Annemiek Willemze

Marked differences in fine specificity and isotype usage of the anti–citrullinated protein antibody in health and disease

OBJECTIVE Anti-citrullinated protein antibodies (ACPAs) display high association with rheumatoid arthritis (RA) and are implicated in its pathogenesis. The presence of ACPAs is known to precede the onset of RA. In order to identify the features that could confer its pathogenicity, we extensively characterized this antibody response in a unique North American native population of patients with RA and their unaffected relatives. METHODS The levels of IgA, IgM, and IgG ACPAs, as well as IgM and IgA rheumatoid factor (RF), were measured in serum samples obtained from 81 patients with RA and 195 of their unaffected relatives. The isotype distribution, the fine specificity of the ACPA response, and its association with RF were compared in health and disease. RESULTS ACPA positivity was observed in 19% of the healthy relatives and approximately 91% of the patients with RA. ACPA isotype usage was strikingly lower in unaffected relatives than in patients with RA (1-2 versus 5-6 isotypes). Fine specificity studies showed that reactivity to citrullinated fibrinogen and vimentin was present in sera from patients with RA, while it was virtually absent in their unaffected relatives. Finally, the ACPA and RF responses were associated in patients with RA but were discordant in their healthy relatives. Extended analyses revealed that the presence of ACPAs was associated with RA irrespective of RF status, while the association of RF with disease relied on its interaction with ACPAs. CONCLUSION The fine specificity and isotype usage of the ACPA response are qualitatively different in health and disease. Epitope spreading and expansion of the isotype repertoire might be necessary for development of RA, and this could be facilitated by the presence of RF antibodies.

Antibodies to Porphyromonas gingivalis Are Associated with Anticitrullinated Protein Antibodies in Patients with Rheumatoid Arthritis and Their Relatives

Objective. Anticitrullinated protein antibodies (ACPA) are relatively specific for rheumatoid arthritis (RA), and predate disease. The oral pathogen Porphyromonas gingivalis may play a role in breaking immune tolerance to citrullinated antigens. We studied a cohort of patients with RA and their relatives looking for associations between anti-P. gingivalis antibodies and ACPA. Methods. Patients with RA (n = 82) and their relatives (n = 205) from a North American Native (NAN) population were studied, along with 47 NAN and 60 non-NAN controls. IgM and IgA rheumatoid factor (RF) were tested by nephelometry and ELISA. Second-generation anticyclic citrullinated peptide (anti-CCP2) isotypes and IgG anti-P. gingivalis lipopolysaccharides were tested by ELISA. HLA-DRB1 typing was performed by sequencing. Oral hygiene and smoking habits were assessed by questionnaires. Results. Autoantibody frequency in patients with RA and relatives: ACPA 91% vs 19%, respectively; IgM RF 82% vs 17%; IgA RF 48% vs 22%. Anti-P. gingivalis levels were higher in patients with RA compared to relatives and controls (p = 0.005) and higher in ACPA-positive patients with RA than in ACPA-negative patients with RA (p = 0.04) and relatives (p < 0.001), but comparable in RF-positive and RF-negative patients and relatives. Poor oral hygiene and smoking were prevalent, but with no clear association with autoantibodies. Relatives with 2 shared-epitope alleles were more likely to be ACPA-positive (OR 2.5, p = 0.02). Conclusion. In a genetically predisposed population of NAN patients with RA and their relatives, anti-P. gingivalis antibodies were associated with ACPA. These findings suggest that immune responses to P. gingivalis may be involved in breaking immune tolerance to citrullinated antigens.

The influence of ACPA status and characteristics on the course of RA

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, and affects 0.5–1% of the population. Although it poses a considerable health problem, relatively little remains known about the disease pathogenesis and etiology. In the past decade, anti-citrullinated protein antibodies (ACPA) have emerged as suspects in the development and/or progression of RA. Citrullinated proteins—containing the amino acid citrulline, generated post-translationally from arginine—are found in the joints of patients with RA, but are not specific for the disease. This situation contrasts with the presence of ACPA, which are mostly found in individuals with RA. Intriguingly, ACPA can also be found in individuals before symptom onset. In these instances the ACPA response seems to be in its infancy, recognizing only a few citrullinated antigens and not using the full isotype repertoire. These characteristics of the ACPA response mature before clinical disease precipitates. Evidence is emerging that ACPA status can further characterize the heterogeneous RA phenotype, not only with respect to outcome, but perhaps also with respect to intervention. This Review summarizes the evolution of the ACPA response and its putative role in disease pathogenesis, as well as its relationship with clinical phenotype and diagnostic potential.

