Perivascular Adventitial Fibroblast Specialization Accompanies T Cell Retention in the Inflamed Human Dermis

Abstract

Perivascular accumulation of lymphocytes can be a prominent histopathologic feature of various human inflammatory skin diseases. Select examples include systemic sclerosis, spongiotic dermatitis, and cutaneous lupus. Although a large body of work has described various aspects of the endothelial and vascular smooth muscle layers in these diseases, the outer adventitial compartment is poorly explored. The goal of the current study was to characterize perivascular adventitial fibroblast states in inflammatory human skin diseases and relate these states to perivascular lymphocyte accumulation. In normal skin, adventitial fibroblasts are distinguished by CD90 expression, and dense perivascular lymphocytic infiltrates are uncommon. In systemic sclerosis, this compartment expands, but lymphocyte infiltrates remain sparse. In contrast, perivascular adventitial fibroblast expression of VCAM1 is upregulated in spongiotic dermatitis and lupus and is associated with a dense perivascular T cell infiltrate. VCAM1 expression marks transitioned fibroblasts that show some resemblance to the reticular stromal cells in secondary lymphoid organs. Expanded adventitial compartments with perivascular infiltrates similar to the human settings were not seen in the inflamed murine dermis. This species difference may hinder the dissection of aspects of perivascular adventitial pathology. The altered perivascular adventitial compartment and its associated reticular network form a niche for lymphocytes and appear to be fundamental in the development of an inflammatory pattern.

FIGURE 1.

Perivascular adventitial fibroblasts express CD90. (A) Immunohistochemical staining of human skin biopsy specimens for CD90 (brown) and a combination of CD34 and SMA (both blue) to define the vascular media and intimal layers. CD90 staining highlights the PA layer. Scale bar, 200 μm. (B) A higher magnification view of the boxed vessels in (A) with adventitial (AF) and dermal (DF) fibroblasts indicated. Scale bar, 50 μm. (C) H&E stain of a serial section showing the equivalent region in (A) with the region shown in (B) boxed. Scale bar, 200 μm. (D) Morphometric analysis of the area of CD90 staining in the PA in healthy skin (107 vascular units, five volunteers), skin from SSc patients (107 vascular units, five patients), and DLE skin lesions (80 vascular units, five patients). Area refers to the square micrometers of CD90⁺ pixels per vascular unit. ****p < 0.0001.

FIGURE 2.

VCAM1 expression defined a different state of the PA. (A) Typical VCAM1 expression (brown) in the PA compartments in healthy, SSc, and DLE biopsy specimens with a combination of CD34 and SMA (blue). The SSc VCAM1⁻ and VCAM1⁺ panels represent the full range of patterns observed in SSc. Scale bar, 50 μm. (B) Morphometric analysis of the area of VCAM1⁺ vascular adventitia in healthy skin (56 vascular units, three volunteers), skin from SSc patients (260 vascular units, four patients), and DLE skin lesions (194 vascular units, three patients). Area refers to the square micrometers of VCAM1+ pixels per vascular unit. (C) Immunofluorescent staining of a human DLE skin lesion for VCAM1 (red: left, goat polyclonal; right, rabbit mAb) and a combination of CD45RO and RA (cyan). (D) Examples of VCAM1⁺ (left) and VCAM1⁻ (right) vessels in DLE, VCAM1 (red), and CD31 (green). (C and D) Scale bar, 25 μm. ***p < 0.001, ****p < 0.0001.

FIGURE 3.

Dense perivascular T cell infiltrates are found within VCAM1⁺ PA fibroblast networks. (A) Low-power immunofluorescence image of DLE skin showing VCAM1⁺ structures (green), CD31/SMA⁺ blood vessels (cyan) and CD45RA/RO⁺ leukocytes (red), “E” marks the epidermis. Scale bar, 100 μm. (B) Higher magnification of the white boxed area from (A). (C) Immunofluorescent staining for VCAM1 (green), CD3 (red), and SMA (cyan) in representative healthy, SSc, CSD, and DLE biopsy specimens. Scale bar, 25 μm in (B) and (C). (D) Counts of perivascular leukocytes in the vascular adventitia and media from H&E-stained skin sections (101 vascular units, eight DLE patients). (E) Morphometric analysis of the area of CD3⁺ T cells in the PA compartment in healthy skin (35 vascular units, four volunteers), SSc (57 vascular units, five patients), CSD (47 vascular units, five patients), and DLE skin lesions (57 vascular units, five patients). Area refers to the square micrometers of CD3⁺ pixels per vascular unit. ****p < 0.0001.

