Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint

Abstract

IntroductionA major subset of patients with rheumatoid arthritis (RA) is characterized by the presence of circulating autoantibodies directed to citrullinated proteins/peptides (ACPAs). These autoantibodies, which are commonly detected by using an enzyme-linked immunosorbent assay (ELISA) based on synthetic cyclic citrullinated peptides (CCPs), predict clinical onset and a destructive disease course. In the present study, we have used plasma and synovial fluids from patients with RA, for the affinity purification and characterization of anti-CCP2 reactive antibodies, with an aim to generate molecular tools that can be used in vitro and in vivo for future investigations into the pathobiology of the ACPA response. Specifically, this study aims to demonstrate that the surrogate marker CCP2 can capture ACPAs that bind to autoantigens expressed in vivo in the major inflammatory lesions of RA (that is, in the rheumatoid joint).MethodsPlasma (n = 16) and synovial fluid (n = 26) samples were collected from RA patients with anti-CCP2 IgG levels of above 300 AU/mL. Total IgG was isolated on Protein G columns and subsequently applied to CCP2 affinity columns. Purified anti-CCP2 IgG was analyzed for reactivity and specificity by using the CCPlus® ELISA, in-house peptide ELISAs, Western blot, and immunohisto-/immunocytochemistry.ResultsApproximately 2% of the total IgG pool in both plasma and synovial fluid was CCP2-reactive. Purified anti-CCP2 reactive antibodies from different patients showed differences in binding to CCP2 and differences in binding to citrullinated peptides from α-enolase, vimentin, fibrinogen, and collagen type II, illustrating different ACPA fine-specificity profiles. Furthermore, the purified ACPA bound not only in vitro citrullinated proteins but, more importantly, in vivo-generated epitopes on synovial fluid cells and synovial tissues from patients with RA.ConclusionsWe have isolated ACPAs from plasma and synovial fluid and demonstrated that the CCP2 peptides, frequently used in diagnostic ELISAs, de facto act as surrogate antigens for at least four different, well-characterized, largely non-cross-reactive, ACPA fine specificities. Moreover, we have determined the concentration and proportion of CCP2-reactive IgG molecules in rheumatoid plasma and synovial fluid, and we have shown that the purified ACPAs can be used to detect both in vitro- and in vivo-generated citrullinated epitopes by various techniques. We anticipate that these antibodies will provide us with new opportunities to investigate the potential pathogenic effects of human ACPAs.

Elevated IgA Plasmablast Levels in Subjects at Risk of Developing Rheumatoid Arthritis

“…Prior studies in patients with established RA have revealed multiple clonal families of antibodies, many of which bind to known RA-related autoantigens. Thus, this method is capable of isolating 2378 KINSLOW ET AL monoclonal autoantibodies and identifying their cognate antigens (23,(40)(41)(42)(43). Similar approaches can be used in preclinical RA to identify plasmablast abnormalities with relevance to the pathogenesis of RA.…”

Characterization of Autoantigens Targeted by Anti-Citrullinated Protein Antibodies In Vivo: Prominent Role for Epitopes Derived from Histone 4 Proteins

“…For example, fibrinogen (subunits α, β, γ) was identified in 16% of ACPA positive synovial immune complexes, but also in 44% of ACPA negative immune complexes isolated using protein-G (data not shown), suggesting that this protein may associate with ACPA both specifically as a citrullinated autoantigen, and as a non-antigen bound protein. There is now considerable evidence incriminating fibrinogen as a specific target for ACPA in RA, and it is widely detectable in the synovial microenvironment [15, 32, 33]. Other well characterized candidate autoantigens include vimentin, α-enolase, and collagen type II [5, 6, 10, 15, 32, 34].…”

“…There is now considerable evidence incriminating fibrinogen as a specific target for ACPA in RA, and it is widely detectable in the synovial microenvironment [15, 32, 33]. Other well characterized candidate autoantigens include vimentin, α-enolase, and collagen type II [5, 6, 10, 15, 32, 34]. Surprisingly, we identified vimentin in only 1/19 ACPA positive immune complexes from RA synovial fluids, and we did not identify α-enolase or collagen type II in any of the synovial immune complexes, whether they were isolated using protein-G or CCP3.…”

Label-free detection of immune complexes with myeloid cells

“…The presence of five or more ACPA antibody types and high levels of total IgG anti-CCP2 were associated with more severe disease progression [44]. In general IgM, IgA and the IgG subclasses all seem to contribute to ACPAmediated clinical symptoms, with the available data suggesting ACPA IgG being the most prominent disease inducer [44,50,51]. In agreement with this published literature, it was difficult to pinpoint a single isotype or IgGsubclass promoting the strongest cellular response, as all show underlying secondary correlations.…”