Association of HLA–DR3 with anti–cyclic citrullinated peptide antibody–negative rheumatoid arthritis

Objective

Recent data have shown that the most prominent and longest known genetic risk factors for rheumatoid arthritis (RA), HLA–DRB1 shared epitope alleles, are only associated with RA that is characterized by the presence of antibodies against cyclic citrullinated peptide (anti‐CCP antibodies) and not with anti‐CCP–negative RA. We undertook this study to investigate whether anti‐CCP–negative RA is associated with other HLA–DRB1 alleles.

Methods

HLA typing was performed for 377 patients from the Leiden Early Arthritis Clinic who were diagnosed as having RA within the first year of followup (206 anti‐CCP–positive patients and 171 anti‐CCP–negative patients), 235 patients who, after 1 year, had undifferentiated arthritis (UA) (28 anti‐CCP–positive patients and 207 anti‐CCP–negative patients), and 423 healthy control subjects. Odds ratios (ORs) with 95% confidence intervals (95% CIs) for HLA–DRB1 allele frequencies were determined for all patient groups compared with the healthy control group.

Results

HLA–DR3 was more frequently present in the anti‐CCP–negative RA group than in the control group (OR 1.84, 95% CI 1.26–2.67). This was not the case for anti‐CCP–positive RA (OR 0.92, 95% CI 0.60–1.40). HLA–DR3 was also more frequently present in anti‐CCP–negative UA patients (OR 1.59, 95% CI 1.10–2.28), but not in anti‐CCP–positive UA patients (OR 0.68, 95% CI 0.17–1.92).

Conclusion

HLA–DR3 is associated with anti‐CCP–negative arthritis and not with anti‐CCP–positive arthritis. These data show that distinct genetic risk factors are associated with the presence of anti‐CCP antibodies in RA and indicate that different pathogenetic mechanisms underlie anti‐CCP–positive and anti‐CCP–negative RA.

     

Specific association of type 1 diabetes mellitus with anti–cyclic citrullinated peptide–positive rheumatoid arthritis

Objective

The co‐occurrence of autoimmune diseases such as rheumatoid arthritis (RA) and type 1 diabetes mellitus (DM) has been reported in individuals and families. In this study, the strength and nature of this association were investigated at the population level in a Swedish case–control cohort.

Methods

For this case–control study, 1,419 patients with incident RA diagnosed between 1996 and 2003 were recruited from university, public, and private rheumatology units throughout Sweden; 1,674 matched control subjects were recruited from the Swedish national population registry. Sera from the subjects were tested for the presence of antibodies to cyclic citrullinated peptide (anti‐CCP), rheumatoid factor (RF), and the 620W PTPN22 allele. Information on a history of diabetes was obtained by questionnaire, telephone interview, and/or medical record review. The prevalence of type 1 DM and type 2 DM was compared between patients with incident RA and control subjects and further stratified for the presence of anti‐CCP, RF, and the PTPN22 risk allele.

Results

Type 1 DM was associated with an increased risk of RA (odds ratio [OR] 4.9, 95% confidence interval [95% CI] 1.8–13.1), and this association was specific for anti‐CCP–positive RA (OR 7.3, 95% CI 2.7–20.0), but not anti‐CCP–negative RA. Further adjustment for the presence of PTPN22 attenuated the risk of anti‐CCP–positive RA in patients with type 1 DM to an OR of 5.3 (95% CI 1.5–18.7). No association between RA and type 2 DM was observed.

Conclusion

The association between type 1 DM and RA is specific for a particular RA subset, anti‐CCP–positive RA. The risk of developing RA later in life in patients with type 1 DM may be attributed, in part, to the presence of the 620W PTPN22 allele, suggesting that this risk factor may represent a common pathway for the pathogenesis of these 2 diseases.

     

Strong combined gene–environment effects in anti–cyclic citrullinated peptide–positive rheumatoid arthritis: A nationwide case–control study in Denmark

Objective

To study the role of shared epitope (SE) susceptibility genes, alone and in combination with tobacco smoking and other environmental risk factors, for risk of subtypes of rheumatoid arthritis (RA) defined by the presence or absence of serum antibodies against cyclic citrullinated peptides (CCPs).

Methods

To address these issues, a nationwide case–control study was conducted in Denmark during 2002–2004, comprising incident cases of RA or patients with recently diagnosed RA (309 seropositive and 136 seronegative for IgG antibodies against CCP) and 533 sex‐ and age‐matched population controls. Associations were evaluated by logistic regression analyses, in which odds ratios (ORs) served as measures of relative risk.

