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

Introduction: It 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. Methods: Blood 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 (Fib alpha 561-583, alpha 580-600, beta 62-81a, beta 62-81b, beta 36-52), vimentin (Vim2-17, 60-75), filaggrin (CCP-1/Fil307-324),alpha-enolase (CEP-1/Eno5-21), collagen type II (citC1359-369), and anti-cyclic citrullinated peptide (CCP) 2 antibodies were analysed. Results: HLA-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 Fib beta 62-81a (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 Fib beta 62-81b, CCP-1/Fil307-324, and Fib beta 36-52. 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 Fib beta 36-52, CEP-1, and Fib alpha 580-600. Conclusions: The 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

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

Introduction: It 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. Methods: Blood 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 (Fib alpha 561-583, alpha 580-600, beta 62-81a, beta 62-81b, beta 36-52), vimentin (Vim2-17, 60-75), filaggrin (CCP-1/Fil307-324),alpha-enolase (CEP-1/Eno5-21), collagen type II (citC1359-369), and anti-cyclic citrullinated peptide (CCP) 2 antibodies were analysed. Results: HLA-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 Fib beta 62-81a (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 Fib beta 62-81b, CCP-1/Fil307-324, and Fib beta 36-52. 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 Fib beta 36-52, CEP-1, and Fib alpha 580-600. Conclusions: The 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

Number of individual ACPA reactivities in synovial fluid immune complexes, but not serum anti-CCP2 levels, associate with inflammation and joint destruction in rheumatoid arthritis

Introduction: Individual patients with rheumatoid arthritis (RA) show divergent specific anti-citrullinated protein/peptide antibodies (ACPA) patterns, but hitherto no individual ACPA specificity has consistently been linked to RA pathogenesis. ACPA are also implicated in immune complexes (IC)-associated joint pathology, but until now, there has been no method to investigate the role of individual ACPA in RA IC formation and IC-associated pathogenesis. Methods: We have developed a new technique based on IC binding to C1q-coated magnetic beads to purify and solubilise circulating IC in sera and synovial fluids (SF) from 77 patients with RA. This was combined with measurement of 19 individual ACPA in serum, SF and in the IC fractions from serum and SF. We investigated whether occurrence of individual ACPA as well as number of ACPA in these compartments was related to clinical and laboratory measures of disease activity and inflammation. Results: The majority of individual ACPA reactivities were enriched in SF as compared with in serum, and levels of ACPA in IC were regulated independently of levels in serum and SF. No individual ACPA reactivity in any compartment showed a dominating association to clinical and laboratory measures of disease activity and severity. Instead, the number of individual ACPA reactivities in the IC fraction from SF associated with a number of markers of joint destruction and inflammation. Conclusions: Our data highlight the polyclonality of ACPA in joint IC and the possibility that a broad ACPA repertoire in synovial fluid IC might drive the local inflammatory and matrix-degrading processes in joints, in analogy with antibody-induced rodent arthritis models

Rheumatoid factor isotypes in relation to antibodies against citrullinated peptides and carbamylated proteins before the onset of rheumatoid arthritis

