Background Anti-citrullinated protein antibodies (ACPA) can be detected many years before disease onset, implicating that not all ACPAs are pathogenic and that the autoantibodies undergo critical maturation steps closer to disease onset. We have studied the ACPA profile of synovial fluid and of monoclonal antibodies generated from plasma cells of the same synovial fluid to address these questions.
Materials and methods IgG-secreting cells from RA synovial fluid were isolated, the variable Ig regions were amplified, and recombinant monoclonal antibodies were expressed. The antibody reactivity to citrullinated peptides were addressed both by a planar multi array of RA associated peptides, as well as on a 1 75 000 peptide micro-array of arginine/citrulline peptides from extracellular matrix proteins. The matching synovial fluid was also analysed on the peptide arrays. Chimeric monoclonal antibodies were produced based on IMGT predicted germline sequences of the ACPAs, to address the importance of hypermutations in the heavy versus the light chain. The induction of osteoclastogenesis by the ACPAs were tested in an in vitro assay.
Results Sera from RA-patients display wide cross-reactivity between many citrullinated proteins and peptides. Synovial fluid replicates this pattern, and a glycine-rich consensus sequence could be deduced. The monoclonal ACPAs recognised different consensus sequences, of which some replicated the dominant polyclonal pattern. To dissect the contribution of the heavy and light chain to antigen recognition, chimeric variants were expressed where either the light or the heavy chain was exchanged with the respective germline sequence, or to an unrelated chain. Surprisingly, the ACPAs with the common consensus epitope were highly dependent on the light chain mutations for citrulline reactivity, while the heavy chain appeared primarily important for stability and conformation, independent on the acquired mutations. Even if these ACPA were highly cross-reactive, they did not cause osteoclastogenesis. The pathogenic ACPA was instead dependent on hypermutations in both heavy and light chains for citrulline reactivity, and had a more restricted citrulline peptide reactivity.
Conclusions Our data lend support to the observed high cross-reactivity of ACPA between citrullinated peptides. We also find differences in citrulline-reactivity patterns and light chain contribution between pathogenic and non-pathogenic ACPA.