Multiple antibody reactivities to citrullinated antigens in sera from patients with rheumatoid arthritis: association with HLA-DRB1 alleles

Background:Autoantibodies to cyclic citrullinated peptides (anti-CCP) are present in most patients with rheumatoid arthritis (RA), and associate with HLA-DRB1 shared epitope (SE) alleles.Objective:To investigate reactivities of anti-CCP to various citrullinated proteins/peptides, which represent potential autoantigens in RA, and to examine the relationship between such antibodies, and their association with genetic variants within HLA-DRB1 SE alleles.Methods:Serum samples from 291 patients with established RA and 100 sex- and age-matched healthy subjects were included in this study. Sera were first analysed for presence of anti-CCP antibodies and further for IgG and IgA antibodies towards candidate autoantigens in both their native and citrullinated form including: fibrinogen, α-enolase peptide-1 and the C1-epitope of type II collagen (C1III). Antibody specificity was confirmed by cross-reactivity tests. HLA-DR genotyping was performed.Results:72% of patients with RA were anti-CCP positive. Among the candidate autoantigens examined, IgG antibodies to citrullinated fibrinogen were found in 66% of patients’ sera and in 41% for both citrullinated α-enolase peptide-1 and citrullinated C1III. These antibodies were mainly seen in the anti-CCP-positive patient group; they were specific for their respective antigen and displayed limited cross reactivity. IgA responses were also detected, but less frequently than IgG. Anti-CCP and anti-citrullinated protein antibodies were associated with HLA-DRB1*04 rather than with HLA-DRB1*01 alleles.Conclusions:Antibodies directed against several citrullinated antigens are present in CCP-positive RA, with many patients displaying multireactivity. All specific reactivities were primarily associated with the HLA-DRB1*04 alleles, suggesting common pathways of anti-citrulline immunity.

Download Full-text

Anti-Citrullinated Protein Antibodies in Patients with Rheumatoid Arthritis: Biological Effects and Mechanisms of Immunopathogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms21114015 ◽

2020 ◽

Vol 21 (11) ◽

pp. 4015 ◽

Cited By ~ 1

Author(s):

Chao-Yi Wu ◽

Huang-Yu Yang ◽

Jenn-Haung Lai

Keyword(s):

Rheumatoid Arthritis ◽

Biological Effects ◽

Cross Reactivity ◽

Neutrophil Extracellular Trap ◽

Gamma Receptor ◽

Increased Risk ◽

Citrullinated Proteins ◽

Loss Of Tolerance ◽

System Activation ◽

Citrullinated Protein

Individuals with high anti-citrullinated protein antibody (ACPA) titers have an increased risk of developing rheumatoid arthritis (RA). Although our knowledge of the generation and production of ACPAs has continuously advanced during the past decade, our understanding on the pathogenic mechanisms of how ACPAs interact with immune cells to trigger articular inflammation is relatively limited. Citrullination disorders drive the generation and maintenance of ACPAs, with profound clinical significance in patients with RA. The loss of tolerance to citrullinated proteins, however, is essential for ACPAs to exert their pathogenicity. N-linked glycosylation, cross-reactivity and the structural interactions of ACPAs with their citrullinated antigens further direct their biological functions. Although questions remain in the pathogenicity of ACPAs acting as agonists for a receptor-mediated response, immune complex (IC) formation, complement system activation, crystallizable fragment gamma receptor (FcγR) activation, cross-reactivity to joint cartilage and neutrophil extracellular trap (NET)-related mechanisms have all been suggested recently. This paper presents a critical review of the characteristics and possible biological effects and mechanisms of the immunopathogenesis of ACPAs in patients with RA.