Background: The presence of rheumatoid factor (RF), anti-carbamylated protein antibodies (anti-CarP) and antibodies against citrullinated protein and peptides (ACPA) precedes the onset of symptoms of rheumatoid arthritis (RA) by several years. Relationships between the development of these antibodies are not obvious. Methods: Three isotypes [immunoglobulin A (IgA), IgG and IgM) of RF were analysed in 321 pre-symptomatic individuals who provided 598 samples collected a median of 6.2 (interquartile range 7.2) years before the onset of symptoms, and in 492 population control subjects. All samples were donated to the Biobank of Northern Sweden. RF isotypes were analysed using the EliA system (Phadia GmbH, Freiburg, Germany) with 96 % specificity according to receiver operating characteristic curves. Ten ACPA specificities were analysed using the ImmunoCAP ISAC system, and anti-CCP2 and anti-CarP antibodies were evaluated using enzyme-linked immunosorbent assays. Results: The frequencies of RF isotypes in pre-symptomatic individuals were significantly increased compared with control subjects (p < 0.0001). In samples collected >= 15 years before the onset of symptoms, the IgA-RF isotype was significantly more prevalent than the most frequent ACPAs. Combinations of IgM- and IgA-RF isotypes with ACPA specificities [a-enolase (CEP-1/Eno(5-21))], fibrinogen (Fib)beta(36-52), Fiba(580-600), filaggrin (CCP-1/Fil(307-324)) and anti-CCP2 antibodies were associated with a significantly shorter time to onset of symptoms (p < 0.001-0.05). Using conditional inference tree analysis, anti-CCP2 in combination with anti-filaggrin antibodies gave the highest probability, 97.5 %, for disease development. Conclusions: RF isotypes predicted the development of RA, particularly in combination with ACPA, anti-CCP2 or anti-CarP antibodies. The highest probability for disease development was the presence of anti-CCP2 and anti-filaggrin antibodies

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 (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. 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 [Fiba563-583, Fib alpha 580-600, Fib beta 36-52, Fib beta 62-81a, Fib beta 62-81b], enolase [Eno5-21], and type II collagen [CitCII355-378]) in serum samples from 4,089 RA patients (1,231 Malaysian and 2,858 Swedish) and 827 healthy control subjects (249 Malaysian and 578 Swedish). The positive reaction threshold for each peptide was set separately for each population based on a specificity of 98%. Results. Distinct differences in the frequencies of 5 ACPA fine specificities (Vim60-75, Vim2-17, Fibb62-81b, Eno5-21, and CitCII355-378) were found between the Malaysian and Swedish RA populations, despite a nearly identical percentage of patients in each population who were positive for anti-cyclic citrullinated peptide 2 ish RA patients, the frequencies of antibodies to Vim60-75 (54% versus 44%, corrected P [P-corr] =1.06 x 10(-8)) and CitCII355-378 (17% versus 13%, P-corr = 0.02) were significantly higher, while the frequencies of antibodies to Vim2-17 (25% versus 32%, P-corr = 1.91 x 10(-4)), Fib beta 62-81b (15% versus 30%, P-corr = 2.47 x 10(-22)), and Eno5-21 (23% versus 50%, P-corr = 3.64 x 10(-57)) were significantly lower. Conclusion. Serum ACPA fine specificities differ between RA patients in different populations, although the total proportions of individuals positive for ACPAs are similar. Differing patterns of ACPA fine specificity could be attributed to variations in genetic and/or environmental factors

Antibodies against citrullinated peptides are associated with clinical and radiological outcomes in patients with early rheumatoid arthritis : a prospective longitudinal inception cohort study

Introduction: Anticitrullinated peptide antibody (ACPA) responses for 22 citrullinated peptides in patients with early rheumatoid arthritis (RA) were analysed and related to radiological and clinical outcome during the first 2 years in a prospective inception cohort. Methods: The ACPA reactivities were assessed in 1022 patients with early RA (symptoms <12 months) using the custom-made microarray chip (Thermo Fisher Scientific, Uppsala, Sweden) in a prospective longitudinal study of observational assessments of Disease Activity Score (DAS28 and its components) and radiology during the first 24 months, accounting for the treatment. Results: Frequency of ACPA reactivities varied between 13.3% and 63.1%. Of the anticyclic citrullinated peptide-2 (anti-CCP2) antibody-negative patients, ACPA reactivities were positive in 32.6%. Smoking, human leucocyte antigen-shared epitope (HLA-SE), anti-CCP2/rheumatoid factor, protein tyrosine phosphatase non-receptor type 22 (1858C/T) and DAS28 were significantly associated with number of ACPA reactivities. The ACPA reactivities modified differently the development of DAS28 over 24 months (identified using trajectories). Anti-Filaggrin307-324, anti-hnRNP (Peptide)-Z1 and anti-F4-CIT-R antibodies anticipated lower DAS28 values (p<0.01–0.05), while positivity for anti-Fibrinogen(Fib)β62-78(74), and anti-Fibα563-583 predicted higher DAS28 (p<0.01 both). Interaction between anti-Fibß36-52, anti-Pept-5 and anti-Bla-26 antibodies, respectively, and DAS28 during 24 months decreased significantly the DAS28 values (p<0.01–0.05). Corticosteroids and biologicals were related to DAS28-area under the curve and Larsen score 24 months. Anti-vimentin2-17 antibodies remained significantly associated with Larsen score at baseline and 24 months, respectively, and radiological progression, besides biologicals at 24 months adjusted for sex and age. Conclusions: Several ACPA reactivities modified significantly the DAS28 development during the first 24 months and were significantly associated with Larsen score at baseline, 24 months and radiological progression

