Background We have described a planar microarray for the determination of antibodies against individual citrullinated peptides (ACPA; Hansson M et al. Arthritis Res Ther 2012;14:R201). We have also developed a method for the quantification of autoantibodies in immune complexes (IC; Sohrabian et al. Ann Rheum Dis 2015;74(Suppl 1):A74). Here we have combined these techniques to determine ACPA profiles in RA IC.
Objectives To investigate if measurement of specific ACPA in synovial fluids (SF) and in IC from sera and SF can provide more prognostic information than conventional measurement of total ACPA and rheumatoid factor (RF) in serum.
Methods Seventy-seven RA patients with knee synovitis were treated with intra-articular triamcinolone hexacetonide, and followed until relapse. DAS28 and radiographic joint damage according to Larson-Dale were recorded. Anti-CCP2, IgM and IgA RF and circulating C1q-binding immune complexes (CIC) were determined in paired sera and SF. IC were purified from sera and SF by binding to C1q-coated beads, and thereafter eluted with a procedure developed in our laboratory. Antibodies against 19 citrullinated peptides were investigated with a custom-made microarray assay based on the ImmunoCAP ISAC system (Phadia AB, Sweden) in sera and SF as well as in IC from sera and SF. The target peptides were filaggrin 307–324 (CCP1), vimentin peptides 60–75 and 2–17, fibrinogen peptides α36–50,, α563–583, α580–600, α621–635, β36–52, β60–74, β62–81 (with citrullination in positions 72 and 74, respectively), α-enolase 5–21 (CEP-1), peptides 1, 5, Z1, Z2 and Bla26 from hnRNP, and histone 4 peptides 14–34 and 31–50. Cutoffs were established in relation to healthy controls. Backward stepwise regression was used to investigate what factors determined Larsen Dale index, DAS28, and duration of remission after steroid treatment. Independent factors were anti-CCP2, IgM RF, IgA RF, CIC, number of ACPA peptide reactivities, and number of ACPA reactivities in IC, all measured both in serum and paired SF.
Results A considerable proportion of anti-CCP2 negative patients had multiple ACPA in SF, and in IC fractions. High DAS28 associated with reactivity against 7/19 peptides in serum and 9/19 in SF. High Larsen score associated with number of specific ACPA in SF IC and with CIC in SF. DAS28 levels associated with IgM RF in SF and with CIC in SF, and steroid response duration with number of specific ACPA in serum and in SF IC.
Conclusions We found ACPA in SF, and especially in the IC fraction of SF, in a sizeable fraction of anti-CCP2 negative patients. Number of peptide-specific ACPA (but not anti-CCP2 levels) associated with radiological destruction and length of remission after intra-articular steroid therapy. Our data do not support a role for any unique ACPA specificity in RA pathogenesis. Instead, the number of individual ACPA specificities may be important.
Disclosure of Interest None declared