Background and Objectives In a previous study, we have identified endogeneously citrullinated sites in fibrinogen from RA synovial tissue (Hermansson, et al, 2010 in Proteomics-Clin Appl). Within the alpha chain, Arg573 and Arg591 were found citrullinated with an occupancy rate in the range of 1–2% and in the β-chain, Arg72 and Arg74 were also found citrullinated. We now demonstrate that these citrullinated residues are autoantigenic as well as demonstrate that peptides containing these epitopes can be used as probes for development of ACPA neutralising compounds.
Materials and Methods The autoantigenic potential was investigated using the Phadia’s ImmunoCAP ISAC® system. Citrullinated and unmodified fibrinogen peptides were immobilised onto a glass slide in an arrayed fashion and serum from 404 CCP positive and 532 CCP negative RA patients and 461 healthy controls from the EIRA cohort were tested. We also assayed the identified citrulline fibrinogen peptides for their ability to prevent purified ACPA (Ossipova, et al, 2012 submitted) to bind to CCP (CCPlus® ELISA, Euro-Diagnostica AB). Peptides were individually or in combinations incubated with different ACPA pools and the blocking efficiency was expressed as percent of inhibition and IC50. Corresponding arginine peptides were used as controls.
Results We found that 31% (87% are CCP positive) of patients were positive to Cit573 peptide. For the Cit591 peptide, the corresponding numbers were 10% (65%), for the Cit74 peptide 28% (68%) and for the Cit72 peptide 20% (68%). Interestingly, citrullinated 573 and Cit591 peptides revealed a maximum of 77% and 48% ACPA inhibition, respectively. When equally mixed, these peptides displayed an additive higher degree of ACPA neutralisation (84%). In contrast, Cit74 and Cit72 peptides reached a more modest maximum inhibition of 26% and 30%, respectively. This experiment was repeated using a different set of ACPA pool and then the efficiencies were lower for Cit573 (47%) but similar for Cit591 (51%). Logically, the efficiency of specific citrullinated compounds will depend on the individual ACPA specificities.
Conclusions Here we demonstrate extensive autoantibody reactivity against in vivo citrullinated fibrinogen epitopes found in RA synovial membranes. These peptides can now be used as additional biomarkers for studies of ACPA sub-specificity profiles as recently reported (Brink, et al, 2012 A&R, in press). We also demonstrate that these citrullinated peptides can be used as neutralising agents blocking a significant portion of ACPA binding to CCP. These results open novel possibilities for the design of personalised ACPA blockers preventing for instance the osteoclastogenesis and bone loss induced by ACPA (Harre, et al, 2012 JCI).