Monika Hansson

Multiplex analyses of antibodies against citrullinated peptides in individuals prior to development of rheumatoid arthritis.

OBJECTIVE The presence of antibodies against cyclic citrullinated peptides has been demonstrated to precede the onset of symptoms of rheumatoid arthritis (RA) by several years. The aim of this study was to analyze antibodies against 10 citrullinated autoantigen-derived peptides for reactivity before the onset of RA symptoms. METHODS A case-control study was conducted within the Medical Biobank of Northern Sweden. The study was performed in 409 individuals, 386 of whom donated 717 blood samples before the onset of symptoms of RA (pre-patients). The median period of time predating the onset of RA was 7.4 years. A total of 1,305 population-based control subjects were also studied. Antibodies to 10 citrullinated peptides, fibrinogen α573 (Fibα573), Fibα591, Fibβ36-52, Fibβ72, Fibβ74, α-enolase (citrullinated α-enolase peptide 1 [CEP-1]), triple-helical type II collagen peptide C1 (citC1III), filaggrin, vimentin 2-17 (Vim2-17), and Vim60-75, were analyzed using a microarray system. RESULTS The fluorescence intensity of antibodies against Fibβ36-52, Fibβ74, CEP-1, citC1III, and filaggrin was significantly increased in pre-patients compared with controls (P<0.001). The levels of the earliest-detectable antibodies (Fibα591 and Vim60-75) fluctuated over time, with only a slight increase after the onset of disease. The frequency of antibodies against Fibβ36-52, CEP-1, and filaggrin increased gradually, reaching the highest levels before symptom onset. The frequency of a cluster of antibodies, citC1III, Fibα573, and Fibβ74, increased only slightly before the onset of symptoms but increased prominently after disease onset. The odds ratio for the development of RA in individuals expressing both CEP-1 and Fibβ36-52 antibodies (using data from samples obtained <3.35 years predating symptom onset) was 40.4 (95% confidence interval 19.8-82.3) compared with having either antibody alone. CONCLUSION Development of an immune response toward citrullinated peptides is initially restricted but expands with time to induce a more specific response, with levels, particularly those of antibodies against CEP-1, Fibβ36-52, and filaggrin, increasing during the predating time period closer to the onset of symptoms.

Features of the Synovium of Individuals at Risk of Developing Rheumatoid Arthritis: Implications for Understanding Preclinical Rheumatoid Arthritis

Findings from previous studies have suggested that subclinical inflammation of the synovium does not coincide with the appearance of rheumatoid arthritis (RA)–specific autoantibodies. This study was undertaken to examine the relationship between the presence of autoantibodies, changes in the synovium, and development of arthritis over time in a markedly larger, prospective study.

Validation of a multiplex chip-based assay for the detection of autoantibodies against citrullinated peptides

IntroductionAutoantibodies directed against citrullinated proteins/peptides (ACPAs) are highly specific and predictive for the development of rheumatoid arthritis (RA). Different subgroups of RA patients, which have different prognoses and may require different treatments, are characterized by different autoantibody profiles. The objective of this study was to develop a microarray for the detection of multiple RA-associated autoantibodies, initially focusing on responses against citrullinated epitopes on candidate autoantigens in RA.MethodsThe microarray is based on Phadias ImmunoCAP ISAC system, with which reactivity to more than 100 antigens can be analyzed simultaneously, by using minute serum volumes (< 10 μl). Twelve citrullinated peptides, and the corresponding native arginine-containing control peptides, were immobilized in an arrayed fashion onto a chemically modified glass slide, allowing a three-dimensional layer with high binding capacity. The assay was optimized concerning serum dilution and glass surface, whereas each individual antigen was optimized concerning coupling chemistry, antigen concentration, and selection of spotting buffer. The performance of each peptide in the ImmunoCAP ISAC system was compared with the performance in enzyme-linked immunosorbent assays (ELISAs). Serum from 927 RA patients and 461 healthy controls from a matched case-control study were applied onto reaction sites on glass slides, followed by fluorescent-labeled anti-human immunoglobulin G (IgG) antibody. Fluorescence intensities were detected with a laser scanner, and the results analyzed by using image-analysis software.ResultsStrong correlations between the ImmunoCAP ISAC system and ELISA results were found for individual citrullinated peptides (Spearman ρ typically between 0.75 and 0.90). Reactivity of RA sera with the peptides was seen mainly in the anticyclic citrullinated peptide 2 (CCP2)-positive subset, but some additional reactivity with single citrullinated peptides was seen in the anti-CCP2-negative subset. Adjusting for reactivity against arginine-containing control peptides did not uniformly change the diagnostic performance for antibodies against the individual citrullinated peptides.ConclusionsThe multiplexed array, for detection of autoantibodies against multiple citrullinated epitopes on candidate RA autoantigens, will be of benefit in studies of RA pathogenesis, diagnosis, and potentially as a guide to individualized treatment.

