Methylation status of a single CpG site in the IL6 promoter is related to IL6 messenger RNA levels and rheumatoid arthritis

Objective. Genetic variation in the gene for interleukin-6 (IL-6) contributes to the pathogenesis of inflammatory arthritis, but the role, if any, of epigenetic variability has not been reported. The aims of this study were to compare the DNA methylation status of the IL6 promoter in rheumatoid arthritis (RA) patients and control subjects and to study the effects on gene expression. Methods. Genomic DNA was isolated from peripheral blood mononuclear cells (PBMCs) obtained from RA patients and healthy controls. Macrophages from healthy controls were isolated and stimulated with lipopolysaccharide (LPS). Methylation status was determined using bisulfite genomic sequencing and IL6 messenger RNA (mRNA) levels by quantitative polymerase chain reaction. Gel shift assays were performed with methylated or unmethylated probes and HeLa cell nuclear extract. Results. The proximal CpG motifs (-666 to +27) were predominantly unmethylated and the upstream motifs (-1099 to -1001) were highly methylated in PBMCs from patients and controls. Methylation of individual CpG motifs was similar, except at -1099C, which was less methylated in the patients than in the controls (58% versus 98%; P = 1 X 10(-6)). To test whether this observation might relate to gene regulation, LPS-stimulated macrophages were grouped according to their IL6 mRNA stimulation index (SI). The level of methylation at -1099C was significantly lower in the group with high (SI > 75) compared with the group with low (SI < 10) induced mRNA levels (71%, versus 93%; P = 0.007). Gel shift assays revealed decreased protein binding to the -1099C unmethylated probe. Conclusion. These data suggest that methylation of a single CpG in the IL6 promoter region may affect IL6 gene regulation and may play a role in the pathogenesis of RA.