OBJECTIVE: To quantify peptidylarginine deiminase 2 (PAD2) in SF of RA patients and OA patients, and to determine the association between PAD2 levels, disease activity and inflammatory markers in RA. METHODS: Blood and SF samples were obtained from 39 RA patients and 40 patients with OA. PAD2 content and PAD activity were measured by means of in-house assays. TNF-α, IL-1β, IL-6, IL-8, IL-10 and IL-12 were measured using flow cytometry. RESULTS: PAD2 levels and PAD activity were higher in SF from RA than OA patients (P < 0.0001 and P = 0.03, respectively), as were all cytokine levels (P < 0.0001-0.05). SF PAD2 levels were higher among anti-CCP-positive patients than among anti-CCP-negative patients (P = 0.005). PAD2 levels in SF from RA patients correlated with disease activity, as assessed by DAS28 (P < 0.005). Moreover, SF PAD2 levels correlated with circulating CRP and anti-CCP levels (P < 0.0006), as well as with leucocyte count, IL-6, IL-8 and IL-10 levels in SF (P < 0.0001-0.02). PAD activity in SF was higher in RA patients than in OA patients, and correlated with PAD2 concentration. CONCLUSION: Extracellular PAD2 levels in SF correlate with disease activity in RA patients. Anti-CCP-positive RA patients have higher PAD2 levels in SF than anti-CCP-negative RA patients and OA patients. Since we could demonstrate enzymatically active PADs in SF, we propose that free, extracellular PAD is of pathogenic relevance.

Institute for Inflammation Research Center for Rheumatology and Spine Diseases Copenhagen University Hospital Rigshospitalet Section for Periodontology Microbiology and Community Dentistry Department of Odontology Faculty of Health and Medical Sciences University of Copenhagen Copenhagen Denmark and

Institute of Rheumatology and Department of Rheumatology 1 Faculty of Medicine Charles University Prague Czech Republic

Zobrazit více v PubMed

Gabriel SE. The epidemiology of rheumatoid arthritis. Rheum Dis Clin North Am 2001;27:269–81. PubMed

Sato K, Takayanagi H. Osteoclasts, rheumatoid arthritis, and osteoimmunology. Curr Opin Rheumatol 2006;18:419–26. PubMed

Pap T, Meinecke I, Muller-Ladner U, Gay S. Are fibroblasts involved in joint destruction? Ann Rheum Dis 2005;64 (Suppl 4):iv52–4.. PubMed PMC

Schellekens GA, de Jong BA, van den Hoogen FH, van de Putte LB, van Venrooij WJ. Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. J Clin Invest 1998;101:273–81. PubMed PMC

Gregersen PK, Silver J, Winchester RJ. The shared epitope hypothesis. An approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis. Arthritis Rheum 1987;30:1205–13. PubMed

Hill JA, Southwood S, Sette A. et al. Cutting edge: the conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class II molecule. J Immunol 2003;171:538–41. PubMed

Lindqvist E, Eberhardt K, Bendtzen K, Heinegard D, Saxne T. Prognostic laboratory markers of joint damage in rheumatoid arthritis. Ann Rheum Dis 2005;64:196–201. PubMed PMC

van der Helm-van Mil AH, Verpoort KN, Breedveld FC, Toes RE, Huizinga TW. Antibodies to citrullinated proteins and differences in clinical progression of rheumatoid arthritis. Arthritis Res Ther 2005;7:R949–R958. PubMed PMC

Syversen SW, Gaarder PI, Goll GL. et al. High anti-cyclic citrullinated peptide levels and an algorithm of four variables predict radiographic progression in patients with rheumatoid arthritis: results from a 10-year longitudinal study. Ann Rheum Dis 2008;67:212–7. PubMed

Ronnelid J, Wick MC, Lampa J. et al. Longitudinal analysis of citrullinated protein/peptide antibodies (anti-CP) during 5 year follow up in early rheumatoid arthritis: anti-CP status predicts worse disease activity and greater radiological progression. Ann Rheum Dis 2005;64:1744–9. PubMed PMC

Miriovsky BJ, Michaud K, Thiele GM. et al. Anti-CCP antibody and rheumatoid factor concentrations predict greater disease activity in men with rheumatoid arthritis. Ann Rheum Dis 2010;69:1292–7. PubMed PMC

Vossenaar ER, Zendman AJ, van Venrooij WJ, Pruijn GJ. PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. Bioessays 2003;25:1106–18. PubMed

Nakayama-Hamada M, Suzuki A, Kubota K. et al. Comparison of enzymatic properties between hPADI2 and hPADI4. Biochem Biophys Res Commun 2005;327:192–200. PubMed

