Identification of novel biomarker as citrullinated inter-alpha-trypsin inhibitor heavy chain 4, specifically increased in sera with experimental and rheumatoid arthritis

Arthritis Res Ther. 2018 Apr 10;20(1):66. doi: 10.1186/s13075-018-1562-7.


Background: Anticitrullinated protein antibodies (ACPA) and citrullinated proteins play key roles in the pathogenesis of rheumatoid arthritis (RA). Many candidate citrullinated antigens have been identified in joints, but citrullinated proteins in sera are mostly uncertain in patients with RA. We explored the expression of citrullinated proteins in joints and sera of experimental arthritis, and we further investigated their specific expression correlated with the disease activity in patients with RA.

Methods: Citrullinated protein expression in tissues was examined by IHC in peptide glucose-6-phosphate isomerase-induced arthritis (pGIA). Serum citrullinated proteins from pGIA were examined by Western blotting, and the sequence was identified by MS. With the same methods, serum citrullinated proteins were analyzed in patients with RA, primary Sjögren's syndrome, systemic lupus erythematosus, and osteoarthritis as well as in healthy subjects, by Western blotting and MS. In patients with RA, the relationship between the expression of the identified protein (inter-alpha-trypsin inhibitor heavy chain 4 [ITIH4]) and clinical features was evaluated, and the levels of citrullinated ITIH4 were compared before and after biological treatment. The antibody response against citrullinated ITIH4 peptide was measured by enzyme-linked immunosorbent assay.

Results: Citrullinated proteins were detected specifically in arthritic joints and sera from pGIA relative to controls. In sera, a common band of citrullinated protein at 120 kDa was revealed, and it fluctuated in parallel with arthritis score of pGIA by Western blotting. Interestingly, in 82% of RA patient sera, similar bands of citrullinated protein were specifically detected. These proteins were identified as citrullinated ITIH4, and especially the R438 site was commonly citrullinated between mice and humans. Citrullinated ITIH4 levels were associated with clinical parameters such as C-reactive protein (CRP), rheumatoid factor, and Disease Activity Score in 28 joints as measured by CRP in patients with RA. Its levels were decreased in correlation with the reduction of disease activity score after effective treatment in patients with RA. Moreover, antibody response to citrullinated epitope in ITIH4 was specifically observed in patients with RA.

Conclusions: Our results suggest that serum citrullinated ITIH4 was specifically increased in patients with RA and could be a novel biomarker for assessing disease activity in patients with RA.

Keywords: Citrullinated proteins; Inter-alpha-trypsin inhibitor heavy chain 4; PAD; Rheumatoid arthritis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Animals
  • Anti-Citrullinated Protein Antibodies / blood
  • Anti-Citrullinated Protein Antibodies / immunology
  • Arthritis, Experimental / blood
  • Arthritis, Experimental / immunology
  • Arthritis, Rheumatoid / blood*
  • Arthritis, Rheumatoid / immunology*
  • Biomarkers / blood*
  • Blood Proteins / immunology
  • Citrullination
  • Female
  • Glycoproteins / blood*
  • Glycoproteins / immunology
  • Humans
  • Male
  • Mice
  • Middle Aged
  • Proteinase Inhibitory Proteins, Secretory / blood*
  • Proteinase Inhibitory Proteins, Secretory / immunology
  • Young Adult


  • Anti-Citrullinated Protein Antibodies
  • Biomarkers
  • Blood Proteins
  • Glycoproteins
  • ITIH4 protein, human
  • Proteinase Inhibitory Proteins, Secretory