Autoantibodies against the catalytic domain of BRAF are not specific serum markers for rheumatoid arthritis

Background

Autoantibodies to the catalytic domain of v-raf murine sarcoma viral oncogene homologue B1 (BRAF) have been recently identified as a new family of autoantibodies involved in rheumatoid arthritis (RA). The objective of this study was to determine antibody responses to the catalytic domain of BRAF in RA and other autoimmune diseases. The association between RA-related clinical indices and these antibodies was also assessed.

Methodology/Principal Findings

The presence of autoantibodies to the catalytic domain of BRAF (anti-BRAF) or to peptide P25 (amino acids 656–675 of the catalytic domain of BRAF; anti-P25) was determined in serum samples from patients with RA, primary Sjögren''s syndrome (pSS), systemic lupus erythematosus (SLE), and healthy controls by using indirect enzyme-linked immunosorbent assays (ELISAs) based on the recombinant catalytic domain of BRAF or a synthesized peptide, respectively. Associations of anti-BRAF or anti-P25 with disease variables of RA patients were also evaluated. Our results show that the BRAF-specific antibodies anti-BRAF and anti-P25 are equally present in RA, pSS, and SLE patients. However, the erythrocyte sedimentation rate (ESR) used to detect inflammation was significantly different between patients with and without BRAF-specific antibodies. The anti-BRAF-positive patients were found to have prolonged disease, and active disease occurred more frequently in anti-P25-positive patients than in anti-P25-negative patients. A weak but significant correlation between anti-P25 levels and ESRs was observed (r = 0.319, p = 0.004).

Conclusions/Significance

The antibody response against the catalytic domain of BRAF is not specific for RA, but the higher titers of BRAF-specific antibodies may be associated with increased inflammation in RA.     

Natural autoantibodies reactive with glycosaminoglycans in rheumatoid arthritis

Introduction

Although natural autoantibodies make up the majority of circulating immunoglobulins and are also present in high numbers in therapeutically used intravenous immunoglobulin preparations, they have received little attention and their precise role remains largely unknown. An increasing awareness of the importance of posttranslational autoantigen modifications and glycobiology led us to explore carbohydrate-reactive natural autoantibodies in patients with rheumatoid arthritis. This study examined systematic antibodies reactive to glycosaminoglycans (GAGs), the carbohydrate components of proteoglycans that are released in large amounts from degrading cartilage.

Methods

To measure antibodies reactive to six different types of GAGs, a specialised ELISA was used in which the carbohydrates were covalently linked to the plastic surface through a 2 nm spacer. Sera from rheumatoid arthritis patients (n = 66), umbilical cord serum samples (n = 11) and adult controls (n = 54) were studied. In order to explore cross-reactivity with microbial antigens, bacterial peptidoglycans and fungal polysaccharides were used. Sera and synovial fluid samples were also tested using a GlycoChip carbohydrate array to characterise individual carbohydrate recognition patterns. We followed a multistep statistical screening strategy for screening GAG-reactive antibodies as predictive disease markers.

Results

While anti-GAG antibodies were absent in the umbilical cord sera, they were readily detectable in adult controls and were significantly elevated in patients with rheumatoid arthritis (p < 0.001). Anti-GAG antibodies showed significant cross-reactivity among different types of GAGs. They also reacted with bacterial peptidoglycans and fungal polysaccharides. Interestingly, anti-chondroitin sulphate C IgM antibody levels showed inverse correlation both with the Disease Activity Score (DAS) 28 scores and C-reactive protein (CRP) levels in rheumatoid arthritis.

Conclusion

The highly abundant and cross-reactive, GAG-specific natural autoantibodies in serum may serve as novel disease-state markers in patients with rheumatoid arthritis.     

Soluble OX40L is associated with presence of autoantibodies in early rheumatoid arthritis

Introduction

OX40 and its ligand OX40L are key components in the generation of adaptive memory response and provide necessary co-stimulatory signals for activated effector T cells. Here we investigate the dual roles of the membrane and soluble (s) forms of OX40 and OX40L in plasma and synovial fluid and their association with autoantibodies and disease activity in rheumatoid arthritis (RA).

Methods

Soluble OX40 and sOX40L plasma levels were measured in treatment-naïve early RA patients (eRA) at baseline and after 3, 6, and 12 months of treatment with methotrexate and adalimumab (n = 39) and with methotrexate alone (n = 37). Adalimumab was discontinued after the first year, and patients were followed for additional 12 months. For comparison, sOX40 and sOX40L were measured in patients with chronic RA (cRA, n = 15) and healthy volunteers (HV, n = 34). Membrane-bound OX40 and OX40L expression on T cells, B cells and monocytes were quantified.

