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. 2015 Jun 10;17(1):155.
doi: 10.1186/s13075-015-0666-6.

Targeting of anti-citrullinated protein/peptide antibodies in rheumatoid arthritis using peptides mimicking endogenously citrullinated fibrinogen antigens

Cátia Fernandes-Cerqueira  1 , Elena Ossipova  2 , Sunithi Gunasekera  3 , Monika Hansson  4 , Linda Mathsson  5 , Anca I Catrina  6 , Yngve Sommarin  7 , Lars Klareskog  8 , Karin Lundberg  9 , Johan Rönnelid  10 , Ulf Göransson  11 , Per-Johan Jakobsson  12
Affiliations

Targeting of anti-citrullinated protein/peptide antibodies in rheumatoid arthritis using peptides mimicking endogenously citrullinated fibrinogen antigens

Cátia Fernandes-Cerqueira et al. Arthritis Res Ther. .

Abstract

Introduction: We have previously identified endogenously citrullinated peptides derived from fibrinogen in rheumatoid arthritis (RA) synovial tissues. In this study, we have investigated the auto-antigenicity of four of those citrullinated peptides, and explored their feasibility to target anti-citrullinated protein/peptide antibodies (ACPA).

Methods: The autoantigenic potential of the fibrinogen peptides was investigated by screening 927 serum samples from the Epidemiological Investigation of RA (EIRA) cohort on a peptide microarray based on the ImmunoCAP ISAC® system. In order to assay for ACPA blocking, two independent pools of purified ACPA were incubated with the respective targeting peptide prior to binding to cyclic citrullinated peptide (CCP)2 using the CCPlus® ELISA kit.

Results: Two peptides derived from the fibrinogen α chain, Arg573Cit (563-583) and Arg591Cit (580-600), referred to as Cit573 and Cit591, and two peptides from the fibrinogen β chain, Arg72Cit (62-81) and Arg74Cit (62-81) (Cit72 and Cit74), displayed 65%, 15%, 35%, and 53% of immune reactivity among CCP2-positive RA sera, respectively. In CCP2-negative RA sera, a positive reactivity was detected in 5% (Cit573), 6% (Cit591), 8% (Cit72), and 4% (Cit74). In the competition assay, Cit573 and Cit591 peptides reduced ACPA binding to CCP2 by a maximum of 84% and 63% respectively. An additive effect was observed when these peptides were combined. In contrast, Cit74 and Cit72 were less effective. Cyclization of the peptide structure containing Cit573 significantly increased the blocking efficiency.

Conclusions: Here we demonstrate extensive autoantibody reactivity against in vivo citrullinated fibrinogen epitopes, and further show the potential use of these peptides for antagonizing ACPA.

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Figures

Fig. 1
Fig. 1
a. Percentage of responses among tested patients. Immune reactivity (%) against the respective peptide in sera from CCP2-positive patients. Positive immune reactivity (%) (dark grey) against each individual citrullinated fibrinogen peptide in sera from CCP2-positive patients; light gray represents negative immune response (%) against each individual citrullinated peptide in CCP2-positive sera tested. b. Relative frequency (%) of patient sera reacting to one, two, three, or four citrullinated fibrinogen peptides
Fig. 2
Fig. 2
Dose-response curves representing the percentage of ACPA targeted by fibrinogen α chain peptides. ACPA was incubated with the respective peptides at different concentrations for one hour at room temperature, before proceeding with the anti-CCP2 ELISA. a. ACPA (pool I) blocking with citrulline or arginine-containing fibrinogen peptides from the α chain, 573 (left panel) and 591 (right panel). b. ACPA (pool II) blocking with citrulline or arginine-containing fibrinogen peptides from the α chain, 573 (left panel) and 591 (right panel). X-axes show peptide amount (nmol). Y-axes show ACPA inhibition levels (%). Circles represent means of two to seven experiments per ACPA pool and error bars represent SEM. * P <0.05, ** P <0.001
Fig. 3
Fig. 3
Dose-response curves representing the percentage of ACPA targeted by fibrinogen 573-derived peptides. ACPA was incubated at different concentrations of the respective peptide for one hour at room temperature before proceeding with the anti-CCP2 ELISA. a. ACPA (pool I) blocking with the Cit573 peptide, with the Cit573 truncated or cyclic forms or with the unmodified arginine-containing 573 peptide as control. b. Corresponding experiment as in (a) but using ACPA pool II. X-axes show peptide amount (nmol). Y-axes show ACPA inhibition percentage levels (%). Circles represent means of two to seven experiments per ACPA pool
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