Associations of antibodies against citrullinated peptides with human leukocyte antigen-shared epitope and smoking prior to the development of rheumatoid arthritis

IntroductionIt has previously been shown that an increased number of antibodies against citrullinated peptides/proteins (ACPA) predate the onset of rheumatoid arthritis (RA). Over time antibody positivity expands, involving more specific responses when approaching the onset of symptoms. We investigated the impact of human leukocyte antigen-shared epitope (HLA-SE) alleles and smoking on the development of ACPA, as well as in combination with ACPA during the state of quiescent autoimmunity (before the onset of symptoms), on the development of RA.MethodsBlood samples donated to the Medical Biobank of Northern Sweden from individuals prior to the onset of symptoms of RA (n = 370) and after onset (n = 203) and from population-based controls (n = 585) were used. Antibodies against 10 citrullinated peptides, fibrinogen (Fibα561-583, α580-600, ß62-81a, ß62-81b, ß36-52), vimentin (Vim2-17, 60-75), filaggrin (CCP-1/Fil307-324), α-enolase (CEP-1/Eno5-21), collagen type II (citC1359-369), and anti-cyclic citrullinated peptide (CCP)2 antibodies were analysed.ResultsHLA-SE-positive individuals were more frequently positive for ACPA compared with HLA-SE-negative individuals prior to the onset of symptoms of RA, particularly for antibodies against CEP-1 and Fibß62-81a (72). Smoking was associated with antibodies against Vim2-17 and citC1359-369. HLA-SE and smoking showed increasing association to the presence of the antibodies closer to disease onset. The highest odds ratio (OR) for development of RA was for the combination of HLA-SE alleles and ACPA positivity, especially for antibodies against Fibß62-81b, CCP-1/Fil307-324, and Fibβ36-52. A gene-environment additive interaction between smoking and HLA-SE alleles for the risk of disease development was found, with the highest OR for individuals positive for antibodies against Fibβ36-52, CEP-1, and Fibα580-600.ConclusionsThe relationships between antibodies against the different ACPA specificities, HLA-SE, and smoking showed a variable pattern in individuals prior to the onset of RA. The combination of smoking and HLA-SE alleles was significantly associated with the development of some of the antibody specificities closer to onset of symptoms, and these associations remained significant at diagnosis. An additive gene-environment interaction was found for several of the antibodies for the development of RA.

The autoantibody repertoire in periodontitis: a role in the induction of autoimmunity to citrullinated proteins in rheumatoid arthritis? Antibodies against uncitrullinated peptides seem to occur prior to the antibodies to the corresponding citrullinated peptides

The autoantibody repertoire in periodontitis : a role in the induction of autoimmunity to citrullinated proteins in rheumatoid arthritis? Antibodies against uncitrullinated peptides seem to occur prior to the antibodies to the corresponding citrullinated peptides

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

IntroductionWe 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).MethodsThe 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.ResultsTwo 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.ConclusionsHere we demonstrate extensive autoantibody reactivity against in vivo citrullinated fibrinogen epitopes, and further show the potential use of these peptides for antagonizing ACPA.