Recognition of citrullinated and carbamylated proteins by human antibodies: specificity, cross-reactivity and the ‘AMC-Senshu’ method

Recently, a novel family of autoantibodies in rheumatoid arthritis (RA) patients was described: anti-carbamylated protein (Anti-CarP) antibodies, which target carbamylated (homocitrulline-containing) epitopes.1 ,2 Since citrulline and homocitrulline have a similar structure, we wished to determine to what extent human autoantibodies can differentiate between them. Unlike human antibodies, the anti-modified citrulline (AMC) antibody developed by Dr Senshu3–8 is able to recognise citrullinated epitopes irrespective of the neighbouring amino acids. Thus, we also aimed to verify whether the AMC assay could distinguish between these two amino acids. To address this, we loaded gels with citrullinated, carbamylated and non-modified forms of foetal calfs serum (FCS) and human fibrinogen (Fib). Gels loaded with equal amounts of these protein preparations (figure 1A) were used for western blotting and staining with the ‘AMC-Senshu’ method. Both the citrullinated and the carbamylated forms of the proteins tested were strongly recognised, whereas, the non-modified form did not reveal any staining (figure 1B). Staining similar western blots with selected human sera1 revealed that sera-positive …

Extensive glycosylation of ACPA-IgG variable domains modulates binding to citrullinated antigens in rheumatoid arthritis

Objectives To understand the molecular features distinguishing anti-citrullinated protein antibodies (ACPA) from ‘conventional’ antibodies in rheumatoid arthritis (RA). Methods Serum of ACPA-positive RA patients was fractionated by size exclusion chromatography and analysed for the presence of ACPA-IgG by ELISA. ACPA-IgG and non-citrulline-specific IgG were affinity purified from serum, plasma and/or synovial fluid and analysed by gel electrophoresis. Electrophoresis bands were excised, enzymatically digested and analysed by mass spectrometry. Binding affinity to citrullinated antigens was measured by ELISA and imaging surface plasmon resonance using recombinant monoclonal ACPA with molecular modifications. Results In all donor samples studied (n=24), ACPA-IgG exhibited a 10–20 kDa higher molecular weight compared with non-autoreactive IgG. This feature also distinguished ACPA-IgG from antibodies against recall antigens or other disease-specific autoantibodies. Structural analysis revealed that a high frequency of N-glycans in the (hyper)variable domains of ACPA is responsible for this observation. In line with their localisation, these N-glycans were found to modulate binding avidity of ACPA to citrullinated antigens. Conclusions The vast majority of ACPA-IgG harbour N-glycans in their variable domains. As N-linked glycosylation requires glycosylation consensus sites in the protein sequence and as these are lacking in the ‘germline-counterparts’ of identified variable domains, our data indicate that the N-glycosylation sites in ACPA variable domains have been introduced by somatic hypermutation. This finding also suggests that ACPA-hyperglycosylation confers a selective advantage to ACPA-producing B cells. This unique and completely novel feature of the citrulline-specific immune response in RA elucidates our understanding of the underlying B cell response.

Distinct ACPA fine specificities, formed under the influence of HLA shared epitope alleles, have no effect on radiographic joint damage in rheumatoid arthritis

Objectives Human leucocyte antigen shared epitope (SE) alleles are associated with joint destruction, the presence of anticitrullinated protein antibodies (ACPA) and the ACPA fine specificity repertoire in rheumatoid arthritis (RA). A large variation in joint destruction is seen within the ACPA-positive patient population, and it is conceivable that certain ACPA reactivities contribute to radiological damage. The authors investigated whether ACPA fine specificities, which are formed under the influence of SE alleles, associate with the extent of radiographic joint damage. Methods Antibodies recognising six citrullinated epitopes were determined in sera of 330 ACPA-positive RA patients genotyped for SE alleles. The association between SE alleles, ACPA fine specificity and radiographic joint damage was assessed using radiographic follow-up data. A second cohort of 154 RA patients with 5 and 10-year radiographic follow-up was used for replication. Results SE alleles predisposed to the recognition of certain citrullinated epitopes. However, none of the ACPA fine specificities studied influenced radiographic joint damage. Importantly, although SE alleles associated with radiographic damage in the total RA population, this association was no longer detectable after stratification for the presence of ACPA. Conclusions SE alleles are instrumental in shaping the ACPA repertoire. However, ACPA fine specificities formed under the influence of SE alleles do not seem to affect joint destruction.

A genetic variant in the region of MMP-9 is associated with serum levels and progression of joint damage in rheumatoid arthritis

Objectives The severity of joint destruction is highly variable between rheumatoid arthritis (RA) patients. The majority of its heritability is still unexplained. Several autoimmune diseases share genetic risk variants that may also influence disease progression. We aimed to identify genetic risk factors for the severity of joint damage in RA by studying genetic susceptibility loci of several autoimmune diseases. Methods In phase 1, 3143 sets of x-rays of 646 Dutch RA patients taken over 7 years (Sharp van der Heijde (SHS) scored) were studied. Genotyping was done by Immunochip. Associations of single-nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) >0.01 and joint destruction were analysed. In phase 2, 686 North American RA patients with 926 SHS-scored x-rays over 15 years of follow-up were evaluated. In both phases multiple testing corrections were done for the number of uncorrelated SNPs; the thresholds for significance were p<1.1×10−6 and p<0.0036. Matrix metalloproteinase 9 (MMP-9) levels were measured with ELISA in baseline serum samples. Results In phase 1, 109 SNPs associated significantly with joint destruction (p<1.1×10−6). Of these, 76 were located in the HLA region; the 33 non-HLA variants were studied in phase 2. Here two variants were associated with the severity of joint destruction: rs451066 on chromosome 14 (p=0.002, MAF=0.20) and rs11908352 on chromosome 20 (p=0.002, MAF=0.21). Rs11908352 is located near the gene encoding MMP-9. Serum levels of MMP-9 were significantly associated with the rs11908352 genotypes (p=0.007). Conclusions These data indicate that two loci that confer risk to other autoimmune diseases also affect the severity of joint destruction in RA. Rs11908352 may influence joint destruction via MMP-9 production.

