Background/Purpose: Rheumatoid arthritis (RA) is a chronic inflammatory disorder featured by synovial hyperplasia, inflammatory cell infiltration and 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 have shown that interleukin 6 (IL-6) can be citrullinated and mediates inflammation in RA. Additional experiments were performed to define the receptor for citrullinated IL-6 (citIL-6).

Methods: Recombinant human (rh) IL-6 was incubated with rhPAD4 and the citrullination of IL-6 was verified by liquid chromatography-mass spectrometry (LC-MS). We identified the presence of citIL-6 and ACPAs against citIL-6 in RA synovial fluids (SFs) and sera by performing Western blotting and immunodot blot assay, respectively. In vitro, monocyte (MN) chemotaxis assays were performed with modified Boyden chambers to examine the effect of citIL-6 on MN migration. The role of citIL-6 in RA fibroblast-like synoviocyte (FLS) proliferation and migration was determined using the IncuCyte S3 live-cell analysis system and scratch wound assays. FLS were stimulated with citIL-6 or noncitIL-6 to examine the phosphorylation of signaling molecules by Western blotting without exogenous IL-6 receptor (IL-6R). To define the receptor for citIL-6, RA FLS were transfected with gp130 siRNA or blocked with sarilumab, an IL-6R neutralizing antibody, and signaling molecules were tested by Western blotting after stimulation with citIL-6 or noncitIL-6. In vivo, citIL-6 or noncitIL-6 was injected into mouse knees and joint circumference was measured at 0 hour and 24 hours after injection.

Results: LC-MS confirmed that all of the arginines in rhIL-6 can be citrullinated by rhPAD4. Western blotting showed that citIL-6 was present in the SFs from RA, and immunodot blot assay showed that sera from RA patients but not healthy controls contained ACPAs reactive with citIL-6 but not noncitIL-6. Compared to noncitIL-6, citIL-6 induced more MN migration (p< 0.001). We found that citIL-6 induced significantly higher FLS proliferation and migration rates after 24 hours than noncitIL-6. CitIL-6, without exogenous IL-6R, consistently upregulated phosphorylation of Erk1/2, Jnk, and Stat3 in RA FLS while noncitIL-6 did not unless exogenous IL-6R was added. Furthermore, gp130 knock down or monoclonal antibody blockade of the IL-6R abrogated the phosphorylation of Stat3 and Erk1/2 in FLS by both citIL-6 and noncitIL-6, indicating IL-6R and gp130 are necessary for function of both forms of IL-6. The change in mouse knee circumference with citIL-6 injection was approximately 7-fold higher than that with noncitIL-6 injection (0.90±0.13 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.

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 appears to be a more potent ligand of IL-6R and play an important role in the pathogenesis of RA by inducing proliferation as well as migration of FLS and recruitment of monocytes.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/citrullination-of-interleukin-6-augments-its-pro-inflammatory-capacity-and-signaling-potency-through-interleukin-6-receptor-in-rheumatoid-arthritis/