A three-dimensional atlas of human dermal leukocytes, lymphatics, and blood vessels

Abstract

Dendritic cells (DCs), macrophages (Mφ), and T cells are major components of the skin immune system, but their interstitial spatial organization is poorly characterized. Using four-channel whole-mount immunofluorescence staining of the human dermis, we demonstrated the three-dimensional distribution of CD31(+) blood capillaries, LYVE-1(+) lymphatics, discrete populations of CD11c(+) myeloid DCs, FXIIIa(+) Mφ, and lymphocytes. We showed phenotypic and morphological differences in situ between DCs and Mφ. DCs formed the first dermal cellular layer (0-20 μm beneath the dermoepidermal junction), Mφ were located deeper (40-60 μm), and CD3(+) lymphocytes were observed throughout (0-60 μm). Below this level, DCs, T cells, and the majority of Mφ formed stable perivascular sheaths. Whole-mount imaging revealed the true extent of dermal leukocytes previously underestimated from cross-section views. The total area of apical dermis (0-30 μm) contained approximately 10-fold more myeloid DCs than the entire blood volume of an average individual. Surprisingly, <1% of dermal DCs occupied lymphatics in freshly isolated skin. Dermal DCs rapidly accumulated within lymphatics, but Mφ remained fixed in skin explants cultured ex vivo. The leukocyte architecture observed in normal skin was distorted in inflammation and disease. These studies illustrate the micro-anatomy of dermal leukocytes and provide further insights into their functional organization.

Figure 1

Dermal lymphatics and blood vessels. (a–c) CD31 (red) and LYVE-1 (green) staining to identify CD31⁺LYVE-1⁻ blood vessels and CD31^loLYVE-1⁺ lymphatic vessels. (d, e) Serial sections of region within inset in c. (d) CD31⁺capillary loops and LYVE-1⁺lymphatic tips at 0–25 μm. (e) Blood and lymphatic vessels between 25 and 50 μm beneath the dermoepidermal junction (DEJ). (f) A three-dimensional projection of d and e. (g) Loss of LYVE-1 (green) expression at the transition from initial to collecting lymphatic vessel (>500 μm beneath the DEJ). (h) LYVE-1⁺podoplanin⁺ initial lymphatic vessels and LYVE-1^lo podoplanin⁺ collecting vessels. (i) Valves (white arrows) within collecting lymphatic vessels. (j) Smooth muscle actin (red) at the start of collecting lymphatic vessel. Data are representative of n>5 from >3 separate donors, maximum Z-stacks projection. Scale bar =100 μm except c =500 μm.

Figure 2

Flow cytometry, cytology, and in situ identification of dermal leukocytes. (a) Dermal leukocytes by flow cytometry as % of CD45⁺ cells. Representative results from >3 experiments from >3 donors. (b) Cytology of dermal leukocytes. (c) CD11c (green; dendritic cell (DC)), CD3 (red; T cells), and FXIIIa (blue; Mφ). (d) CD11c⁺ DCs (red) are HLA-DR^br (green), whereas FXIIIa⁺ Mφ (blue) are HLA-DR^lo/neg. Cropped images on right. (e) LYVE-1 (green) is expressed by Mφ. (f) Some FXIIIa⁺Mφ express LYVE-1. (g) CD11c (green), CD14 (red), and FXIIIa (blue) distinguishes CD11c⁺CD14⁻FXIIIa^neg DCs (green arrow) from CD11c⁺CD14⁺FXIIIa^lo−neg DCs (CD14⁺ DCs; red arrow) and CD11c⁻CD14⁺ FXIIIa^br Mφ (blue arrow). Scale bar =50 μm. (a–f) Representative of n>5 from >3 separate donors. (h) CLEC9A⁺(red) HLA-DR⁺ (green) DCs (white arrows). Representative images from two donors. Maximum projection of Z-stacks shown.

Figure 3

Anisotropic distribution of dermal leukocytes. Distribution of CD11c⁺dendritic cells (DCs; green), FXIIIa⁺ Mφ (blue), and CD3⁺ T cells (red) at (a) 0–20 μm, (b) 20–40 μm, and (c) 40–60 μm beneath the dermoepidermal junction (DEJ). (d) Three-dimensional reconstruction of a–c; DCs (green), Mφ (blue), and T cells (red). (e) Distribution of CD11c⁺ DCs, FXIIIa⁺ Mφ, and CD3⁺ T cells at >150 μm beneath the DEJ. Scale bar=200 μm except d=50 μm. (f) Scatter plot of number of DCs, Mφ, and T cells per mm² of dermis up to 30 μm depth. Each point is from one × 20 field; three fields per donor and 3–4 donors analyzed. (g) CD8⁺ and CD3⁺CD8⁻ (“CD4⁺”) T cells at <30 μm and >150 μm beneath the DEJ. P<0.001. Mean±SEM of six donors.

Figure 4

Relationship of dermal leukocytes to vascular structures. Distribution of (a) CD11c⁺ dendritic cells (DCs; green), (b) FXIIIa⁺ Mφ (blue), and (c) CD3⁺ T cells (red) in relation to blood vessels at <30 and >150 μm beneath the dermoepidermal junction (DEJ). Accompanying Imaris software representation is shown on the right. Bar graphs showing number of cells in × 20 field found in perivascular (peri; <15 μm from vessel) and interstitial (inter; >15 μm from vessel) distribution; mean±SEM. (d) Distribution of CD11c⁺ DC(green), LYVE-1⁺ Mφ (blue), and CD3⁺ T cells (red) with lymphatic vessels (blue). Scale bar =50 μm except d =200 μm. Images are representative of >8 experiments, from >5 separate skin donors. (e) Schematic diagram illustrating the distribution of dermal leukocytes.

Figure 5

Dermal dendritic cells (DCs) migrate into lymphatics. (a) Apical CD11c⁺ DCs (green) are rounded cells (yellow rectangle) with clusters of branching cells (white square; higher magnification in right panel). (b) CD11c⁺ DCs (green) at >150 μm are round. (c) Branching apical CD11c⁺ DCs (green) express fascin (red). (d) DCs (green) at >150 μm beneath the dermoepidermal junction (DEJ) very rarely express fascin (red). (e) FXIIIa⁺Mφ (cyan) are fascin⁻ (green). (f) <1% of CD11c⁺ DCs (red) are inside LYVE-1⁺lymphatic vessels (green) in freshly isolated skin, but (g) >90% are detectable after 32 hours of skin explant culture. LYVE-1⁺Mφ (green) are in the interstitium. White arrows indicate cells inside lymphatics in the orthogonal plane. Right panels are higher magnification of region within white rectangles. (h) Cells within LYVE-1⁺lymphatics (green) are FXIIIa⁻ (cyan) and fascin⁺(red). Scale bar =20 μm except a, b =50 μm. Representative images from >3 experiments and donors.

Figure 6

Dermal leukocyte architecture is distorted in disease. (a) Psoriasis, (b) atopic dermatitis, (c) cutaneous T cell lymphoma (CTCL), and (d) graft-versus-host disease (GVHD) skin showing CD11c⁺ dendritic cells (DCs; green), FXIIIa⁺Mφ (blue), and CD3⁺T cells (red); (scale bar=200 μm except d=100 μm left panel) and × 20 of white inset (scale bar=100 μm except d=50 μm right panel). Representative images from two to six independent donors.