Darrah E, Rosen A, Giles JT, Andrade F. Peptidylarginine deiminase 2, 3 and 4 have distinct specificities against cellular substrates: novel insights into autoantigen selection in rheumatoid arthritis. Ann Rheum Dis 2012;71:92–8. PubMed PMC

Assohou-Luty C, Raijmakers R, Benckhuijsen WE. et al. The human peptidylarginine deiminases type 2 and type 4 have distinct substrate specificities. Biochim Biophys Acta 2014;1844:829–36. PubMed

Damgaard D, Senolt L, Nielsen M, Pruijn G, Nielsen CH. Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen. Arthritis Res Ther 2014;16:498. PubMed PMC

Lundberg K, Nijenhuis S, Vossenaar ER. et al. Citrullinated proteins have increased immunogenicity and arthritogenicity and their presence in arthritic joints correlates with disease severity. Arthritis Res Ther 2005;7:R458–67. PubMed PMC

Foulquier C, Sebbag M, Clavel C. et al. Peptidyl arginine deiminase type 2 (PAD-2) and PAD-4 but not PAD-1, PAD-3, and PAD-6 are expressed in rheumatoid arthritis synovium in close association with tissue inflammation. Arthritis Rheum 2007;56:3541–53. PubMed

Van SK, Tilleman K, De Ceuleneer M. et al. Citrullinated vimentin as an important antigen in immune complexes from synovial fluid of rheumatoid arthritis patients with antibodies against citrullinated proteins. Arthritis Res Ther 2010;12:R132. PubMed PMC

Feitsma AL, van der Voort EI, Franken KL. et al. Identification of citrullinated vimentin peptides as T cell epitopes in HLA-DR4-positive patients with rheumatoid arthritis. Arthritis Rheum 2010;62:117–25. PubMed

Kinloch A, Lundberg K, Wait R. et al. Synovial fluid is a site of citrullination of autoantigens in inflammatory arthritis. Arthritis Rheum 2008;58:2287–95. PubMed

Vossenaar ER, Smeets TJ, Kraan MC. et al. The presence of citrullinated proteins is not specific for rheumatoid synovial tissue. Arthritis Rheum 2004;50:3485–94. PubMed

Makrygiannakis D, Af KE, Lundberg IE. et al. Citrullination is an inflammation-dependent process. Ann Rheum Dis 2006;65:1219–22. PubMed PMC

Chapuy-Regaud S, Sebbag M, Baeten D. et al. Fibrin deimination in synovial tissue is not specific for rheumatoid arthritis but commonly occurs during synovitides. J Immunol 2005;174:5057–64. PubMed

Gudmann NS, Hansen NU, Jensen AC, Karsdal MA, Siebuhr AS. Biological relevance of citrullinations: diagnostic, prognostic and therapeutic options. Autoimmunity 2014;48:73–9. PubMed

Cherrington BD, Zhang X, McElwee JL. et al. Potential role for PAD2 in gene regulation in breast cancer cells. PLoS One 2012;7:e41242. PubMed PMC

Mohanan S, Cherrington BD, Horibata S. et al. Potential role of peptidylarginine deiminase enzymes and protein citrullination in cancer pathogenesis. Biochem Res Int 2012;2012:895343. PubMed PMC

Suzuki A, Yamada R, Chang X. et al. Functional haplotypes of PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are associated with rheumatoid arthritis. Nat Genet 2003;34:395–402. PubMed

Plenge RM, Padyukov L, Remmers EF. et al. Replication of putative candidate-gene associations with rheumatoid arthritis in > 4,000 samples from North America and Sweden: association of susceptibility with PTPN22, CTLA4, and PADI4. Am J Hum Genet 2005;77:1044–60. PubMed PMC

Hoppe B, Haupl T, Gruber R. et al. Detailed analysis of the variability of peptidylarginine deiminase type 4 in German patients with rheumatoid arthritis: a case-control study. Arthritis Res Ther 2006;8:R34. PubMed PMC

Barton A, Bowes J, Eyre S. et al. A functional haplotype of the PADI4 gene associated with rheumatoid arthritis in a Japanese population is not associated in a United Kingdom population. Arthritis Rheum 2004;50:1117–21. PubMed

Martinez A, Valdivia A, Pascual-Salcedo D. et al. PADI4 polymorphisms are not associated with rheumatoid arthritis in the Spanish population. Rheumatology 2005;44:1263–6. PubMed

Burr ML, Naseem H, Hinks A. et al. PADI4 genotype is not associated with rheumatoid arthritis in a large UK Caucasian population. Ann Rheum Dis 2010;69:666–70. PubMed PMC

