J Clin Immunol. 2013 Apr;33(3):558-66.

Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells.

Lu MC, Lai NS, Yin WY, Yu HC, Huang HB, Tung CH, Huang KY, Yu CL.

Division of Allergy, Immunology and Rheumatology, Buddhist Dalin Tzu Chi General Hospital, Chia-Yi, Taiwan.



In a previous study, we found that anti-citrullinated protein antibodies (ACPAs) enhance nuclear factor (NF)-κB activity and tumor necrosis factor (TNF)-α production by normal human peripheral blood mononuclear cells (PBMCs) and U937 cells via binding to surface-expressed citrullinated glucose-regulated protein 78 (cit-GRP78). However, the downstream signaling pathways remain unclear after binding. In the present study, we firstly measured the effects of different kinase inhibitors on ACPA-mediated TNF-α production from normal PBMCs and monocytes. Then, the native and phosphorylated mitogen-activated protein kinases (MAPKs) were detected in ACPA-activated U937 cells by Western blotting. We also explored the role of the phosphoinositide 3-kinase (PI3K)-Akt pathway in activating IκB kinase alpha (IKK-α) in ACPA-stimulated U937 cells. Finally, we measured the amount of cit-GRP78 from PBMC membrane extracts in RA patients and controls. We found that MAPK and Akt inhibitors, but not PI3K inhibitor, remarkably suppressed ACPA-mediated TNF-α production. Interestingly, ACPAs selectively activated extracellular signal-regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase (JNK), but not p38 MAPK, in U937 cells. This activation was suppressed by cit-GRP78, but not GRP78. The JNK activation further enhanced the phosphorylation of Akt and IKK-α. The expression of cit-GRP78 on cell membrane was higher in RA than normal PBMCs. Taken together; these results suggest that through binding to surface, over-expressed cit-GRP78 on RA PBMCs, ACPAs selectively activate ERK1/2 and JNK signaling pathways to enhance IKK-α phosphorylation, which leads to the activation of NF-κB and the production of TNF-α .

PMID: 23188524


[General background]

Rheumatoid arthritis (RA) is a common chronic inflammatory disease with potential disability. The prevalence of RA is around 0.4-1% in the world. Although the etiopathogenesis of RA remains elucidation, the presence of anti-citrullinated protein antibodies (ACPA) in the serum is identified as the unique biomarker for RA diagnosis. Both sensitivity and specificity of ACPA for RA diagnosis are even higher than rheumatoid factors or other antibodies (1). In addition, ACPA can be used as a useful predictor of the development to RA in pre-clinical or undifferentiated arthritis stages. However, the most valuable application of ACPA in clinical practice is that the serum title of ACPA can reflect the severity of joint inflammation and disease activity of patients with RA. Accordingly, the measurement of serum ACPA can be used as an accurate therapeutic monitoring parameter for drug efficacy in RA treatment. In short, ACPA is a unique biomarker for RA in consideration that many different autoimmune diseases (i.e. SLE, Sjogren’s syndrome, systemic sclerosis) or chronic infections (i.e. chronic viral hepatitis, tuberculosis or leprosy) may produce rheumatoid factors, but not ACPA.

[The findings of our studies]

It is conceivable that the cognate autoantigens for ACPA are the citrullinated proteins in the joints. Citrulline is an amino acid not belongs to the regular 20 amino acid residues. It is a neutral-charged amino acid from positive charged arginine after post-translational modification by peptidylarginine deiminase (PAD) in the presence of high Ca2+environment (Fig.1). The matrical proteins such as collagenous proteins or fibrinogen are the targets for tissue PAD. The conjugation of ACPA and citrullinated-matrical proteins becomes immune complex that may activate complement system to induce inflammation and tissue damage. To further elucidate the immunopathological roles of APCAs in rheumatoid arthritis especially focusing on the correlation with disease activity, we co-cultured normal human mononuclear cells (MNC) with ACPA purified from active RA sera. We demonstrated that ACPA potently stimulates MNC to produce TNF-α in our previous report (2). Furthermore, we identified the surface-expressed citrullinated-glucose regulated protein 78 (cit-GRP78) as the target for ACPA. Based on these findings, we argue that the immunopathological roles of ACPA in rheumatoid synovitis may include two parts: (1) immune complex-mediate inflammation, (2) direct stimulation of monocytes/ macrophages to produce TNF-α (Fig.1). For further exploring the molecular bases of ACPA on monocytes/macrophages TNF-α gene expression, we used different inhibitors to block MAPKs and PI3K signaling pathways. We noted two of the MAPK signaling molecules, ERK1/2 and JNK, are phosphorylated by ACPA after binding to surface-expressed cit-GRP78 on monocytes/macrophages. However, P38-MAPK is not involved in TNF-α expression.


Fig.1: The possible mechanism of ACPA genesis and their roles in rheumatoid synovitis


f2Fig.2: Signaling pathway of ACPAs to induce TNF-α production from monocytes/macrophages


[The importance of the studies]

Two aspects are considered:

  1. We are the first to explore the molecular bases of the close correlation between the title of ACPA and disease activity in the patients with rheumatoid arthritis. We identified the cognate autoantigen for ACPA is the surface-expressed cit-GRP78 on monocytes/macrophages different from the findings of other authors. As depicted in Fig.1, we hypothesize that a least two mechanisms many elicit inflammation and tissue damage in RA; (a) immune complex-complement– mediated model (2) direct induction of TNF-α production model by macrophages.
  2. Potential clinical applications in the therapy of RA:
  3. In the present study, we clearly demonstrate that P38- and ERK1/2-MAPK -Akt-NF-kB signaling pathways are activated by ACPA to induce TNF-α gene expression. It is anticipated that a specific Akt inhibitor may be a potential therapeutic agent for RA treatment. On the other hand, blocking antibody against membrane expressed cit-GRP78 may become another candidate to prevent TNF-α production by monocytes/macrophages. However, more and more investigations are mandatory to confirm it.


  1. Lu MC, Hsieh SC, Lai NS, Li KJ, Yu CH, Yu CL. Comparison of anti-agalactosyl IgG antibodies, rheumatoid factors, and anti-cyclic citrullinated peptide antibodies in the differential diagnosis of rheumatoid arthritis and its mimics. Clin Exp Rheumatol 2007; 25: 716-21.
  2. Lu MC, Lai NS, Yu HC, Huang HB, Hsieh SC, Yu CL. Anti-citrullinated protein antibodies bind surface-expressed citrullinated Grp78 on monocytes/macrophages and stimulate tumor necrosis factor alpha production. Arthritis Rheum 2010; 62: 1213-23.
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