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Abstract

Background: Plasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon.

Methods: We isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo.

Results: Proteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (±SD) level of CXCL4 in patients with systemic sclerosis was 25,624±2652 pg per milliliter, which was significantly higher than the level in controls (92.5±77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346±1011 pg per milliliter), ankylosing spondylitis (1368±1162 pg per milliliter), or liver fibrosis (1668±1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 down-regulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis.

Conclusions: Levels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.).

Figures

Figure 1
Figure 1. Identification of CXCL4 as the Major Protein Product of Plasmacytoid Dendritic Cells in Systemic Sclerosis
Panel A shows the results of proteome-wide analysis of supernatants from plasmacytoid dendritic cells (pDC) obtained from patients with various subtypes of systemic sclerosis (SSc) and from healthy controls (HC). Highlighted in the larger view is the analysis of samples obtained from patients with early diffuse systemic sclerosis, showing peaks for CXCL4, connective tissue-activating peptide III (CTAP-III), S100A8/A9 (MRP8/14), and lysozyme. Panel B shows the level of CXCL4 in supernatants from plasmacytoid dendritic cells from the different groups that were investigated — 20 healthy donors, 16 patients with limited disease, 19 with late diffuse disease, and 18 with early diffuse disease — on CXCL4-specific enzyme-linked immunosorbent assay. The horizontal lines indicate means, and I bars standard deviations. Panel C shows frozen skin sections from a representative patient with early diffuse disease (at left) and from a healthy control (at right), which were stained for the plasmacytoid dendritic-cell marker BDCA2 (red), CXCL4 (green), and DAPI-labeled nuclei (blue). BDCA2+ cells expressing CXCL4 are seen as orange (merged, lower panels).
Figure 2
Figure 2. Increased Levels of Circulating CXCL4 in Systemic Sclerosis and the Association with Lung Fibrosis and Pulmonary Arterial Hypertension
High levels of CXCL4 were observed in the circulation of patients with limited systemic sclerosis (SSc), late diffuse SSc, and early diffuse SSc in the identification cohort at Boston University (Panel A) and in 161 patients with corresponding subtypes of systemic sclerosis in two independent (Dutch and Swedish) replication cohorts, as compared with 129 age- and sex-matched healthy controls, 109 patients with systemic lupus erythematosus (SLE), 93 with ankylosing spondylitis (AS), and 93 with hepatic fibrosis (Panel B). The horizontal lines indicate means, and I bars standard deviations. Shown are the estimated times until the development of lung fibrosis (Panel C) and pulmonary arterial hypertension (PAH) (Panel D) within the first 24 months after the diagnosis of systemic sclerosis among patients with a high circulating CXCL4 level (≥10 ng per milliliter) and among those with a low CXCL4 level (<10 ng per milliliter).
Figure 3
Figure 3. Changes in Endothelial Cells and Augmented Responses in Toll-Like Receptors Induced by CXCL4
The addition of lipopolysaccharide (LPS), low and high levels of CXCL4, and plasma obtained from a patient with diffuse systemic sclerosis (SSc) induce the secretion of endothelin 1 by human umbilical-vein endothelial cells, reactions that were attenuated by the addition of a neutralizing antibody against CXCL4 (Panel A). In five independent experiments, CXCL4 was shown to reduce the expression of transcription factor FLI1 (P<0.001) and induce the expression of endothelin 1 (ET-1) (P =0.003), both on the RNA and protein level, in human dermal microvascular endothelial cells (Panel B). The expression level for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was set at 1. On stimulation with ligands of toll-like receptors (TLR) (including R848, CpG, and CpG control), plasmacytoid dendritic cells from patients with systemic sclerosis secreted more interferon α (a type I interferon) than did controls (Panel C). This reaction was fully reversed by the addition of anti-CXCL4 antibody (Panel D). The culture medium was RPMI-1640 with 10% fetal-calf serum. All values are expressed as means; T bars represent standard deviations.
Figure 4
Figure 4. (facing page). Inflammatory Skin Changes Mimicking Those in Systemic Sclerosis Induced by CXCL4 In Vivo in Mice
Panels A and B show the results of histologic analyses of skin from wild-type C57BL/6 mice that were treated with phosphate-buffered saline (PBS) as a control (Panel A) or with CXCL4 (Panel B) for 7 days with the use of a subcutaneous-pump model, shown at 4× and 20× magnification (hematoxylin and eosin, left; CD45 immunohistochemical staining, right). The murine sample that was treated with CXCL4 shows marked infiltration of inflammatory cells in the dermis and subdermis, as compared with the control sample. Panel C shows quantification of the thickening of skin layers after CXCL4 treatment for 7 days with the use of the pump model, as compared with the PBS controls. Shown are mean values for three analyses in each group, with T bars indicating standard errors. Panel D shows the quantification of infiltrating immune cells after 7-day exposure to CXCL4 or PBS, in which CD45+ cells in the dermis were counted after immunohistochemical staining; the means of three analyses per group are shown (at left). In addition, the influx of inflammatory cells is confirmed by increased CD45 messenger RNA (mRNA) expression in CXCL4-exposed skin isolated from the distal or proximal (local) area to the pump outlet, as measured on quantitative polymerasechain-reaction (PCR) assay (at right). In Panels C and D, a single asterisk denotes P<0.05 for the between-group comparison; double asterisks denote P<0.01. Panel E shows the mRNA expression of proinflammatory marker CCL2 induced after 7-day exposure to CXCL4, as compared with PBS, also measured on quantitative PCR assay. In Panels D and E, the mRNA analyses included 4 samples for PBS and 7 samples for CXCL4. The horizontal lines indicate means, and I bars standard errors.

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