Download Full-text

Relatedness of Antibodies to Peptides Containing Homocitrulline or Citrulline in Patients with Rheumatoid Arthritis

The Journal of Rheumatology ◽

10.3899/jrheum.170446 ◽

2017 ◽

Vol 45 (3) ◽

pp. 302-309 ◽

Cited By ~ 4

Author(s):

Patrick Lac ◽

Maud Racapé ◽

Lillian Barra ◽

David A. Bell ◽

Ewa Cairns

Keyword(s):

Rheumatoid Arthritis ◽

Lupus Erythematosus ◽

Healthy Subjects ◽

Cross Reactivity ◽

Strongly Correlated ◽

Spearman Correlation ◽

Peptide Backbone ◽

The Relationship ◽

Citrullinated Protein ◽

Homologous Peptide

Objective.Antibodies that target citrullinated protein/peptide (ACPA) and homocitrullinated/carbamylated protein/peptide (AHCPA) are associated with rheumatoid arthritis (RA). The relationship between ACPA and AHCPA remains unclear. We examined the expression and cross-reactivity of these antibodies using citrulline- and homocitrulline-containing synthetic peptides, CitJED and HomoCitJED, respectively, which have equal numbers of citrulline or homocitrulline residues on the same peptide backbone.Methods.Serum from healthy subjects (n = 51) and patients with RA (n = 137), systemic lupus erythematosus (SLE; n = 37), and psoriatic arthritis (PsA; n = 37) were screened for IgG anti-CitJED and anti-HomoCitJED antibodies by ELISA. Cross-reactivity of these antibodies was examined by inhibition with various concentrations of CitJED and HomoCitJED.Results.Out of 137 patients with RA, antibodies to CitJED and HomoCitJED were detected in 69 (50%) and 78 (57%), respectively. Anti-CitJED and HomoCitJED antibodies were 77% concordant and their levels were strongly correlated [Spearman correlation coefficient (rs) = 0.6676]. Sera from 25/27 patients (93%) with RA were inhibited by both CitJED and HomoCitJED with equal or higher affinity for the cognate (homologous) peptide.Conclusion.Antibodies to CitJED and HomoCitJED frequently occurred in RA, but were not found in SLE or PsA, suggesting that these antibodies are specific to RA. Cross-reactivity between anti-HomoCitJED and anti-CitJED antibodies suggests that ACPA and AHCPA are derived from the same B cell population and both may contribute to the pathogenesis of RA.

Download Full-text

Identification of potential autoantigens in anti-CCP-positive and anti-CCP-negative rheumatoid arthritis using citrulline-specific protein arrays

Scientific Reports ◽

10.1038/s41598-021-96675-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Thomas B. G. Poulsen ◽

Dres Damgaard ◽

Malene M. Jørgensen ◽

Ladislav Senolt ◽

Jonathan M. Blackburn ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Protein Microarray ◽

Specific Protein ◽

Immune Reactivity ◽

Igg Antibodies ◽

Native Proteins ◽

Array Technology ◽

Citrullinated Proteins ◽

Folded Proteins

AbstractThe presence or absence of autoantibodies against citrullinated proteins (ACPAs) distinguishes two main groups of rheumatoid arthritis (RA) patients with different etiologies, prognoses, disease severities, and, presumably, disease pathogenesis. The heterogeneous responses of RA patients to various biologics, even among ACPA-positive patients, emphasize the need for further stratification of the patients. We used high-density protein array technology for fingerprinting of ACPA reactivity. Identification of the proteome recognized by ACPAs may be a step to stratify RA patients according to immune reactivity. Pooled plasma samples from 10 anti-CCP-negative and 15 anti-CCP-positive RA patients were assessed for ACPA content using a modified protein microarray containing 1631 different natively folded proteins citrullinated in situ by protein arginine deiminases (PADs) 2 and PAD4. IgG antibodies from anti-CCP-positive RA plasma showed high-intensity binding to 87 proteins citrullinated by PAD2 and 99 proteins citrullinated by PAD4 without binding significantly to the corresponding native proteins. Curiously, the binding of IgG antibodies in anti-CCP-negative plasma was also enhanced by PAD2- and PAD4-mediated citrullination of 29 and 26 proteins, respectively. For only four proteins, significantly more ACPA binding occurred after citrullination with PAD2 compared to citrullination with PAD4, while the opposite was true for one protein. We demonstrate that PAD2 and PAD4 are equally efficient in generating citrullinated autoantigens recognized by ACPAs. Patterns of proteins recognized by ACPAs may serve as a future diagnostic tool for further subtyping of RA patients.