Anti-Citrullinated Protein Antibody Specificities, Rheumatoid Factor Isotypes, and Incident Cardiovascular Events in Patients With Rheumatoid Arthritis

Objective To investigate the relationship between anti–citrullinated protein antibodies (ACPAs), specific ACPA subspecificities, rheumatoid factor (RF) isotypes, and incident cardiovascular (CV) events in patients with rheumatoid arthritis (RA). Methods Serum samples from Swedish patients with new-onset RA (diagnosed within 1 year of symptom onset between 1996 and 2009) were centrally typed for anti–cyclic citrullinated peptide 2 (anti-CCP2) antibodies, 20 ACPA subspecificities, and RF isotypes. Patients were followed up longitudinally in nationwide registers to monitor the occurrence of acute coronary syndrome (ACS), stroke, CV-related death, and major adverse CV events (MACE). The association between each serologic marker and CV outcome, and the impact of adjustment for the Disease Activity Score in 28 joints (DAS28), smoking status, and income at baseline, were assessed using Cox proportional hazards models. In addition, associations of serologic markers with all-cause mortality were explored. Results In total, 2,814 patients with RA were included in the study. The median follow-up was 13 years, during which the CV end points of ACS, stroke, or CV-related death were reported to occur in 375 patients. Occurrence and/or levels of anti-CCP2 were associated with risk of incident ACS (hazard ratio [HR] 1.46, 95% confidence interval [95% CI] 1.03–2.06), stroke (HR 1.47, 95% CI 1.03–2.10), CV-related death (P = 0.024 for association with anti-CCP2 levels), and MACE (HR 1.34, 95% CI 1.06–1.70). Similarly, an association with the number of ACPA subspecificities was observed; however, this could not be attributed to any individual or group of ACPA subspecificities. Presence of IgM-RF was associated with all CV end points except ACS, and IgA-RF was exclusively associated with CV-related death. Adjustment for smoking status, income, and DAS28 scores decreased most of the HRs, whereas IgA-RF remained associated with CV-related death (HR 1.61, 95% CI 1.05–2.48). All of the assessed serologic makers were associated with all-cause mortality. Conclusion RF isotypes and ACPAs are associated with future CV events in patients with RA. ACPA levels and number of subspecificities seem more important than the occurrence of particular subspecificities, and these associations were not explained by a history of ever smoking

Presence of autoantibodies in "seronegative" rheumatoid arthritis associates with classical risk factors and high disease activity.