Low levels of antibodies against common viruses associate with anti-citrullinated protein antibody-positive rheumatoid arthritis; implications for disease aetiology

BackgroundInfection by common viruses has long been discussed in the aetiology of a number of autoimmune diseases, including rheumatoid arthritis (RA). However, studies investigating this hypothesis in RA show conflicting results. These studies often lack well-matched control populations, and many do not include data on autoantibodies, genetic risk factors and other environmental factors, which are known to contribute to disease only in subgroups of patients. In the present study, we have therefore examined the role of Epstein-Barr virus (EBV), cytomegalovirus (CMV) and parvovirus B19 (B19) in RA aetiology, by analysing anti-viral antibodies in relation to anti-citrullinated protein antibodies (ACPA), smoking, HLA-DRB1 shared epitope (SE) alleles, and clinical parameters, in both RA patients and matched controls.MethodsAnti-viral antibodies were measured by ELISA in serum samples from 990 RA patients and 700 controls from the Swedish population-based Epidemiological Investigation of RA (EIRA) cohort. Data on ACPA, smoking, SE, inflammation (C-reactive protein) and disease activity score in 28 joints (DAS28) was obtained from the EIRA database. Fisher’s exact test, the chi-squared test, and the Mann-Whitney U test were used to calculate differences in anti-viral antibody frequencies and levels; unconditional logistic regression was used to determine the association of anti-viral antibodies with different RA subsets.ResultsAntibodies against all viruses were highly prevalent in EIRA, with no major differences detected between ACPA-positive RA, ACPA-negative RA and controls. However, both anti-B19 and anti-EBV IgG levels were significantly lower in ACPA-positive RA compared to controls, and there were significant interactions between low levels of anti-B19 and anti-EBV antibodies and SE in the development of ACPA-positive RA.ConclusionWe could not detect an association between RA and elevated anti-viral antibody levels, for any of the three common viruses, EBV, CMV or B19. On the contrary, our study demonstrated association between low anti-EBV/anti-B19 antibody levels and ACPA-positive RA, in particular when HLA-DRB1 SE was present. These data could potentially suggest that high anti-viral antibody levels would be protective against ACPA-positive RA. Further investigations are required to address the mechanisms behind these findings.

Differences in the Spectrum of Anti-Citrullinated Protein Antibody Fine Specificities Between Malaysian and Swedish Patients With Rheumatoid Arthritis: Implications for Disease Pathogenesis.

Objective: Antibodies to the citrullinated protein antigens (ACPA) are important in the diagnosis and pathogenesis of rheumatoid arthritis (RA). However, the prevalence of ACPAs with different specificities in different populations is unclear. We report the fine specificity of the antibody responses towards citrullinated proteins in RA patients from Malaysia, an area where genetic and environmental determinants of RA are different from those in the more frequently studied Caucasians. Methods: A multiplex analytic microarray system was used to analyze the occurrence of antibodies to 10 different citrullinated peptides [filaggrin (fil307-324), vimentin (Vim2-17, Vim60-75), fibrinogen (Fibα563-583, Fibα580-600, Fibβ36-52, Fibβ62-81a, Fibβ62-81b), enolase (Eno5-21) and collagen type II (citCII355-378)] in 4,089 RA serum samples (1,231 Malaysian and 2,858 Swedish) and 827 normal controls samples (249 Malaysian and 578 Swedish). The positive reaction threshold was set for each population with each peptide separately based on 98% specificity. Results: Distinct differences in five ACPA fine specificities (Vim60-75, Vim2-17, Fibβ62-81b, Eno5-21 and CitCII355-378) were found between the Malaysian and Swedish RA populations despite identical frequency of anti-CCP2-positive individuals. In comparison with the Swedish RA patients, the Malaysian RA patients had significantly higher frequencies for antibodies towards Vim60-75 (54% vs 44%, Pcorrected=1.06x10−08), CitCII355-378(17% vs 13%, Pcorrected =0.02) and significantly lower frequencies towards Vim2-17 (25% vs 32%, Pcorrected=1.91x10−04), Fibβ62-81b (15% vs 30%, Pcorrected=2.47x10−22) and Eno5-21(23% vs 50%, Pcorrected=3.64x10−57). Conclusion: RA patients in different populations are different concerning ACPA fine specificities and similar concerning total frequencies of ACPAs, which could be due to variant genetic and/or environmental factors. This article is protected by copyright. All rights reserved.Antibodies to the citrullinated protein antigens (ACPAs) are important in the diagnosis and pathogenesis of rheumatoid arthritis (RA). However, the prevalence of ACPAs with different fine specificities in different populations is unclear. This study sought to examine the fine specificity of the antibody responses toward citrullinated proteins in RA patients from Malaysia, an area where genetic and environmental determinants of RA are different from those in more frequently studied cohorts of Caucasian subjects.