Chang X, Xia Y, Pan J. et al. PADI2 is significantly associated with rheumatoid arthritis. PLoS One 2013;8:e81259. PubMed PMC

Vossenaar ER, Radstake TR, van der Heijden A. et al. Expression and activity of citrullinating peptidylarginine deiminase enzymes in monocytes and macrophages. Ann Rheum Dis 2004;63:373–81. PubMed PMC

Asaga H, Yamada M, Senshu T. Selective deimination of vimentin in calcium ionophore-induced apoptosis of mouse peritoneal macrophages. Biochem Biophys Res Commun 1998;243:641–6. PubMed

De Rycke L, Nicholas AP, Cantaert T. et al. Synovial intracellular citrullinated proteins colocalizing with peptidyl arginine deiminase as pathophysiologically relevant antigenic determinants of rheumatoid arthritis-specific humoral autoimmunity. Arthritis Rheum 2005;52:2323–30. PubMed

Takizawa Y, Suzuki A, Sawada T. et al. Citrullinated fibrinogen detected as a soluble citrullinated autoantigen in rheumatoid arthritis synovial fluids. Ann Rheum Dis 2006;65:1013–20. PubMed PMC

Arnett FC, Edworthy SM, Bloch DA. et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24. PubMed

Damgaard D, Palarasah Y, Skjodt K. et al. Generation of monoclonal antibodies against peptidylarginine deiminase 2 (PAD2) and development of a PAD2-specific enzyme-linked immunosorbent assay. J Immunol Methods 2014;405:15–22. PubMed

Nielsen CH, Hegedus L, Rieneck K. et al. Production of interleukin (IL)-5 and IL-10 accompanies T helper cell type 1 (Th1) cytokine responses to a major thyroid self-antigen, thyroglobulin, in health and autoimmune thyroid disease. Clin Exp Immunol 2007;147:287–95. PubMed PMC

Zhao X, Okeke NL, Sharpe O. et al. Circulating immune complexes contain citrullinated fibrinogen in rheumatoid arthritis. Arthritis Res Ther 2008;10:R94. PubMed PMC

van Venrooij WJ, Pruijn GJ. An important step towards completing the rheumatoid arthritis cycle. Arthritis Res Ther 2008;10:117. PubMed PMC

Nielsen CH, Leslie RG, Jepsen BS. et al. Natural autoantibodies and complement promote the uptake of a self antigen, human thyroglobulin, by B cells and the proliferation of thyroglobulin-reactive CD4(+) T cells in healthy individuals. Eur J Immunol 2001;31:2660–8. PubMed

Brimnes MK, Hansen BE, Nielsen LK, Dziegiel MH, Nielsen CH. Uptake and presentation of myelin basic protein by normal human B cells. PLoS One 2014;9:e113388. PubMed PMC

Vossenaar ER, van Venrooij WJ. Citrullinated proteins: sparks that may ignite the fire in rheumatoid arthritis. Arthritis Res Ther 2004;6:107–11. PubMed PMC

Arandjelovic S, McKenney KR, Leming SS, Mowen KA. ATP induces protein arginine deiminase 2-dependent citrullination in mast cells through the P2X7 purinergic receptor. J Immunol 2012;189:4112–22. PubMed PMC

Spengler J, Lugonja B, Ytterberg AJ. et al. Release of active peptidyl arginine deiminases by neutrophils can explain production of extracellular citrullinated autoantigens in RA synovial fluid. Arthritis Rheumatol 2015;67:3135–45. PubMed PMC

de Lange-Brokaar BJ, Ioan-Facsinay A, van Osch GJ. et al. Synovial inflammation, immune cells and their cytokines in osteoarthritis: a review. Osteoarthritis Cartilage 2012;20:1484–99. PubMed

Sanchez-Pernaute O, Filkova M, Gabucio A. et al. Citrullination enhances the pro-inflammatory response to fibrin in rheumatoid arthritis synovial fibroblasts. Ann Rheum Dis 2013;72:1400–6. PubMed

Doss F, Menard J, Hauschild M. et al. Elevated IL-6 levels in the synovial fluid of osteoarthritis patients stem from plasma cells. Scand J Rheumatol 2007;36:136–9. PubMed

Applicability of Small-Molecule Inhibitors in the Study of Peptidyl Arginine Deiminase 2 (PAD2) and PAD4

Relative efficiencies of peptidylarginine deiminase 2 and 4 in generating target sites for anti-citrullinated protein antibodies in fibrinogen, alpha-enolase and histone H3

Calgizzarin (S100A11): a novel inflammatory mediator associated with disease activity of rheumatoid arthritis