Download Full-text

A Novel High Sensitivity Type II Collagen Blood-Based Biomarker, PRO-C2, for Assessment of Cartilage Formation

International Journal of Molecular Sciences ◽

10.3390/ijms19113485 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3485 ◽

Cited By ~ 10

Author(s):

Yunyun Luo ◽

Yi He ◽

Ditte Reker ◽

Natasja Gudmann ◽

Kim Henriksen ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Growth Factor ◽

Knee Osteoarthritis ◽

High Sensitivity ◽

Type Ii Collagen ◽

Cartilage Explants ◽

Type Ii ◽

Serum Samples ◽

Human Cartilage ◽

Cartilage Formation

N-terminal propeptide of type II collagen (PIINP) is a biomarker reflecting cartilage formation. PIINP exists in two main splice variants termed as type IIA and type IIB collagen NH2-propeptide (PIIANP, PIIBNP). PIIANP has been widely recognized as a cartilage formation biomarker. However, the utility of PIIBNP as a marker in preclinical and clinical settings has not been fully investigated yet. In this study, we aimed to characterize an antibody targeting human PIIBNP and to develop an immunoassay assessing type II collagen synthesis in human blood samples. A high sensitivity electrochemiluminescence immunoassay, hsPRO-C2, was developed using a well-characterized antibody against human PIIBNP. Human cartilage explants from replaced osteoarthritis knees were cultured for ten weeks in the presence of growth factors, insulin-like growth factor 1 (IGF-1) or recombinant human fibroblast growth factor 18 (rhFGF-18). The culture medium was changed every seven days, and levels of PIIBNP, PIIANP, and matrix metalloproteinase 9-mediated degradation of type II collagen (C2M) were analyzed herein. Serum samples from a cross-sectional knee osteoarthritis cohort, as well as pediatric and rheumatoid arthritis samples, were assayed for PIIBNP and PIIANP. Western blot showed that the antibody recognized PIIBNP either as a free fragment or attached to the main molecule. Immunohistochemistry demonstrated that PIIBNP was predominately located in the extracellular matrix of the superficial and deep zones and chondrocytes in both normal and osteoarthritic articular cartilage. In addition, the hsPRO-C2 immunoassay exhibits acceptable technical performances. In the human cartilage explants model, levels of PIIBNP, but not PIIANP and C2M, were increased (2 to 7-fold) time-dependently in response to IGF-1. Moreover, there was no significant correlation between PIIBNP and PIIANP levels when measured in knee osteoarthritis, rheumatoid arthritis, and pediatric serum samples. Serum PIIBNP was significantly higher in controls (KL0/1) compared to OA groups (KL2/3/4, p = 0.012). The hsPRO-C2 assay shows completely different biological and clinical patterns than PIIANP ELISA, suggesting that it may be a promising biomarker of cartilage formation.

Download Full-text

The shared epitope revisited: shared epitope negative HLA-DR alleles influence susceptibility to rheumatoid arthritis

Arthritis Research & Therapy ◽

10.1186/ar272 ◽

2001 ◽

Vol 3 (S2) ◽

Author(s):

D Reviron ◽

A Perdriger ◽

E Toussirot ◽

D Wendling ◽

N Balandraud ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Shared Epitope ◽

Hla Dr

Download Full-text

Hierarchy in the Avidity and Cross-reactivity of Anti-citrullinated Protein Antibodies in the Serum of Patients With Rheumatoid Arthritis

10.21203/rs.3.rs-32917/v1 ◽

2020 ◽

Author(s):

Akihisa Haraguchi ◽

Hisakata Yamada ◽

Takahide Sakuragi ◽

Tomomi Tsuru ◽

Masakazu Kondo ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Immune Response ◽

