Clinically suspect arthralgia and rheumatoid arthritis: patients' perceptions of illness.

OBJECTIVES: Clinically suspect arthralgia (CSA) is an at-risk stage of rheumatoid arthritis (RA), in which patients experience symptoms and physical limitations. Perceptions of CSA-patients have remained largely unknown. Therefore, we aimed to map perceptions of CSA-patients and compare them to RA-patients. Additionally, we studied changes in perceptions in CSA over time. METHODS: 399 consecutively included CSA-patients from the Leiden and Rotterdam CSA-cohorts and 100 recently diagnosed RA-patients from the Leiden Early Arthritis Clinic were included. Patients' illness perceptions (IP) were assessed using the Brief Illness Perception Questionnaire (BIPQ), consisting of 8 questions (scale 0-10; higher score indicating more negative IP) covering cognitive, emotional and comprehensibility domains, and one open question on causes of disease. IP were measured at baseline in both populations and during 2 years follow-up in the CSA-cohorts. RESULTS: Total BIPQ-scores were comparable at CSA-presentation and RA-diagnosis (40±11 and 40±10; range 0-80). Comparing dimensions separately revealed that CSA-patients were less worried on physical complaints compared to RA-patients. However, CSA-patients were more negative about expected treatment-effect on symptoms. IP over time in CSA improved in patients without development of clinical arthritis (38±11 to 34±14; p=0.005) but remained similar in CSA-patients who progressed to arthritis/RA (mean 40 at both timepoints). CSA-patients mainly reported physical strain and heredity as perceived causes of their complaints. CONCLUSIONS: Although CSA-patients have not developed clinical arthritis, illness perceptions at CSA-presentation and RA-diagnosis are equally severe. Knowledge on worries and expectations may contribute to improving patient-contact and care in patients at risk of RA.

Ultrasound of the forefeet besides the hands in patients at risk for rheumatoid arthritis: is it worth the effort? A longitudinal cohort study.

OBJECTIVE: Ultrasound (US) can detect subclinical joint-inflammation in patients with clinically suspect arthralgia (CSA), which is valuable as predictor for rheumatoid arthritis (RA) development. In most research protocols both hands and forefeet are scanned, but it is unclear if US of the forefeet has additional value for predicting RA, especially since synovial hypertrophy in MTP-joints of healthy individuals is also common. To explore the possibility to omit scanning of the forefeet we determined if US of the forefeet is of additional predictive value for RA-development in CSA patients. METHODS: CSA patients of two independent cohorts underwent US of the hands and forefeet. We analyzed the association between RA-development and US-positivity for the full US-protocol, the full US-protocol with correction for Gray Scale(GS)-findings in the forefeet of healthy and the protocol without-forefeet. RESULTS: In total, 298 CSA patients were studied. In patients with a positive US, subclinical joint-inflammation was mostly present in the hands (90-86%). Only 10-14% of patients had subclinical joint-inflammation solely in the forefeet. US-positivity was associated with inflammatory arthritis development in both cohorts, with HRs 2.6(95%CI 0.9-7.5) and 3.1(95%CI 1.5-6.4) for the full protocol, 3.1(95%CI 1.3-7.7) and 2.7(95%CI 1.3-5.4) for the full US-protocol with correction, and 3.1(95%CI 1.4-6.9) and 2.8(95%CI 1.4-5.6) without the forefeet. AUROCs were equal across both cohorts. CONCLUSION: The forefeet can be omitted when US is used for the prediction of RA-development in CSA patients. This is due to the finding that subclinical joint-inflammation in the forefeet without concomitant inflammation in the hands is infrequent.

The Natural Sequence in Which Subclinical Inflamed Joint Tissues Subside or Progress to Rheumatoid Arthritis: A Study of Serial MRIs in the TREAT EARLIER Trial.