International audienceRheumatoid arthritis (RA) is classified as seropositive or seronegative, depending on the presence/absence of rheumatoid factor (RF), primarily IgM RF, and/or anti-citrullinated protein antibodies (ACPA), commonly detected using anti-cyclic citrullinated peptide (CCP) assays. Known risk factors associate with the more severe seropositive form of RA; less is known about seronegative RA. Here, we examine risk factors and clinical phenotypes in relation to presence of autoantibodies in the RA subset that is traditionally defined as seronegative.Methods: Anti-CCP2 IgG, 19 ACPA fine-specificities, IgM/IgG/IgA RF, anti-carbamylated-protein (CarP) antibodies, and 17 other autoantibodies, were analysed in 2755 RA patients and 370 controls. Antibody prevalence, levels, and co-occurrence were examined, and associations with risk factors and disease activity during 5 years were investigated for different antibody-defined RA subsets.Results:Autoantibodies were detected in a substantial proportion of the traditionally defined seronegative RA subset, with ACPA fine-specificities found in 30%, IgA/IgG RF in 9.4%, and anti-CarP antibodies in 16%, with a 9.6% co-occurrence of at least two types of RA-associated autoantibodies. HLA-DRB1 shared epitope (SE) associated with the presence of ACPA in anti-CCP2-negative RA; in anti-CCP2-positive RA, the SE association was defined by six ACPA fine-specificities with high co-occurrence. Smoking associated with RF, but not with ACPA, in anti-CCP2-negative RA. Presence of ACPA and RF, but not anti-CarP antibodies, in conventionally defined “seronegative” RA, associated with worse clinical outcome.Conclusions:“Seronegative” RA is not truly a seronegative disease subset. Additional screening for ACPA fine-specificities and IgA/IgG RF defines a group of patients that resembles seropositive patients with respect to risk factors and clinical picture and may contribute to earlier diagnosis for a subset of anti-CCP2−/IgM RF− patients with a high need for active treatment.Keywords: Rheumatoid arthritis (RA), Autoantibodies, Anti-citrullinated protein antibodies (ACPA), Cyclic citrullinated peptide (CCP), Rheumatoid factor (RF), Anti-carbamylated protein antibodies (anti-CarP), Disease activity score for 28 joints (DAS28), HLA-DRB1 shared epitope (SE), Smokin

The role of anti-citrullinated protein antibody reactivities in an inception cohort of patients with rheumatoid arthritis receiving treat-to-target therapy

Background: Anti-citrullinated protein antibody (ACPA) reactivities precede clinical onset of rheumatoid arthritis (RA), and it has been suggested that ACPA reactivities towards distinct target proteins may be associated with differences in RA phenotypes. We aimed to assess the prevalence of baseline ACPA reactivities in an inception cohort of patients with early RA, and to investigate their associations with disease activity, treatment response, ultrasound findings and radiographic damage. Methods: Disease-modifying antirheumatic drug (DMARD)-naïve patients with early RA, classified according to the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria, were included in the ARCTIC trial and assessed in the present analysis. During follow up, patients were monitored frequently and treatment was adjusted according to a predetermined protocol, starting with methotrexate monotherapy with prednisolone bridging. Analysis of 16 different ACPA reactivities targeting citrullinated peptides from fibrinogen, alpha-1 enolase, vimentin, filaggrin and histone was performed using a multiplex chip-based assay. Samples from 0, 3, 12 and 24 months were analysed. Controls were blood donors with similar characteristics to the patients (age, gender, smoking status). Results: A total of 217 patients and 94 controls were included. Median [25, 75 percentile] number of ACPA reactivities in all patients was 9 [4, 12], and were most prevalent in anti-cyclic citrullinated peptide /rheumatoid factor-positive patients 10 [7, 12]. Disease activity measures and ultrasound scores at baseline were lower in ACPA reactivity-positive compared to ACPA reactivity-negative patients. ACPA reactivity levels decreased after 3 months of DMARD treatment, most pronounced for fibrinogenβ 60–74 to 62% of baseline antibody level, with least change in filaggrin 307–324 to 81% of baseline antibody level, both p < 0.001. However, outcomes in disease activity measures, ultrasound and radiographic scores after 12 and 24 months were not associated with baseline levels or changes in ACPA reactivity levels and/or seroreversion after 3 months. Conclusions: The clinical relevance of analysing ACPA reactivities in intensively treated and closely monitored early RA was limited, with no apparent associations with disease activity, prediction of treatment response or radiographic progression. Further studies in larger patient materials are needed to understand the role of ACPA reactivities in patients with RA classified according to the 2010 ACR/EULAR criteria and treated according to modern treatment strategies