ACPA fine-specificity profiles in early rheumatoid arthritis patients do not correlate with clinical features at baseline or with disease progression.

IntroductionAutoantibodies against citrullinated peptides/proteins (ACPA) are found in approximately 75% of the sera of patients with rheumatoid arthritis (RA). The RA-specific ACPA are frequently present prior to disease onset and their presence associates with a more erosive disease course. ACPA can therefore be used to aid the diagnosis and prognosis of RA. Recently, it became clear that ACPA are very heterogeneous, both in an individual patient and among different patients. The aim of this study was to investigate whether clinically meaningful ACPA profiles exist in early RA patients.MethodsTwenty citrullinated peptides and the corresponding non-citrullinated control peptides were immobilized on microarray sensor chips. Sera from 374 early arthritis patients were analyzed by surface plasmon resonance imaging (i SPR) of biomolecular interactions on the sensor chip.ResultsCluster analysis of the reactivities with the citrullinated peptides, after subtraction of the reactivities with the corresponding control peptides confirmed the heterogeneity of the ACPA response in RA and revealed 12 distinct ACPA profiles. The association of the 5 most frequent profiles with clinical features at diagnosis and during the disease course was examined, showing no statistically significant associations.ConclusionsCompared to the detection of ACPA in RA sera by CCP-based assays, ACPA profiling in early arthritis patients did not reveal associations with disease activity and progression scores.

Anti-citrullinated fibronectin antibodies in rheumatoid arthritis are associated with human leukocyte antigen-drb1 shared epitope alleles

IntroductionFibronectin is one of the most abundant proteins present in the inflamed joint. Here, we characterized the citrullination of fibronectin in the joints of rheumatoid arthritis (RA) patients and studied the prevalence, epitope specificity and human leukocyte antigen (HLA) association of autoantibodies against citrullinated fibronectin in RA.MethodsCitrullinated residues in fibronectin isolated from RA patient synovial fluid were identified by mass spectrometry. The corresponding citrullinated and non-citrullinated peptides were synthesized and used to analyze the presence of autoantibodies to these peptides in RA sera and sera from other diseases and healthy controls by ELISA. The data were compared with risk factors like shared epitope HLA alleles and smoking, and with clinical features.ResultsFive citrullinated residues were identified in fibronectin from RA synovial fluid. RA sera reacted in a citrulline-dependent manner with two out of four citrullinated fibronectin peptides, one of which contains two adjacent citrulline residues, in contrast to non-RA sera, which were not reactive. The most frequently recognized peptide (FN-Cit1035,1036, LTVGLTXXGQPRQY, in which × represents citrulline) was primarily targeted by anti-CCP (cyclic citrullinated peptide) 2-positive RA patients. Anti-FN-Cit1035,1036 autoantibodies were detected in 50% of established anti-CCP2-positive RA patients and in 45% of such patients from a early arthritis clinic. These antibodies appeared to be predominantly of the immunoglobulin G (IgG) isotype and to be associated with HLA shared epitope alleles (odds ratio = 2.11).ConclusionsFibronectin in the inflamed synovia of RA patients can be citrullinated at least at five positions. Together with the flanking amino acids, three of these citrullinated residues comprise two epitopes recognized by RA autoantibodies. Anti-citrullinated fibronectin peptide antibodies are associated with HLA shared epitope alleles.

The concentration of anticitrullinated protein antibodies in serum and synovial fluid in relation to total immunoglobulin concentrations

Background Anticitrullinated protein antibodies (ACPA) are one of the best predictors for the development of rheumatoid arthritis. Nonetheless, relatively little information is present on the absolute concentration of ACPA in relation to total immunoglobulin (Ig) concentrations. Such information would be of relevance to compare ACPA levels to other antibody levels. Here, we estimated the relative abundance of ACPA Ig in serum and synovial fluid using a quantitative approach. Methods ACPA were purified using HiTrap Streptavidin columns coupled with biotinylated cyclic citrullinated peptide (CCP2). Total Ig and anti-CCP2 isotype reactivities were measured by ELISA. Results ACPA were successfully isolated as substantial antibody amounts were eluted from sera of ACPA-positive patients and neglectable antibody amounts were eluted from sera of ACPA-negative patients. Up to 1 in 80 IgG-molecules were estimated to be ACPA. Strikingly, IgM-ACPA was most abundant in synovial fluid (with the highest enrichment in the range of one IgM-ACPA for every eight IgM-antibodies). Conclusions ACPA-IgG levels are estimated to be within the range of peak levels of protective antibody responses against recall antigens. IgM-ACPA is abundantly present in synovial fluid, suggesting the presence of a continuous ongoing autoimmune response in the synovial compartment.