Results

Compared with individuals without SE susceptibility genes, SE homozygotes had an elevated risk of anti‐CCP–positive RA (OR 17.8, 95% confidence interval [95% CI] 10.8–29.4) but not anti‐CCP–negative RA (OR 1.07, 95% CI 0.53–2.18). Strong combined gene–environment effects were observed, with markedly increased risks of anti‐CCP–positive RA in SE homozygotes who were heavy smokers (OR 52.6, 95% CI 18.0–154), heavy coffee drinkers (OR 53.3, 95% CI 15.5–183), or oral contraceptive users (OR 44.6, 95% CI 15.2–131) compared with SE noncarriers who were not exposed to these environmental risk factors.

Conclusion

Persons who are homozygous for SE susceptibility genes, notably those who are also exposed to environmental risk factors, have a markedly and selectively increased risk of anti‐CCP–positive RA. A distinction between anti‐CCP–positive RA and anti‐CCP–negative RA seems warranted, because these RA subtypes most likely represent etiologically distinct disease entities.

     

Synovial fluid levels of anti–cyclic citrullinated peptide antibodies and IgA rheumatoid factor in rheumatoid arthritis,psoriatic arthritis,and osteoarthritis

Objective

To assess the levels of anti–cyclic citrullinated peptide (anti‐CCP) and IgA rheumatoid factor (IgA‐RF) in synovial fluids of patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), and osteoarthritis (OA).

Methods

Knee effusions of 29 patients with RA (23 women, 6 men; mean ± SD age 60 ± 15 years), 20 with PsA (6 women, 14 men; mean age 51 ± 12 years), and 19 with OA (9 women, 10 men; mean age 73 ± 11.8 years) were aspirated, tested for white blood cell (WBC) counts, centrifuged, and stored at ?20°. Sera of 22, 11, and 12 of these patients with RA, PsA, and OA, respectively, were similarly stored. IgG anti‐CCP and IgA‐RF were detected by enzyme‐linked immunosorbent assay. Erythrocyte sedimentation rate and C‐reactive protein levels were used as measures of disease activity.

Results

Mean levels of synovial fluid anti‐CCP and IgA‐RF were significantly increased in RA joint effusions compared with PsA and OA (anti‐CCP: 150 ± 134, 34 ± 29, and 24 ± 26 units, respectively [P < 0.003]; IgA‐RF: 76 ± 77, 15.7 ± 10, and 18 ± 20 units, respectively). No significant difference was noted between OA and PsA. A significant correlation was found between synovial fluid anti‐CCP and serum anti‐CCP and IgA‐RF. In patients with RA, a significant correlation was found between synovial fluid WBC counts and IgA‐RF (P = 0.03) and serum IgA‐RF (P = 0.008), but not between synovial fluid and serum anti‐CCP levels. In RA patients, C‐reactive protein correlated with serum IgA‐RF.

Conclusion

Anti‐CCP and IgA‐RF were significantly increased in synovial fluid of RA in comparison with PsA and OA patients.

     

Smoking increases rheumatoid arthritis susceptibility in individuals carrying the HLA–DRB1 shared epitope,regardless of rheumatoid factor or anti–cyclic citrullinated peptide antibody status

Objective

Smoking is associated with rheumatoid arthritis (RA) in individuals with the HLA–DRB1 shared epitope (SE). SE alleles have been shown to be predominantly associated with anti–cyclic citrullinated peptide (anti‐CCP)–positive RA. These risk factors have not been identified for anti‐CCP–negative RA. The aim of this study was to investigate whether SE‐containing HLA–DRB1 alleles, smoking, or the combination of these factors contributes to the development of RA, depending on the presence or absence of serologic markers, in a Korean population.

Methods

All of the patients with RA (n =1,482) and all of the control subjects (n = 1,119) were Korean. Four‐digit HLA–DRB1 typing was performed by a conventional polymerase chain reaction–sequence‐based typing method. Information about smoking history was obtained through a questionnaire. The patients with RA were tested for anti‐CCP antibodies and rheumatoid factor (RF).