Results

Soluble OX40 plasma levels of eRA patients were not different at the time of treatment initiation, but were significantly higher after 12 months of treatment, compared with HV or cRA patients. Soluble OX40L was significantly elevated throughout the first 12 months of treatment compared with HVs and patients with cRA. Adalimumab treatment did not influence sOX40 or sOX40L plasma levels. At baseline, sOX40L levels were strongly associated with the presence of anti-citrullinated protein antibodies (ACPA) (P <0.001) and IgM-RF (P <0.0001). The sOX40/sOX40L ratio was decreased in eRA, and a low ratio at the time of adalimumab discontinuation was associated with increased DAS28CRP and risk of flare the following year. T cells in the synovial fluid had the highest expression of OX40, while monocytes and B cells were the main expressers of OX40L in the joint.

Conclusions

Plasma levels of sOX40 and sOX40L were increased in eRA and sOX40L was correlated with ACPA and IgM-RF. Further, expression of membrane-bound OX40 and OX40L was increased in eRA and cRA. Combined, these findings could reflect that increased activity in the OX40 systems facilitate to drive disease activity and autoantibody production in RA.

Trial registration

Clincaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00660647","term_id":"NCT00660647"}}NCT00660647, 10 April 2008.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0474-4) contains supplementary material, which is available to authorized users.     

New autoantibodies in early rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease causing articular cartilage and bone destruction. Since irreversible joint destruction can be prevented by intervention at the early stages of disease, early diagnosis of RA is important. In this study, we identified new autoantibodies in the sera of patients with early (less than one year) RA.

Methods

We screened the sera of 20 RA patients with disease duration less than one year, 19 RA patients with disease duration more than five years and 23 controls on 8,268 human protein arrays. We confirmed the validity of protein array detection by ELISA assays. We then performed epitope mapping with overlapping 15-mers to analyze RA sera reactivity.

Results

WIBG (within BGCN homolog (Drosophila)), GABARAPL2 (GABA(A) receptor associated protein like 2) and ZNF706 (zinc finger protein 706) proteins are preferentially recognized by autoantibodies from early RA patients. Of interest, autoantibodies to WIBG are very specific for early RA. Indeed, 33% of early RA patients'' sera recognize WIBG versus 5% of RA patients with disease duration more than 5 years and 2% of controls. We identified three linear peptides on WIBG GABARAPL2 and ZNF706 that are preferentially recognized by sera of early RA patients.

Conclusions

We identified new autoantibodies associated with RA with disease duration less than one year. These autoantibodies could be used as diagnosis markers in RA patients.     

Tissue autoantibodies during therapy with levamisole for rheumatoid arthritis

In 20 patients affected with rheumatoid arthritis, tissue autoantibodies were studied during a year of treatment with levamisole. Before therapy, antinuclear antibodies were present in 11 cases, anti-thyroid microsomes in 1 and anti-smooth muscle in 6. During therapy, autoantibodies remained almost unchanged in all patients, thus indicating that levamisole does not influence this aspect of humoral immunity in rheumatoid arthritis. Furthermore, no correlation was found between autoantibody pattern before treatment and effects of levamisole on clinical course of disease.     

Autoantibodies in rheumatoid arthritis and their clinical significance

Autoantibodies are proven useful diagnostic tools for a variety of rheumatic and non-rheumatic autoimmune disorders. However, a highly specific marker autoantibody for rheumatoid arthritis (RA) has not yet been determined. The presence of rheumatoid factors is currently used as a marker for RA. However, rheumatoid factors have modest specificity (~70%) for the disease. In recent years, several newly characterized autoantibodies have become promising candidates as diagnostic indicators for RA. Antikeratin, anticitrullinated peptides, anti-RA33, anti-Sa, and anti-p68 autoantibodies have been shown to have >90% specificity for RA. These autoantibodies are reviewed and the potential role of the autoantibodies in the pathogenesis of RA is briefly discussed.     

