Global chemokine expression in systemic sclerosis (SSc): CCL19 expression correlates with vascular inflammation in SSc skin

Abstract

Objective: To characterise global chemokine expression in systemic sclerosis (SSc) skin in order to better understand the relationship between chemokine expression and vascular inflammation in this disease.

Methods: We investigated chemokine mRNA expression in the skin through quantitative PCR analysis comparing patients with diffuse cutaneous (dcSSc) or limited cutaneous (lcSSc) disease with healthy controls. We tested correlations between the most regulated chemokines and vascular inflammation and macrophage recruitment. CCL19 expression was examined in human primary immune cells treated with innate immune activators.

Results: The chemokines, CCL18, CCL19 and CXCL13, were upregulated in dcSSc skin, and CCL18 in lcSSc skin. Expression of CCL19 in dcSSc skin correlated with markers of vascular inflammation and macrophage recruitment. Immunofluorescence data showed CCL19 colocalisation with CD163 macrophages in dcSSc skin. In vitro studies on human primary cells demonstrated that CCL19 expression was induced after toll-like receptor activation of peripheral blood mononuclear cells and separated populations of CD14 monocytes.

Conclusions: CCL18, CCL19 and CXCL13-chemoattractants for macrophage and T cell recruitment-were three of six chemokines with the highest expression in dcSSc skin. Increased CCL19 expression in the skin suggests a role for CCL19 in the recruitment of immune cells to the peripheral tissue. Induction of CCL19 in macrophages but not structural cells indicates a role for skin-resident or recruited immune cells in perivascular inflammation. This study demonstrates that CCL19 is a sensitive marker for the perivascular inflammation and immune cell recruitment seen in dcSSc skin disease.

Keywords: Chemokines; Inflammation; Systemic Sclerosis.

Figure 1

Chemokine expression in systemic sclerosis (SSc) skin. (A) Data from chemokine array analysis of healthy controls (HC) (n=4), patients with diffuse cutaneous SSc (dcSSc) (n=10) and patients with limited cutaneous SSc (n=6). Only genes with a >2-fold change in expression comparing SSc with HC are shown. Data analysis of all genes on the chemokine array can be found in online supplementary table S1. (B) Quantitative PCR Taqman gene expression analysis of CCL19, CCL18 and CXCL13. HC (n=12); dcSSc (n=26). Horizontal lines represent the mean ±SEM.

Figure 2

CCL19 expression correlates with vascular inflammation in systemic sclerosis (SSc) skin. (A) Correlation between vascular inflammation score and change in skin score. Vascular inflammation was scored in a blinded fashion according to histological representation in online supplementary figure S2. Change in skin score was calculated as the difference in modified Rodnan Skin Score (MRSS) over 6 months (n=12). (B) Correlation of vascular inflammation with CCL19 expression (n=14). (C) Serum CCL19 levels in patients with diffuse cutaneous SSc (dcSSc) (n=28) compared with healthy controls (HC) (n=12). (D) Immunofluorescence staining of CCL19 (red) with CD163 (green) co-stain for both HC and dcSSc frozen skin sections. 10× magnification. (E) Double staining for CCL19 (red) and CD163 (green) for HC and dcSSc frozen skin sections. 20× magnification. (F) Gene expression analysis of CCL19-associated genes: CCL21 and CCR7. HC (n=12); dcSSc (n=26). Horizontal bars represent the mean±SEM.

Figure 3

CCL19 expression correlates with macrophage markers and vascular injury. (A) Correlation of CCL19 expression with expression of markers of vascular injury and activation: angiopoietin 2, junctional adhesion molecule 2 ( JAM2) and von Willebrand factor. (B) Correlation of CCL19 expression with expression of macrophage markers: Siglec-1, CD163 and CCL2. For all gene analysis, n=26.

Figure 4

Microarray analysis shows association between increased expression of CCL19 and macrophage markers. (A) Gene clustering in accordance with changes in expression in systemic sclerosis (SSc) skin compared with healthy controls. (B) Cluster of genes containing CCL19 including macrophage markers (*). Data were clustered by unsupervised, hierarchic clustering of genes and arrays. Colour variations green to red represent levels of gene expression from lower to higher, respectively (see inset). These data include four arrays of skin samples from healthy controls and 20 arrays of skin samples from patients with SSc.

Figure 5

CCL19 expression induced in peripheral blood mononuclear cells (PBMCs) and macrophages after toll-like receptor (TLR) activation. (A) PBMCs were isolated from blood of healthy donors and immediately treated with TLR ligands (poly(I:C) (PIC), TLR3; lipopolysaccharide (LPS), TLR4; CpG, TLR9). Cells were collected for RNA expression analysis at 0, 2, 6 and 16 h. For all samples, (n=2) (B) PBMCs were isolated from the blood of healthy donors and separated into CD14, CD3 and CD14^–, CD3^– populations using positive selection. Cells were treated with TLR ligands (PIC, TLR3; LPS, TLR4; CpG, TLR9) overnight and then collected for gene expression analysis. For CD14 and CD14^–, CD3^– cells: media, PIC, LPS, n=4; CpG, n=2. For CD3 cells: media, PIC, LPS, n=3; CpG, n=1. Data are expressed as the mean fold change after normalisation to mRNA expression of media-only treatment. Each bar represents the mean±SEM.