OBJECTIVE: The natural trajectory of clinical arthritis progression at the tissue level remains elusive. We hypothesized that subclinical inflammation in different joint tissues (synovitis, tenosynovitis, osteitis) increases in a distinct temporal order in patients with clinically suspect arthralgia (CSA) who develop rheumatoid arthritis (RA) and subsides in a different sequence when CSA spontaneously resolves. METHODS: We studied 185 serial magnetic resonance images (MRIs) from CSA patients with subclinical joint inflammation from the placebo arm of the TREAT EARLIER trial: 52 MRIs from 21 RA progressors (MRIs conducted at 1 year before, at 4 months before, and upon RA development), and 133 MRIs from 35 patients with spontaneous resolution of pain (MRIs conducted at baseline and at 4, 12, and 24 months). MRIs were scored for osteitis, synovitis, and tenosynovitis. We used cross-lagged models to evaluate 2 types of time patterns between pairs of inflamed tissues: a simultaneous pattern (coinciding changes) and a subsequent pattern (inflammatory changes in 1 tissue preceding changes in another tissue). RESULTS: In patients who developed RA, synovitis, tenosynovitis, and osteitis increased simultaneously. Increasing osteitis occurred in the final 4 months before RA diagnosis, following incremental tenosynovitis and synovitis changes during the 1 year to 4 months before diagnosis (P < 0.01). In anti-citrullinated protein antibody (ACPA)-positive and ACPA-negative patients who progressed to RA, osteitis increased just before RA development. In patients with pain resolution, simultaneous decreases in synovitis, tenosynovitis, and osteitis occurred, with tenosynovitis decreasing in the first 4 months after CSA onset preceding decreasing synovitis and osteitis during 4-12 months (P = 0.02 and P < 0.01). CONCLUSION: We identified natural sequences of subclinical inflammation in different joint tissues, which deepens our understanding of clinical arthritis and RA development. During RA progression, increasing osteitis followed previous increases in tenosynovitis and synovitis. During pain resolution, tenosynovitis decreased first, followed by decreasing synovitis and osteitis.

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.

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

INTRODUCTION: Autoantibodies 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. METHODS: Twenty 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 (iSPR) of biomolecular interactions on the sensor chip. RESULTS: Cluster 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. CONCLUSIONS: Compared 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.

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.

Anti-citrullinated fibronectin antibodies in rheumatoid arthritis are associated with human leukocyte antigen-DRB1 shared epitope alleles.

INTRODUCTION: Fibronectin 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. METHODS: Citrullinated 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. RESULTS: Five 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). CONCLUSIONS: Fibronectin 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 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.

The ACPA recognition profile and subgrouping of ACPA-positive RA patients.

OBJECTIVE: Anticitrullinated protein antibodies (ACPA) are the most predictive factor for the development of rheumatoid arthritis (RA). Epitope spreading towards more citrullinated epitopes occurs before the onset of RA. Here, the authors investigated whether specific epitope recognition allows the identification of specific RA subgroups and whether it is associated with clinical features of RA. METHODS: The reactivity of 661 patients with RA from the Leiden Early Arthritis Clinic against several citrullinated antigens was determined by ELISA. Cluster analyses were performed to identify subgroups of patients on the basis of their ACPA recognition profile. The association of the specific reactivities with clinical characteristics was studied. RESULTS: ACPA-positive patients displayed a heterogeneous ACPA recognition profile. After performing cluster analyses, no apparent clustering of patients was found, and on the basis of the reactivities analysed, 64 different subgroups could already be identified. The extent of epitope recognition was associated with anticyclic citrullinated peptide-2 levels. The recognition of specific citrullinated epitopes was not associated with baseline characteristics. Likewise, patients with an extended fine specificity repertoire did not display differences in baseline characteristics or joint damage after 7 years of follow-up using cyclic citrullinated peptide-2 levels as a proxy, compared to ACPA-positive patients recognising fewer peptides. CONCLUSION: These data show that the ACPA response is highly diverse with respect to recognition of specific citrullinated epitopes. Furthermore, the authors' data indicate that clinical correlates in established ACPA-positive RA are independent from the specific (group of) citrullinated peptides recognised.

The fine specificity of IgM anti-citrullinated protein antibodies (ACPA) is different from that of IgG ACPA.