Non-HLA genes PTPN22, CDK6 and PADI4 are associated with specific autoantibodies in HLA-defined subgroups of rheumatoid arthritis

Introduction

Genetic susceptibility to complex diseases has been intensively studied during the last decade, yet only signals with small effect have been found leaving open the possibility that subgroups within complex traits show stronger association signals. In rheumatoid arthritis (RA), autoantibody production serves as a helpful discriminator in genetic studies and today anti-citrullinated cyclic peptide (anti-CCP) antibody positivity is employed for diagnosis of disease. The HLA-DRB1 locus is known as the most important genetic contributor for the risk of RA, but is not sufficient to drive autoimmunity and additional genetic and environmental factors are involved. Hence, we addressed the association of previously discovered RA loci with disease-specific autoantibody responses in RA patients stratified by HLA-DRB1*04.

Methods

We investigated 2178 patients from three RA cohorts from Sweden and Spain for 41 genetic variants and four autoantibodies, including the generic anti-CCP as well as specific responses towards citrullinated peptides from vimentin, alpha-enolase and type II collagen.

Results

Our data demonstrated different genetic associations of autoantibody-positive disease subgroups in relation to the presence of DRB1*04. Two specific subgroups of autoantibody-positive RA were identified. The SNP in PTPN22 was associated with presence of anti-citrullinated enolase peptide antibodies in carriers of HLA-DRB1*04 (Cochran-Mantel-Haenszel test P = 0.0001, P_corrected <0.05), whereas SNPs in CDK6 and PADI4 were associated with anti-CCP status in DRB1*04 negative patients (Cochran-Mantel-Haenszel test P = 0.0004, P_corrected <0.05 for both markers). Additionally we see allelic correlation with autoantibody titers for PTPN22 SNP rs2476601 and anti-citrullinated enolase peptide antibodies in carriers of HLA-DRB1*04 (Mann Whitney test P = 0.02) and between CDK6 SNP rs42041 and anti-CCP in non-carriers of HLA-DRB1*04 (Mann Whitney test P = 0.02).

Conclusion

These data point to alternative pathways for disease development in clinically similar RA subgroups and suggest an approach for study of genetic complexity of disease with strong contribution of HLA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0414-3) contains supplementary material, which is available to authorized users.     

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

Kiddo, a new transposable element family closely associated with rice genes

The promoter region of the rice ubiquitin2 (rubq2) gene was found to be polymorphic between japonica (T309) and indica (IR24) lines as the result of a 270-bp deletion in T309. A TTATA footprint in the T309 rubq2 promoter suggested that an excision event had occurred, and inspection of the 270-bp region present in IR24 revealed that it had all the characteristics of a miniature inverted repeat transposable element (MITE). Database searches showed that this element is a member of a new MITE family, which we have named Kiddo. Thirty-five complete Kiddo sequences were identified in existing rice genomic sequence databases. They could be arranged into four groups, within-group sequence identity was over 90%, with 65-75% identity between groups. The high sequence similarity within a group indicates that some Kiddo members were recently mobile and may still be active. An additional 24 decayed Kiddo sequences were detected. Interestingly, approximately 80% of 18 Kiddo members from annotated accessions lie within 530 bp of a coding sequence. That approximately 40% of Kiddo members present in genic regions reside in introns suggests that Kiddo transposition entails the use of both DNA and RNA intermediates, and may provide some insight into the origins of individual groups. DNA blot analysis showed that Kiddo is a rice-specific element, although one sequence with limited (72%) similarity to Kiddo group A was detected as a wheat EST. Kiddo family members may represent new molecular and phylogenetic markers, as well as representing valuable materials for studying the molecular mechanisms of MITE transposition.     

Polymorphism in a T-cell receptor variable gene is associated with susceptibility to a juvenile rheumatoid arthritis subset

This report demonstrates a T-cell receptor (Tcr) restriction fragment length polymorphism, defined by a Tcrb-V6.1 gene probe and Bgl II restriction enzyme, to be absolutely correlated with allelic variation in the coding sequence of a Tcrb-V6.1 gene. A pair of non-conservative amino acid substitutions distinguish the Tcrb-V6.1 allelic variants. An association of this Tcrb-V6.1 gene allelic variant with one form of juvenile rheumatoid arthritis (JRA) was established in a cohort of 126 patients. The association was observed in patients possessing the HLA-DQA1*0101 gene. Among HLA-DQA*0101 individuals, 19 of 26 patients (73.1%) carried one particular Tcrb-V6.1 gene allele as opposed to 11 of 33 controls (33%; p<0.005). Haplotypes carrying this HLA gene have previously been shown to confer increased risk for progression of arthritis in JRA. This demonstration of a disease-associated Tcrb-V gene allelic variant has not, to our knowledge, been previously reported and supports the contribution of polymorphism in the Tcr variable region genomic repertoire to human autoimmune disease.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M67511 for V6.1A and M67512 for V6.1B.     

