Fibrinogen Induced Inhibition of Osteoclastogenesis Is Reversed by Citrullination of Fibrinogen by Peptidylarginine Deiminase

Eun Young Lee¹, Ji Soo Kim², Hye Won Kim³, Sung Hae Chang⁴, Jin-Su Song⁵, Hie-Joon Kim⁵, Kyung-Hyun Park-Min⁶, Lionel B. Ivashkiv⁷, Eun Bong Lee⁸ and Yeong Wook Song⁹, ¹Division of Rheumatology, Department of Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea, ²Seoul National University College of Medicine, Seoul, South Korea, ³Rheumatology, Seoul National University College of Medicine, Seoul, South Korea, ⁴Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea, ⁵Department of Chemistry, Seoul National University, Seoul, South Korea, ⁶Rheumatology, Hospital for Special Surgery, New York, NY, ⁷Arthritis and Tissue Degeneration Program and the David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, NY, ⁸Seoul National University, Seoul, South Korea, ⁹Division of Rheumatology, Department of Internal Medicine,, Seoul National University College of Medicine, Seoul, South Korea

Meeting: 2012 ACR/ARHP Annual Meeting

Keywords: Citrulline and osteoclastogenesis

Session Information

Title: Rheumamtoid Arthritis - Human Etiology and Pathogenesis

Session Type: Abstract Submissions (ACR)

Background/Purpose: There are increasing evidences that autoantibodies and immune complexes (ICs) containing citrullinated fibrinogen are present in rheumatoid arthritis (RA) patients’ sera and contribute to synovitis. We have in vitro data that fibrinogen can inhibit osteoclastogenesis from precursor cells. In this experiment, we investigated the effect of citrullinated fibrinogen, which are abundant in RA, on the in vitro osteoclastogenesis.

Methods: Fibrinogen (human and bovine; 1mg/ml) was citrullinated in vitro by reacting with rabbit skeletal muscle peptidylarginine deiminase (rmPAD2). Soluble fibrinogen, citrullinated or non-citrullinated, were applied with increasing doses to buffy coat–derived CD14+ cells, and osteoclastogenesis was induced in the presence of M-CSF (20 ng/ml) and RANKL (40 ng/ml). To confirm a migration shift due to citrullination, western blotting was performed. The citrullinated sites of fibrinogen were analyzed using mass spectrometry.

Results: Fibrinogen inhibited osteoclastogenesis in a dose-dependent manner. In contrast, citrullinated fibrinogen via rmPAD2 did not show inhibition of osteoclastogenesis, which were evident with non-citrullinated fibrinogen (Figure). Several osteoclastogeneisis related genes, especially DC-STAMP, were suppressed by fibrinogen during osteoclastoegenesis, but restored by citrullinated fibrinogen. Citrullination of fibrinogen was confirmed by Western blot analysis and Mass spectrometry, showing peak changes between citrullinated and non-citrullinated fibrinogen at 1576 and 1593 m/z. Western blot with anti-citrullinated antibody showed that proteins from RA synovial fluid were more citrullinated than those from osteoarthritis (OA) synovial fluid.

Conclusion: Fibrinogen was successfully citrullinated by PAD and confirmed by Western blot and Mass spectrometry. In contrast to fibrinogen, citrullinated fibrinogen did not show inhibition of osteoclastogenesis. These results may suggest that citrullinated fibrinogen can contribute to osteoclastogenesis in RA patients.

Disclosure:

E. Y. Lee,
None;

J. S. Kim,
None;

H. W. Kim,
None;

S. H. Chang,
None;

J. S. Song,
None;

H. J. Kim,
None;

K. H. Park-Min,
None;

L. B. Ivashkiv,
None;

E. B. Lee,
None;

Y. W. Song,
None.

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