Aberrant T Cell DNA Methylation Gene Expression and Cellular Function in Idiopathic Lupus

4.1. DNA Methylation

Initial studies compared total genomic dmC content in T cells from patients with inactive and active lupus with controls using reverse-phase HPLC. These studies demonstrated that T cells from patients with active lupus have decreased genomic dmC levels as well as decreased levels of DNA methyltransferase activity. Intracellular pools of SAM and S-adenosylhomocysteine, both regulators of transmethylation reactions, were normal (Richardson et al., 1990). Subsequent studies demonstrated decreased Dnmt1 mRNA levels, implicating decreased maintenance DNA methylation in the DNA hypomethylation (Richardson et al., 1990; Deng et al., 2001). Dnmt3a, but not Dnmt3b, transcripts were also decreased in lupus T cells (C. Deng and B. Richardson, unpublished data). Mechanisms contributing to the decreased DNA methyltransferase expression and consequent DNA hypomethylation were sought.

Others have reported that signaling is abnormal in lupus T cells (Kammer et al., 2002), and our group reported that Dnmt1 and Dnmt3a levels are regulated in part by the JNK and ERK pathways (Deng et al., 2003). Furthermore, inhibiting either ERK or JNK signaling in proliferating cells demethylates DNA (Deng et al., 1998). We therefore examined JNK and ERK pathway signaling in lupus T cells. JNK pathway signaling was found to be intact. ERK pathway signaling, however, is decreased in T cells from patients with active lupus, with levels of Dnmt1 mRNA decreased to the same degree as in T cells treated with ERK

pathway inhibitors (Deng et al., 2001). Since hydralazine and MEK inhibitors decrease Dnmt1 and Dnmt3a expression, demethylate DNA, and because T cells treated with these drugs induce autoimmunity (Deng et al., 2003), decreased T cell ERK pathway signaling may contribute to DNA hypomethylation and autoimmunity in idiopathic lupus by similar mechanisms.

4.2. Gene Expression and Cellular Function

As noted above, CD11a, perforin, and CD70 have been identified as methy-lation-sensitive T cell genes that may contribute to the pathogenesis of DIL. Evidence for overexpression of these genes was therefore sought in T cells from lupus patients.

A subset of T cells was found to express high levels of LFA-1 in lupus patients compared with controls, which included normal individuals, patients with active infections, and patients with multiple sclerosis (Richardson et al., 1992). The degree of overexpression correlated directly with clinical disease activity. In addition, the cells overexpressing LFA-1 were found to be autoreactive and spontaneously lysed autologous, but not allogeneic, macrophages, similar to T cells treated with DNA hypomethylating agents (Richardson et al., 1992). Bisulfite sequencing of the CD11a promoter and flanking regions in T cells from active lupus patients showed that the sequences demethylated are the same as those demethylated in T cells treated with 5-azaC and procainamide (Lu et al., 2002). Figure 6.3 compares the effects of 5-azaC and lupus on CD11a promoter methylation in T cells.

Perforin is also overexpressed in lupus T cells. CD4+ T cells from patients with active, but not inactive, lupus aberrantly express perforin, and the abnormal expression is related to demethylation of the same sequences suppressing perforin transcription in primary CD4+ T cells, which are demethylated by DNA methylation inhibitors (Kaplan et al., 2004) (Figure 6.4). Furthermore, the perforin inhibitor concanamycin A blocks autologous monocyte killing by CD4+ lupus T cells, suggesting that aberrant perforin expression in CD4+ lupus T cells may contribute to autoreactive monocyte/macrophage killing (Kaplan et al., 2004).

CD70 was recently found to be overexpressed in lupus T cells as well. CD4+ SLE T cells overexpress CD70 and stimulate B cell IgG production, similar to T cells treated with DNA methylation inhibitors (Oelke et al., 2004), or transfected with CD70 (Kobata et al., 1995). The abnormal T cell-dependent IgG secretion that characterizes lupus B cells is inhibited by anti-CD70 (Liossis and Tsokos, 1999), as predicted by the in vitro model. Preliminary work has shown that DNA methylation inhibitors demethylate a region ~500 bp 5' to the CD70 transcription start site and that CD4+ T cells from patients with active lupus exhibit hypomethy-lation in the same region (Q. Lu and B. Richardson, unpublished data).

Together, these studies demonstrate that lupus T cells aberrantly overexpress LFA-1, perforin, and CD70, that the same genes are overexpressed in the hypomethylation model, and that the same sequences are demethylated as in T cells treated with DNA methyltransferase or ERK pathway inhibitors. These observations strongly suggest that similar mechanisms may be contributing to overexpression of these genes in the DNA hypomethylation model and in lupus.

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