Serum Antibody ◽

Cross Reactivity ◽

Sodium Thiocyanate ◽

Fine Specificity ◽

Thiocyanate Solution ◽

Citrullinated Protein ◽

Citrullinated Peptide ◽

Substantial Extent

Abstract BackgroundFine specificity of anti-citrullinated protein antibodies (ACPAs), in which cross-reactivity exists, varies among patients with rheumatoid arthritis (RA), but it is unclear whether the mechanism of ACPA production is same or different among individuals. Since avidity of serum antibody reflects the direction of immune response, we compared the levels of avidity and cross-reactivity between various ACPAs in a cohort of RA patient.MethodsSera from 180 RA patients positive for anti-cyclic citrullinated peptide (CCP) 2 antibody were screened for positivity of antibodies against CCP1, and citrullnated fibrinogen (cFib), enolase (cEno), and vimentin (cVim) peptides. Avidity of the four ACPAs, and some autoantibodies and antibodies against foreign antigens was determined by an elution assay using sodium thiocyanate solution. Cross-reactivity between different ACPAs was estimated by measuring the inhibition of binding by competitor peptides. ResultsThe prevalence of anti-CCP1, anti-cFib, anti-cEno, and anti-cVim antibodies in the anti-CCP2-positive RA cohort were 37.7%, 38.3%, 15.6%, and 23.9%, respectively. The avidity of ACPAs, except for anti-cVim antibody, was significantly lower than that of antibodies against foreign antigens, while there was a large variety in the avidity of other autoantibodies. At individual levels, the avidity of anti-cVim was significantly higher than that of other ACPAs, and there was a significant correlation in the avidity of anti-CCP and anti-cFib antibodies. Substantial extent of cross-reactivity was seen between different ACPAs, which also showed a fixed hierarchy.ConclusionThe fixed hierarchy in the avidity and cross-reactivity between different ACPAs suggests that the mechanism underlying ACPA production is common to all RA patients. Presence of a dominant antigen that induces whole ACPA response is speculated.

Download Full-text

Different classes of anti-modified protein antibodies are induced on exposure to antigens expressing only one type of modification

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2018-214950 ◽

2019 ◽

Vol 78 (7) ◽

pp. 908-916 ◽

Cited By ~ 11

Author(s):

Arieke Suzanna Berendina Kampstra ◽

Jacqueline Stephanie Dekkers ◽

Mikhail Volkov ◽

Annemarie L Dorjée ◽

Lise Hafkenscheid ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

B Cell ◽

Cell Response ◽

Cross Reactivity ◽

Single Type ◽

Mutual Relationship ◽

Post Translational Modifications ◽

Modified Proteins ◽

Relationship Of ◽

Citrullinated Protein

ObjectivesAutoantibodies against post-translationally modified proteins (anti-modified protein antibodies or AMPAs) are a hallmark of rheumatoid arthritis (RA). A variety of classes of AMPAs against different modifications on proteins, such as citrullination, carbamylation and acetylation, have now been described in RA. At present, there is no conceptual framework explaining the concurrent presence or mutual relationship of different AMPA responses in RA. Here, we aimed to gain understanding of the co-occurrence of AMPA by postulating that the AMPA response shares a common ‘background’ that can evolve into different classes of AMPAs.MethodsMice were immunised with modified antigens and analysed for AMPA responses. In addition, reactivity of AMPA purified from patients with RA towards differently modified antigens was determined.ResultsImmunisation with carbamylated proteins induced AMPAs recognising carbamylated proteins and also acetylated proteins. Similarly, acetylated proteins generated (autoreactive) AMPAs against other modifications as well. Analysis of anti-citrullinated protein antibodies from patients with RA revealed that these also display reactivity to acetylated and carbamylated antigens. Similarly, anti-carbamylated protein antibodies showed cross-reactivity against all three post-translational modifications.ConclusionsDifferent AMPA responses can emerge from exposure to only a single type of modified protein. These findings indicate that different AMPA responses can originate from a common B-cell response that diversifies into multiple distinct AMPA responses and explain the presence of multiple AMPAs in RA, one of the hallmarks of the disease.

Download Full-text