Background/Purpose:

Rheumatoid arthritis (RA) is a chronic inflammatory disorder characterized by synovial hyperplasia, progressive joint destruction and the presence of anti-citrullinated peptide antibodies (ACPAs) in sera. Citrullination, a post-translational modification of arginine to citrulline by peptidylarginine deiminase (PAD), contributes to the development of RA. We and others have shown that citrullination can modulate biological roles of key inflammatory molecules in RA. Interleukin 6 (IL-6) is highly expressed in synovial fluids (SFs) and sera of RA patients and plays an important role in RA synovitis. But no information is yet available regarding whether IL-6 is citrullinated in RA.

Methods: Recombinant human (rh) IL-6 was citrullinated by rhPAD4. Liquid chromatography-mass spectrometry (LC-MS) was performed to verify citrullination of IL-6. The role of citrullinated IL-6 (citIL-6) in RA fibroblast-like synoviocytes (FLS) proliferation and migration was determined using the IncuCyte S3 live cell analysis system and scratch wound assays. We performed monocyte (MN) chemotaxis assays using a modified Boyden chamber to examine the effect of citIL-6 on MN migration. To evaluate the arthritogenic properties of citIL-6 in vivo, noncitrullinated IL-6 (noncitIL-6) or citIL-6 was injected into mouse knees and joint circumference was measured at 0 hour and 24 hours after injection. We also identified the presence of citIL-6 and ACPAs against citIL-6 in RA SFs and sera by performing Western blotting and immunodot blot assay, respectively. FLS were stimulated with citIL-6 or noncitIL-6 to examine the phosphorylation of downstream signaling molecules by Western blotting without the addition of exogenous IL-6 receptor (IL-6R).

Results:

LC-MS confirmed that all of the arginines in rhIL-6 can be citrullinated by rhPAD4. Both scratch wound assay and live cell imaging analysis showed that FLS proliferation and migration rates were significantly higher in the citIL-6 group compared to the noncit-IL-6 group after 24 hours. We also found that citIL-6 induced more MN migration than noncitIL-6 (p<0.001). The change in mouse knee circumference with citIL-6 injection was approximately 7-fold higher than that with noncitIL-6 injection (0.90±0.27 mm vs 0.13±0.13 mm; n=14; p<0.05), indicating that citIL-6 induced much more severe inflammation in mouse knees compared to noncitIL-6. Western blot assays showed that citIL-6 was present in the SFs from RA. Immunodot blot assay showed that sera from RA patients but not healthy controls contained ACPAs react with citIL-6 but not noncitIL-6. CitIL-6, without exogenous IL-6R, upregulated phosphorylation of Erk1/2, Jnk, and Stat3 in RA FLS while noncitIL-6 did not.

Conclusion:

IL-6 can be citrullinated by PAD, citIL-6 was present in RA SFs, and citIL-6 ACPAs were present in RA sera. CitIL-6 may play an important role in the pathogenesis of RA by inducing proliferation as well as migration of FLS and recruiting monocytes via a special mechanism that is different from noncitIL-6.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/expression-and-function-of-a-novel-citrullinated-form-of-interleukin-6-in-rheumatoid-arthritis/