FIGURE 4.

VCAM1 RNA correlates with T cell infiltration, ITGA4, and IL-15 but not IL-7 RNA. (A) Relative VCAM1 RNA expression from microarray analyses of whole skin biopsy specimens. L, lesioned skin; NL, nonlesioned skin. (B) Relative total T cell levels as determined by CIBERSORT analysis of the whole biopsy specimen transcriptome. (C–F) Relationship between the levels of VCAM1 RNA and total T cells as determined by CIBERSORT. (G–I) Relationship between the levels of VCAM1 RNA and ITGA4, IL-15, and IL-7 RNA as assessed by microarray. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5.

Perivascular T cell infiltrates occur regardless of PA fibroblast PDPN expression. (A) Representative examples of PDPN staining (brown) in the PA compartment in healthy, SSc, and DLE biopsy specimens with CD34 and SMA (both blue). (B) Low- and high-power images of a PDPN⁺ reticular network (red) that costained with VCAM1 (green). (C) Contrast between CSD and DLE biopsy specimens showing PDPN (red), CD3 (cyan), and VCAM1 (green). In the CSD panels a PDPN⁺ lymphatic vessel is seen below the VCAM1⁺ network. The DLE panels have a dilated PDPN⁺ lymphatic on the right side. (D) Correlation of the CD3⁺ area and the corresponding VCAM1⁺ or PDPN⁺ areas within individual vascular units. Red dots are both nonlesioned and lesioned DLE biopsy specimens whereas the remainder are healthy, SSc, and CSD samples. (A–C) Low-power scale bar in (B), 100 μm; all other scale bars, 25 μm.

FIGURE 6.

VCAM⁺SMA^– PA fibroblasts associated with MFAP5⁺ fibers. (A) Immunofluorescence images of a human lymph node stained for VCAM (green), MFAP5 (red), and SMA (cyan). A low-power view is magnified showing the approximate colocalization of VCAM1 and MFAP5 as well as MFAP5 and SMA. (B and C) High magnification image of VCAM1⁺ or SMA⁺ cells with a cross-sectional intensity analysis of an individual fiber/reticular cell complex. (D–F) Identical analysis of a VCAM1⁺ network in the PA from a DLE biopsy specimen. Cross-sectional analysis was consistent with fiber wrapping, but SMA was not present. (A and D) Low-power scale bar, 100 μm; high-power scale bar, 25 μm.

FIGURE 7.

PA compartment in mouse skin. (A) Low-power images of skin sections from nonlesioned MRL/lpr skin, C57BL/6 skin 4 d after incisional wound (Day 4 Wound), and an MRL/lpr skin lesion stained for PDGFRβ (brown) and CD31 (blue). (B and C) Higher magnification images of the indicated regions in (A) showing blood vessels in the dermis (B) and hypodermis (C). (D) A serial section with the PA regions from the vessels in (C) were stained with CD90 (brown) and CD31 (blue). (E) Serial skin sections to those in (A) stained for H&E. Epidermis is at the top of the pictures. Images were representative of four MRL/lpr mice with lesioned skin, two mice 4 d postwounding, and two healthy mice. (A and E) Scale bar, 100 μm. (B–D) Scale bar, 25 μm.

FIGURE 8.

T cells colocalized with VCAM⁺ perivascular stromal cells in the diabetic NOD pancreas and the livers of gld.ApoE mice. (A) Immunohistochemical staining for VCAM (brown) and CD31 (blue) in the indicated organs. VCAM1⁺ Kupffer cells are visible in the liver sinusoids. (B) Serial sections to those in (A) stained for CD3 (brown) and CD31 (blue). Healthy organs were from C57BL/6 mice. All scale bars, 50 μm.