IL2RA is associated with persistence of rheumatoid arthritis

IntroductionAlthough rheumatoid arthritis (RA) is generally a chronic disease, a proportion of RA-patients achieve disease-modifying antirheumatic drug (DMARD)-free sustained remission, reflecting loss of disease-persistence. To explore mechanisms underlying RA-persistence, we performed a candidate gene study. We hypothesized that variants associating with lack of radiographic progression also associate with DMARD-free sustained remission.Methods645 Dutch RA-patients were studied on DMARD-free sustained remission during a maximal follow-up duration of 10-years. Variants associated with radiographic progression under an additive model in the total RA-population (Human Leukocyte Antigens (HLA)-DRB1-shared epitope (SE), Dickkopf-1 (DKK1)-rs1896368, DKK1-rs1896367, DKK1-rs1528873, C5Orf30-rs26232, Interleukin-2 receptor-α (IL2RA)-rs2104286, Matrix metalloproteinase-9 (MMP-9)-rs11908352, rs451066 and Osteoprotegerin (OPG)-rs1485305) were studied. Cox-regression analyses were performed and Bonferroni correction applied. Soluble IL2Rα (sIL2Rα)-levels were studied. For replication, 622 RA-patients included in the French Evaluation et Suivi de POlyarthrites Indifférenciées Récentes cohort (ESPOIR)-cohort were investigated. Results were combined in inverse-variance weighted meta-analysis.ResultsSimilar as previously reported, the SE-alleles associated with less remission (hazard ratio (HR) = 0.57, 95 % confidence interval (95 % CI) = 0.42-0.77, p = 2.72×10⁻⁴). Variants in DKK-1, C5orf30, MMP-9 and OPG were not associated with remission. The IL2RA-rs2104286 minor allele associated with a higher chance on remission (HR = 1.52, 95 % CI = 1.16-1.99, p = 2.44×10⁻³). The rs2104286 minor allele associated with lower sIL2Rα-levels (p = 1.44×10⁻³); lower sIL2Rα-levels associated with a higher chance on remission (HR per 100 pg/L = 0.81, 95 % CI = 0.68-0.95, p = 0.012). When including rs2104286 and sIL2Rα-levels in one analysis, the HR for rs2104286 was 2.27 (95 % CI = 1.06-4.84, p = 0.034) and for sIL2Rα 0.83 (95 % CI = 0.70-0.98, p = 0.026). Within ESPOIR, the HR of rs2104286 was 1.31 (95 % CI = 0.90-1.90). The meta-analysis revealed a p-value of 1.01×10⁻³.ConclusionIL2RA-rs2104286 and sIL2Rα-level associated with RA-persistence. IL2RA variants are known to protect against multiple sclerosis, diabetes mellitus and RA. Besides HLA-SE, IL2RA-rs2104286 is thus far the only known genetic variant associated with both joint destruction and RA-persistence. This underlines the relevance of IL2RA for RA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0739-6) contains supplementary material, which is available to authorized users.     

Detection of autoantibodies to citrullinated BiP in rheumatoid arthritis patients and pro-inflammatory role of citrullinated BiP in collagen-induced arthritis

Introduction

Anti-citrullinated protein/peptide antibodies (ACPAs) are highly specific to rheumatoid arthritis (RA) patients and are thought to have a close relationship with the pathogenesis of arthritis. Several proteins, including fibrinogen, vimentin, and alpha-enolase, were reported as ACPA-target antigens, and their importance in RA pathogenesis was widely proposed. We identified citrullinated immunoglobulin binding protein (citBiP) as another ACPA target in RA patients and examined its pro-inflammatory role in arthritis.

Methods

We measured the levels of anti-citBiP, anti-BiP, and anti-cyclic citrullinated peptide (CCP) antibodies in the serum of RA patients (n = 100), systemic lupus erythematosus (SLE) patients (n = 60), and healthy controls (n = 30) using ELISA and immunoblotting. Epitope mapping was performed using 27 citBiP-derived peptides. In the mouse study, after DBA/1J mice were immunized with BiP or citBiP, serum titers of ACPAs were measured by ELISA and immunohistochemistry. The development of collagen-induced arthritis (CIA) was observed in BiP- or citBiP-pre-immunized mice.