INTRODUCTION: The antigen recognition pattern of immunoglobulin M (IgM) could, when directed against protein antigens, provide an indication of the antigenic moieties triggering new B cells. The half-life of IgM is short and memory B cells against T-cell-dependent protein antigens typically produce IgG and not IgM antibodies. In this study, we analyzed whether a difference exists between the fine specificity of IgM versus IgG anti-citrullinated protein antibodies (ACPAs). METHODS: We determined the fine specificity of IgM and IgG ACPAs in 113 ACPA-positive rheumatoid arthritis patients with IgM cyclic citrullinated peptide 2 (CCP2) levels above 100 AU/ml. Fine specificity was assessed by performing ELISA using citrullinated peptides derived from vimentin, fibrinogen-ß, fibrinogen-α and α-enolase, as well as citrullinated proteins fibrinogen and myelin basic protein. The arginine counterparts were used as controls. RESULTS: Recognition of defined citrullinated antigens by IgM ACPA was confined to samples that also displayed recognition by IgG ACPA. However, the IgM ACPA response displayed a more restricted antigen recognition profile than IgG ACPA (OR = 0.26, P < 0.0001). CONCLUSION: Our data show that several defined citrullinated antigens are recognized only by IgG ACPA, whereas others are also recognized by IgM ACPA. These observations suggest that not all citrullinated antigens are able to activate new B cells despite concurrent recognition by IgG ACPA.

Can anti-cyclic citrullinated peptide antibody-negative RA be subdivided into clinical subphenotypes?

INTRODUCTION: Studies investigating genetic risk factors for susceptibility to rheumatoid arthritis (RA) studied anti-citrullinated peptide antibody (CCP)-positive RA more frequently than anti-CCP-negative RA. One of the reasons for this is the perception that anti-CCP-negative RA may include patients that fulfilled criteria for RA but belong to a wide range of diagnoses. We aimed to evaluate the validity of this notion and explored whether clinical subphenotypes can be discerned within anti-CCP-negative RA. METHODS: The 318 patients with anti-CCP-negative RA (1987 ACR criteria), included in the Leiden Early Arthritis Clinic between 1993 and 2006, were studied for baseline characteristics and radiologic progression data during a mean follow-up of 5 years. Grouping was studied both at variable and patient levels. Principal components analysis and partial least-squares regression were applied to study for clustering of variables. A cluster analysis was performed to look for clustering of patients. RESULTS: The simultaneous presence of patient characteristics at disease presentation was observed for several groups; however, the three largest groups of patients' characteristics explained only 26.5% of the total variance. Plotting the contribution of each patient to these three groups did not reveal clustering of patients. Comparable observations were made when data on progression of joint destruction were studied in relation to baseline clinical data. A cluster analysis, evaluating whether patients resemble each other, revealed no grouping of patients. Altogether, no clinically distinguishable subphenotypes were observed. CONCLUSIONS: The current data provide evidence that, for risk-factor studies, anti-CCP-negative RA patients can be studied as one group.

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.

The interaction between HLA shared epitope alleles and smoking and its contribution to autoimmunity against several citrullinated antigens.

OBJECTIVE: Recent data suggest that a gene-environment interaction between smoking and the HLA shared epitope alleles plays a role in shaping the autoimmune reaction to specific citrullinated antigens. This study was undertaken to determine the effects of HLA shared epitope alleles and tobacco exposure on the immune response against various citrullinated antigens. These associations were analyzed in the anti-citrullinated protein antibody (ACPA)-positive stratum to control for the possibility that the associations found are explained by the known interaction between HLA shared epitope alleles and tobacco exposure on ACPA status. METHODS: In 661 patients with rheumatoid arthritis, reactivity against several citrullinated antigens from vimentin, fibrinogen, enolase, and myelin basic protein was determined by enzyme-linked immunosorbent assay. The effects of the HLA shared epitope alleles and tobacco exposure were assessed by logistic regression analysis. Biologic interaction was analyzed by investigating whether the effects of the risk factors combined exhibited departure from additivity. RESULTS: A significant interaction between tobacco exposure and HLA shared epitope alleles was found for the presence of ACPA as reported previously. When these interaction effects were studied for several ACPA "fine specificities," significant interactions were noted for several citrullinated peptides. However, these interactions were not present after stratification for ACPA status, indicating that the interaction between tobacco exposure and HLA shared epitope alleles influences autoimmunity not to specific citrullinated antigens, but rather to ACPA development. CONCLUSION: Our data indicate that the gene-environment interaction between HLA shared epitope alleles and smoking does not appear to shape the reactivity of the ACPA response. These data suggest that smoking promotes nonspecific citrullination rather than citrullination of specific antigens.

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.

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.