Exposing genetically predisposed individuals to certain environmental agents is believed to cause human lupus. How environmental agents interact with the host to cause lupus is poorly understood. Procainamide and hydralazine are drugs that cause lupus in genetically predisposed individuals. Understanding how these environmental agents cause lupus may indicate mechanisms relevant to the idiopathic disease. Abnormal T cell DNA methylation, a repressive epigenetic DNA modification, is implicated in procainamide and hydralazine induced lupus, as well as idiopathic lupus. Procainamide is a competitive DNA methyltransferase (Dnmt) inhibitor, hydralazine inhibits ERK pathway signaling thereby decreasing Dnmt expression, and in lupus T cells decreased ERK pathway signaling causing a similar Dnmt decrease. T cells treated with procainamide, hydralazine, and other Dnmt and ERK pathway inhibitors cause lupus in mice. Whether the same genetic regulatory elements demethylate in T cells treated with Dnmt inhibitors, ERK pathway inhibitors, and in human lupus is unknown. CD70 (TNFSF7) is a B cell costimulatory molecule overexpressed on CD4 lupus T cells as well as procainamide and hydralazine treated T cells, and contributes to excessive B cell stimulation in vitro and in lupus. In this report we identify a genetic element that suppresses CD70 expression when methylated, and which demethylates in lupus and in T cells treated with Dnmt and ERK pathway inhibitors including procainamide and hydralazine. The results support a model in which demethylation of specific genetic elements in T cells, caused by decreasing Dnmt expression or inhibiting its function, contributes to drug-induced and idiopathic lupus through altered gene expression.

Chromatin structure, determined in part by DNA methylation, is established during differentiation and prevents expression of genes unnecessary for the function of a given cell type. We reported that DNA methylation and chromatin structure contributes to lymphoidspecific ITGAL (CD11a) and PRF1 (perforin) expression. We used bisulfite sequencing to compare methylation patterns in the ITGAL promoter and 5' flanking region of T cells and fibroblasts, and in the PRF1 promoter and upstream enhancer of CD4+ and CD8+ T cells with fibroblasts. The effects of methylation on promoter function were tested using regional methylation of reporter constructs, and confirmed by DNA methyltransferase inhibition. The relationship between DNA methylation and chromatin structure was analyzed by DNaseI hypersensitivity. Herein we described the methods and results in greater detail.

Inhibiting DNA methylation in CD4+ T cells causes aberrant gene expression and autoreactive monocyte/macrophage killing in vitro, and the hypomethylated cells cause a lupus-like disease in animal models. Similar decreases in T cell DNA methylation occur in idiopathic lupus, potentially contributing to disease pathogenesis. The genes affected by DNA hypomethylation are largely unknown. Using DNA methylation inhibitors and oligonucleotide arrays we have identified perforin as a methylation-sensitive gene. Our group has also reported that DNA methylation inhibitors increase CD4+ T cell perforin by demethylating a conserved methylation-sensitive region that is hypomethylated in primary CD8+ cells, which express perforin, but is largely methylated in primary CD4+ cells, which do not. As lupus T cells also have hypomethylated DNA and promiscuously kill autologous monocytes/macrophages, we hypothesized that perforin may be similarly overexpressed in lupus T cells and contribute to the monocyte killing. We report that CD4+ T cells from patients with active, but not inactive, lupus overexpress perforin, and that overexpression is related to demethylation of the same sequences suppressing perforin transcription in primary CD4+ T cells and demethylated by DNA methylation inhibitors. Further, the perforin inhibitor concanamycin A blocks autologous monocyte killing by CD4 lupus T cells, suggesting that the perforin is functional. We conclude that demethylation of specific regulatory elements contributes to perforin overexpression in CD4+ lupus T cells. Our results also suggest that aberrant perforin expression in CD4+lupus T cells may contribute to monocyte killing.

Objective. Generalized DNA hypomethylation contributes to altered T cell function and gene expression in systemic lupus erythematosus (SLE). Some of the overexpressed genes participate in the disease process, but the full repertoire of genes affected is unknown. Methylation-sensitive T cell genes were identified by treating T cells with the DNA methyltransferase inhibitor 5-azacytidine and comparing gene expression with oligonucleotide arrays. CD70, a costimulatory ligand for B cell CD27, was one gene that reproducibly increased. We then determined whether CD70 is overexpressed on T cells treated with other DNA methylation inhibitors and on SLE T cells, and determined its functional significance. Methods. Oligonucleotide arrays, real-time reverse transcription–polymerase chain reaction, and flow cytometry were used to compare CD70 expression in T cells treated with 2 DNA methyltransferase inhibitors (5-azacytidine and procainamide) and 3 ERK pathway inhibitors known to decrease DNA methyltransferase expression (U0126, PD98059, and hydralazine). The consequences of CD70 overexpression were tested by coculture of autologous T and B cells with and without anti-CD70 and measuring IgG production by enzymelinked immunosorbent assay. The results were compared with those of T cells from lupus patients. Results. SLE T cells and T cells treated with DNA methylation inhibitors overexpressed CD70 and overstimulated B cell IgG production. The increase in IgG synthesis was abrogated by anti-CD70. Conclusion. SLE T cells and T cells treated with DNA methyltransferase inhibitors and ERK pathway inhibitors overexpress CD70. This increased B cell costimulation and subsequent immunoglobulin overproduction may contribute to drug-induced and idiopathic lupus.

Perforin is a cytotoxic effector molecule expressed in NK cells and a subset of T cells. The mechanisms regulating its expression are incompletely understood. We observed that DNA methylation inhibition could increase perforin expression in T cells, so we examined the methylation pattern and chromatin structure of the human perforin promoter and upstream enhancer in primary CD4+ and CD8+ T cells as well as in an NK cell line that expresses perforin, compared with fibroblasts, which do not express perforin. The entire region was nearly completely unmethylated in the NK cell line and largely methylated in fibroblasts. In contrast, only the core promoter was constitutively unmethylated in primary CD4 and CD8 cells, and expression was associated with hypomethylation of an area residing between the upstream enhancer at-1 kb and the distal promoter at-0.3 kb. Treating T cells with the DNA methyltransferase inhibitor 5-azacytidine selectively demethylated this area and increased perforin expression. Selective methylation of this region suppressed promoter function in transfection assays. Finally, perforin expression and hypomethylation were associated with localized sensitivity of the 5- flank to DNase I digestion, indicating an accessible configuration. These results indicate that DNA methylation and chromatin structure participate in the regulation of perforin expression in T cells.