Results

The serum levels of anti-BiP and anti-citBiP antibodies were significantly increased in RA patients, although only anti-BiP antibodies were slightly increased in SLE patients. Interestingly, anti-citBiP antibody levels were higher than anti-BiP antibody levels in 72% of RA patients, whereas no significant increase in anti-citBiP antibody levels was detected in SLE patients and healthy controls. The serum levels of anti-CCP antibodies were correlated with those of anti-citBiP antibodies in RA patients (R² = 0.41). Several citrulline residues of citBiP were determined to be major epitopes of anti-citBiP antibodies, one of which showed cross-reactivity with CCP. Immunization of DBA/1J mice with citBiP induced several kinds of ACPAs, including anti-CCP and anti-citrullinated fibrinogen antibodies. Pre-immunization with citBiP exacerbated CIA, and anti-CCP antibody levels were increased in citBiP-pre-immunized CIA mice.

Conclusions

CitBiP is a newly described ACPA target that may play a pro-inflammatory role in arthritis.     

A combination of autoantibodies to cyclic citrullinated peptide (CCP) and HLA-DRB1 locus antigens is strongly associated with future onset of rheumatoid arthritis

Antibodies against cyclic citrullinated peptide (CCP) and rheumatoid factors (RFs) have been demonstrated to predate the onset of rheumatoid arthritis (RA) by years. A nested case-control study was performed within the Northern Sweden Health and Disease study cohort to analyse the presence of shared epitope (SE) genes, defined as HLA-DRB1*0404 or DRB1*0401, and of anti-CCP antibodies and RFs in individuals who subsequently developed RA. Patients with RA were identified from among blood donors whose samples had been collected years before the onset of symptoms. Controls matched for age, sex, and date of sampling were selected randomly from the same cohort. The SE genes were identified by polymerase chain reaction sequence-specific primers. Anti-CCP2 antibodies and RFs were determined using enzyme immunoassays. Fifty-nine individuals with RA were identified as blood donors, with a median antedating time of 2.0 years (interquartile range 0.9-3.9 years) before presenting with symptoms of RA. The sensitivity for SE as a diagnostic indicator for RA was 60% and the specificity was 64%. The corresponding figures for anti-CCP antibodies were 37% and 98%, and for RFs, 17-42% and 94%, respectively. In a logistic regression analysis, SE (odds ratio [OR] = 2.35), anti-CCP antibodies (OR = 15.9), and IgA-RF (OR = 6.8) significantly predicted RA. In a combination model analysis, anti-CCP antibodies combined with SE had the highest OR (66.8, 95% confidence interval 8.3-539.4) in predicting RA, compared with anti-CCP antibodies without SE (OR = 25.01, 95% confidence interval 2.8-222.2) or SE without anti-CCP antibodies (OR = 1.9, 95% confidence interval 0.9-4.2). This study showed that the presence of anti-CCP antibodies together with SE gene carriage is associated with a very high relative risk for future development of RA.     

Demonstration of neutralizing autoantibodies against IL-1 alpha in sera from patients with rheumatoid arthritis

We have recently reported the presence of IgG which has a potent inhibitory activity against IL-1 alpha in some sera from patients with rheumatoid arthritis. The mechanism of this inhibition by IgG against IL-1 alpha is now elucidated. IgG with IL-1 alpha-inhibitory activity inhibited the binding of 125I-IL-1 alpha to receptors on rheumatoid synovial cells. In addition, preincubation of synovial cells with the inhibitory IgG did not block the binding of 125I-IL-1 alpha to receptors, suggesting a direct interaction between IgG and IL-1 alpha. To examine which region of the IgG, namely Fab or Fc region, has the inhibitory activity, the IgG was digested with papain, and Fab and Fc fragments were purified. Fab fragments, but not Fc fragments, inhibited both IL-1 alpha-induced thymocyte-proliferation and the binding of 125I-IL-1 alpha to receptors. We further demonstrated that the inhibitory IgG which was bound to protein A Sepharose could bind a significant amount of 125I-IL-1 alpha, whereas only a negligible binding of the radiolabeled ligand was detected when IgG without the inhibitory activity was used as control. Moreover, the binding of 125I-IL-1 alpha to IgG with the inhibitory activity was clearly blocked by Fab fragments of IgG having the inhibitory activity. Finally, affinity-purified IgG over an IL-alpha affinity column showed approximately 100-fold more potent inhibitory activity on IL-1 alpha-induced thymocyte proliferation compared with untreated IgG. From these results, we conclude that IgG molecules with IL-1-alpha-inhibitory activity are neutralizing autoantibodies against IL-1 alpha.