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 downregulated 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.)

Immune activation of fibrosis likely plays a crucial role in the pathogenesis of systemic sclerosis (SSc). The goal of this study was to better understand innate immune regulation and associated IFN- and TGFβ-responsive gene expression in SSc skin and dermal fibroblasts, in particular the effect of different Toll-like receptor (TLR) ligands. To better understand the relationship between inflammation and fibrosis in vivo we developed a murine model for chronic innate immune stimulation. We found that expression of both IFN- and TGFβ-responsive genes are increased in SSc skin and in SSc fibroblasts when stimulated by TLR ligands. In contrast, cutaneous lupus skin showed much more highly upregulated IFN-responsive and much less highly upregulated TGFβ-responsive gene expression. The TLR3 ligand, Poly(I:C), mostly highly increased fibroblast expression of both IFN- and TGFβ-responsive genes as well as TLR3. Chronic subcutaneous immune stimulation by Poly(I:C) stimulated inflammation, and IFN- and TGFβ-responsive gene expression. However, in this model type I IFNs played no apparent role regulating TGFβ activity in the skin. These results suggest that TLR agonists may be important stimuli of dermal fibrosis, potentially mediated by TLR3 or other innate immune receptors.

To understand the mechanism of transcriptional down-regulation of BRCA1 by promoter methylation, we screened 51 breast cancer cell lines and identified HCC38 as another BRCA1 promoter-methylated cell line in addition to UACC3199. There was low expression of BRCA1 mRNA and BRCA1 protein in both cell lines as measured by quantitative RT-PCR and western blot analysis. After transient treatment with 5-aza-2'-deoxycytidine (5-aza-CdR) and trichostatin A (TSA), re-expression of BRCA1 mRNA and BRCA1 protein was detected in UACC3199 cells, but not in HCC38 cells. Another demethylating agent, zebularine, did not induce BRCA1 re-expression in either cell line. To test the hypothesis that methylation of CpG sites may affect accessibility of the BRCA1 promoter to transcription factors and consequently cause down-regulation of BRCA1, we analyzed the binding of four transcription factors (CTCF, Sp1, E2F1 and E2F6) to the BRCA1 promoter using chromatin immunoprecipitation assay (ChIP) and quantitative PCR. CTCF and E2F1 were enriched at the unmethylated BRCA1 promoter in MCF-7 cells. In contrast, these two transcription factors were not enriched at the methylated BRCA1 promoter in UACC3199 and HCC38 cells. Following demethylating drug treatment, E2F1 was enriched at the BRCA1 promoter in the demethylated UACC3199 cells. This indicates that reduced accessibility of transcription factors to the methylated promoter is one of the mechanisms for down-regulation of BRCA1 in heavily methylated cancer cells.

Cross-reactive anti-DNA antibody idiotypes have been identified on tissue-bound immunoglobulins in a study of renal biopsies from 26 systemic lupus erythematosus (SLE) patients. 12 (46%) biopsies were shown to have one or both the idiotypes tested for by anti-idiotypic reagents. The idiotypes were identified in the glomerular basement membrane, the mesangial cell cytoplasm, and in focal tuft proliferations. In contrast, in none of 24 immunoglobulin-positive disease control biopsies could either idiotype be demonstrated. Blocking studies in two patients indicated that the idiotypes were on anti-DNA antibodies. These findings indicate that some tissue-bound autoantibodies are derived from related families of high-frequency germ line genes that are expressed in SLE patients. The potential role of anti-idiotypic therapy in SLE is discussed.