Anticitrullinated protein/peptide antibody multiplexing defines an extended group of ACPA-positive rheumatoid arthritis patients with distinct genetic and environmental determinants

Introduction The second generation anticycliccitrullinated peptide (anti-CCP2) assay detects the majority but not all anticitrullinated protein/peptide antibodies (ACPA). Anti-CCP2-positive rheumatoid arthritis (RA) is associated with HLA-DRB1* shared epitope (SE) alleles and smoking. Using a multiplex assay to detect multiple specific ACPA, we have investigated the fine specificity of individual ACPA responses and the biological impact of additional ACPA reactivity among anti-CCP2-negative patients. Methods We investigated 2825 patients with RA and 551 healthy controls with full data on anti-CCP2, HLA-DRB1* alleles and smoking history concerning reactivity against 16 citrullinated peptides and arginine control peptides with a multiplex array. Results The prevalence of the 16 ACPA specificities ranged from 9% to 58%. When reactivity to arginine peptides was subtracted, the mean diagnostic sensitivity increased by 3.2% with maintained 98% specificity. Of the anti-CCP2-negative patients, 16% were found to be ACPA positive. All ACPA specificities associated with SE, and all but one with smoking. Correction for arginine reactivity also conveyed a stronger association with SE for 13/16 peptides. Importantly, when all ACPA specificities were analysed together, SE and smoking associated with RA in synergy among ACPA positive, but not among ACPA-negative subjects also in the anti-CCP2-negative subset. Conclusions Multiplexing detects an enlarged group of ACPA-positive but anti-CCP2-negative patients with genetic and environmental attributes previously assigned to anti-CCP2-positive patients. The individual correction for arginine peptide reactivity confers both higher diagnostic sensitivity and stronger association to SE than gross ACPA measurement.

Antibody responses to de novo identified citrullinated fibrinogen peptides in rheumatoid arthritis and visualization of the corresponding B cells

BackgroundAntibodies against citrullinated proteins (ACPA) are common in patients with rheumatoid arthritis (RA). ACPA can appear before disease onset and target many self-antigens. Citrullinated fibrin/fibrinogen represents a classical ACPA target antigen, and mass spectrometry of RA synovial fluid reveals elevated citrullinated (cit) fibrinogen (Fib) peptides compared to non-RA controls. We investigated the extent to which these less-studied peptides represent autoantibody targets and sought to visualize the corresponding cit-Fib-reactive B cells in RA patients.MethodsAn in-house ELISA was established against four cit-Fib α-subunit peptides (cit-Fib α-35; cit-Fib α-216,218; cit-Fib α-263,271 and cit-Fib α-425,426) and serum from patients with established RA (n = 347) and disease controls with psoriatic arthritis (PsA) or ankylosing spondylitis (AS) (n = 236) were analyzed. RA patients were genotyped for HLA-DR alleles, PTPN22 R620W and screened for anti-CCP2 and cit-Fib protein antibodies. The cit-Fib peptides were also used to assemble antigen tetramers to identify cit-Fib-reactive B cells in peripheral blood by flow cytometry.ResultsThe frequencies of autoantibodies against different cit-Fib epitopes in RA patients compared to PsA/AS patients were: cit-Fib α-35 (RA 20%, vs PsA/AS 1%); cit-Fib α-216,218 (13% vs 0.5%); cit-Fib α-263,271 (21% vs 0.5%) and cit-Fib α-425,426 (17% vs 1%). The presence of autoantibodies against these peptides was associated with presence of anti-CCP2 and anti-cit-Fib protein antibodies. No association was found between HLA-DR shared epitope and antibodies to the different cit-Fib peptides. However, association was observed between the PTPN22 risk allele and positivity to cit-Fib α-35 and cit-Fib α-263,271. B cells carrying surface Ig reactive to these cit-Fib peptides were found in RA peripheral blood and these tend to be more common in PTPN22 risk allele carriers.ConclusionsOur data show that several cit-Fib peptides are targeted by autoantibodies in RA, but not in PsA/AS, implicating that these are not due to arthritis but more specific for RA etiology. The RA-associated anti-cit protein response is broad with many parallel immune responses. The association between cit-Fib autoantibodies and the PTPN22 R620W risk allele supports the hypothesis of altered B cell regulation, such as autoreactive B cells evading tolerance checkpoints.