Date: Sunday, November 5, 2017
Session Type: ACR Poster Session A
Session Time: 9:00AM-11:00AM
Anti-citrullinated protein antibodies (ACPA) display broad cross-reactivities and target proteins including a-enolase, filaggrin, vimentin, fibrinogen, and histones. However, every monoclonal ACPA has a distinct recognition pattern. Citrullination occurs during physiological processes such as NETosis and apoptosis, yet the interaction of ACPA with nuclear antigens in apoptotic cells has not been previously investigated. Apoptotic cells, and especially defects in clearance of apoptotic and dead cells resulting in increased exposure to nuclear antigens, have been postulated to play a pivotal role in the pathogenesis of autoimmune disease.
We screened a total of 12 recombinant monoclonal human ACPA-IgG, derived from synovial plasma cells or circulating memory B cells, and two control mAb for binding to full-length citrullinated histones by ELISA. Apoptotic cell binding was determined by flow cytometry and Western blotting, followed by mass spectrometry to identify key antigen targets. Fluorescent microscopy was performed to screen ACPA staining in anti-nuclear antibody (ANA)-Hep-2 tests and for binding to human thymus tissue. A total of 210 seropositive and 50 seronegative RA patients and 157 population controls from the EIRA case-control cohort, were screened for IgG reactivity against full-length native and citrullinated histone 2B (Cit-H2B) by ELISA. Reactivity to citrullinated peptides was determined by antigen microarray multiplex assay.
A distinct subset of ACPA bound apoptotic cells of human and murine origin. We could also observe nuclear staining for these ACPA in human thymus tissue, and ANA positivity with a nuclear dense fine speckled staining pattern. Mass spectrometry revealed that H2B was the pre-dominant target within apoptotic cells. The ANA-positive ACPA had strong recognition of citrullinated histones (H2B, H4). Among CCP2-positive RA patients, 26% were positive for elevated anti-cit-H2B normalized for reactivity to native H2B, and the titer correlated the strongest with binding to citrullinated filaggrin and fibrinogen peptides (R=0.4, p<0.0001). We also observed a weak correlation with disease activity in seropositive RA (R=0.20, p=0.02). Interestingly, 32% seronegative RA patients had autoreactivity to native H2B compared to 4.5% (p<0.0001) among controls, while there was no elevation in reactivity to cit-H2B normalized for native H2B (4% compared to 4.5% in controls).
Interactions between ACPA and citrullinated histones facilitate apoptotic cell binding. We hypothesize that this may be a key functional feature in RA pathogenesis.
To cite this abstract in AMA style:Lloyd KA, Sahlström P, Steen J, Titcombe PJ, Zhou D, Lundqvist C, Ekwall O, Ytterberg J, Rönnelid J, Mueller DL, Klareskog L, Malmström V, Grönwall C. Novel Interaction between Anti-Citrulline Monoclonal Antibodies and Apoptotic Cells Is Mediated through Citrullinated Nuclear Antigens [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/novel-interaction-between-anti-citrulline-monoclonal-antibodies-and-apoptotic-cells-is-mediated-through-citrullinated-nuclear-antigens/. Accessed April 16, 2021.
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