Background/Purpose

Interleukin (IL)-6 is an important driver of rheumatoid arthritis (RA) pathology, and synovial fibroblasts are a major source of IL-6 in the RA synovium.  We noted large differences in IL-6 expression across several human synovial fibroblast lines.  Our objective was to better define the variation in IL-6 expression between synovial fibroblasts and identify mechanisms mediating these differences.

Methods

Human synovial fibroblast lines were derived from discarded surgical specimens by serial passage from ten RA or osteoarthritis (OA) donors.  Human CD14+ monocytes were isolated from healthy donor peripheral blood mononuclear cells by magnetic bead positive selection. IL-6 was measured in cell culture media by ELISA.  IL-6 mRNA expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR).  IL-6 transcript stability was determined by measuring IL-6 mRNA decay after actinomycin D inhibition of transcription.  Cell genotype at the IL-6 proximal promoter single nucleotide polymorphism (SNP) rs18000795 (otherwise known as IL-6 -174 G/C) was determined by restriction fragment length polymorphism analysis.

Results

Human synovial fibroblast lines reproducibly segregated into low, medium, and high IL-6 producers after tumor necrosis factor-alpha (TNF-α) stimulation, independent of cell passage and disease state.  The IL-6 expression pattern after TNF-α stimulation correlated significantly with the expression pattern observed in unstimulated cells or cells stimulated with IL-1 or lipopolysaccharide (LPS), indicating that this pattern was not secondary to differences in TNF-α signaling pathways.  The IL-6 expression pattern also correlated strongly with total mRNA expression, but not with differences in IL-6 mRNA stability, suggesting it was driven by transcriptional rather than post-transcriptional mechanisms.  Given that the proximal promoter SNP rs18000795 has been associated with diverse effects on IL-6 levels and disease expression in many systems, we analyzed synovial fibroblast IL-6 production as a function of rs18000795 genotype.  We found that high IL-6 expression was significantly associated with the homozygous minor allele (CC) genotype.  We then tested if a similar genotype effect was seen in stimulated CD14+ monocytes, since macrophage lineage cells are another major IL-6 producer in the RA synovium.  We found, in contrast to synovial fibroblasts, that the rs18000795 genotype had a modest and opposite effect on CD14+ monocyte IL-6 expression, with a trend toward higher production in the major allele (GG) homozygotes.

Conclusion

This study reports that synovial fibroblast IL-6 expression is significantly influenced by genetic differences in linkage with the IL-6 proximal promoter SNP, rs18000795.  In contrast, little association between rs18000795 and monocyte IL-6 expression was found in this study, showing that SNP associations may have divergent effect when analyzed in different cell types.  These results highlight that analysis of the role of disease-associated SNPs on gene expression and pathologic processes must consider the effect of that genetic variation in different cell types.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/il-6-proximal-promoter-snp-rs18000795-genotype-strongly-correlates-with-synovial-fibroblast-il-6-expression/