled left and right data from a single mouse, and the bar shows the median normalized rate of fluorescence decay. **p<0.01, assessed using a one-tailed Student’s t-test. Measurements are from 1–4 mice/group in 2 independent experiments.

Figure 6. Classical inflammation drives limited lymphangiogenesis but fails to rescue DC mobilization in Chy mice

(A–B) 4 hr after PBS (white bars) or CFA (black bars) injection in the ear (A) or back skin (B), skin was digested and the total number of live CD45⁺CD11b⁺Ly6G⁺ neutrophils are shown. (C) Graph depicts ear thickness of WT and Chy mice after injection of PBS or CFA into the ear dermis. (D) Plot shows the total cellularity of the draining auricular LN 18 days after CFA treatment. (E) Plot depicts the area of the ear dermis covered by LYVE-1⁺ vessels 18 days after injection of PBS or CFA. (F–G) Results shown are the proportion (F) and total number (G) of green bead⁺ cells present in the auricular LN of WT and Chy mice injected with green fluorescent beads on day 15 post-injection with PBS or CFA. LNs were harvested 3 days after injection of green beads. Open circles indicate WT mice while closed circles depict Chy mutant mice. Each circle represents one mouse. (H–I) 4µg OVA mixed with 25µg Ultrapure 0111:B4 LPS (InvivoGen, Nunningen, Switzerland) or 50µg OVA in emulsified CFA (diluted 1:1) was injected into the footpad (H) or back skin dermis (I) of Chy and littermate WT mice. 14–21 days later, serum was harvested and assessed for total anti-OVA IgG antibody by ELISA. Results show the OD value of plasma at various dilutions. n=7–10. *p<0.05; **p<0.01; ***p=0.001; ns=not significant. Results are representative of 2 independent experiments.

Figure 7. DC mobilization from regions with no previous drainage can be rescued by skin transplantation

(A) Representative photo after WT (or Chy) dorsal ear skin was transplanted onto the dorsal ear dermis of Chy recipient mice. 5 regions (as indicated) were examined for the presence of LYVE⁺ lymphatic capillaries. (B) Representative LYVE-1 (green) and SMA (red) staining of ear dermis within (left panel; Region 1) and outside (right panel; Region 4) the transplanted region from Chy mice transplanted with WT donor skin. (C) Lymphangiogenesis was assessed 17 days after surgery using Volocity software and the area of ear dermis covered by LYVE-1⁺ vessels was measured in unmanipulated WT and Chy mice, Chy recipients of WT and Chy donor skin, and mice undergoing sham surgery. n=3–19. (D–F) Representative images of ear dermis of Chy recipients of β-actin-eGFP donor skin 2 weeks post-transplant. Images show LYVE-1 (red; left panel), eGFP signal (green; middle panel) and the colors merged (right panel). Note the presence of LYVE-1⁺ vessels which are GFP⁺ (D), GFP⁻ (E) and a mix of the two (F). Bar= 28µm (D), 80µm (E), 37µm (F). (G) LYVE-1⁺ vessels present in the recipient ear dermis were assessed for GFP signal, quantified and the percentage of vessels in each group are shown. (H) Representative dot plot of MHC-II⁺ green bead⁺ cells in the auricular LN of Chy recipients of WT donor skin 3 days after i.d. injection of 1µm green fluorescent beads into the ear. Beads were injected 14 days post-transplant. (I–J) Results shown are the proportion (I) and total number (J) of green bead⁺ cells present in the auricular LN of transplant recipients or mice undergoing sham surgery. Each circle represents one individual transplant and 2 transplants per mouse were performed. n=11–17. *p<0.05; **p<0.01. Results are representative of 4 independent experiments.