Results

The SE alleles had significant effects on anti‐CCP antibody and RF formation. The DRB1*0901 allele was associated with the presence of anti‐CCP antibodies (odds ratio [OR] 2.49) and RF (OR 2.09). SE alleles and smoking were associated with both anti‐CCP–positive and anti‐CCP–negative RA. The combination of smoking and double copies of the SE allele increased the risk of anti‐CCP–positive RA 36.11‐fold and increased the risk of anti‐CCP–negative RA 12.29‐fold, compared with the risk among nonsmokers not carrying SE alleles. Interactions between SE alleles and smoking were observed for both anti‐CCP–positive and RF‐positive RA, although the associations of RF‐positive RA could be consequences of the underlying anti‐CCP antibody status.

Conclusion

We demonstrated that the combination of SE alleles and smoking is associated with RA susceptibility regardless of anti‐CCP antibody or RF status, but that the combination shows stronger effects in anti‐CCP–positive/RF‐positive patients with RA than in anti‐CCP–negative/RF‐negative patients with RA. The SE–smoking interactions were present in anti‐CCP–positive and RF‐positive RA.

     

The HLA–DRB1 shared epitope alleles are primarily a risk factor for anti–cyclic citrullinated peptide antibodies and are not an independent risk factor for development of rheumatoid arthritis

Objective

The shared epitope (SE)–containing HLA–DRB1 alleles represent the most significant genetic risk factor for rheumatoid arthritis (RA). Recent studies indicate that the SE alleles are associated with only RA that is characterized by the presence of anti–cyclic citrullinated peptide (anti‐CCP) antibodies, and not with anti‐CCP–negative disease. In this study we investigated whether the SE alleles contribute to the development of anti‐CCP–positive RA, or whether they are associated solely with the presence of anti‐CCP antibodies. We therefore determined the influence of the SE alleles and anti‐CCP antibodies on the progression from recent‐onset undifferentiated arthritis (UA) to RA.

Methods

Patients with recent‐onset UA at the 2‐week visit (n = 570) were selected from the Leiden Early Arthritis Cohort. SE alleles, rheumatoid factor (RF) status, and anti‐CCP antibody levels were determined. Progression to RA or other diagnoses was monitored.

Results

One hundred seventy‐seven patients with UA developed RA during the 1‐year followup, whereas the disease in 393 patients remained unclassified or was given other diagnoses. The SE alleles correlated with the presence of anti‐CCP antibodies, but not with the presence of RF. Both in SE‐positive and in SE‐negative patients with UA, the presence of anti‐CCP antibodies was significantly associated with the development of RA. More intriguingly, however, no apparent contribution of the SE alleles to the progression to RA was found when analyses were stratified according to the presence of anti‐CCP antibodies. In patients with anti‐CCP–positive disease, the presence of SE alleles was associated with significantly higher levels of anti‐CCP antibodies, suggesting that the SE alleles act as classic immune response genes.

Conclusion

The SE alleles do not independently contribute to the progression to RA from UA, but rather contribute to the development of anti‐CCP antibodies.

     

Association of smoking with the constitution of the anti–cyclic citrullinated peptide response in the absence of HLA–DRB1 shared epitope alleles

Objective

Smoking is a risk factor for anti–cyclic citrullinated peptide (anti‐CCP) antibody–positive rheumatoid arthritis (RA) in patients with HLA–DRB1 shared epitope (SE) alleles. It is unknown whether smoking influences not only the presence of these antibodies, but also other characteristics of the anti‐CCP response, such as isotype usage. The aim of this study was to determine the influence of smoking on anti‐CCP isotypes in RA patients, and to determine whether this influence is observed in the presence and/or absence of SE alleles.

Methods

IgA, IgM, and IgG subclasses of anti‐CCP antibodies were measured by enzyme‐linked immunosorbent assay in serum obtained at the first visit to the Leiden Early Arthritis Clinic from 216 patients with anti‐CCP–positive RA whose smoking habits were also assessed. HLA genotyping data were available for 202 of these patients.

Results

IgA and IgM anti‐CCP were more frequent in RA patients who were smokers than in those who were nonsmokers (odds ratio 2.8 and 1.8, respectively). In addition, levels of all isotypes of anti‐CCP, except IgG3, were significantly higher (P < 0.05) in smokers. The number of anti‐CCP isotypes was higher in smokers compared with nonsmokers, both in SE‐negative RA (P = 0.04) and in SE‐positive RA (P = 0.07).

Conclusion

Patients with anti‐CCP–positive RA who have a current or former tobacco exposure display a more extensive anti‐CCP isotype usage in general, and IgA and IgM in particular, compared with patients with anti‐CCP–positive RA who have never smoked. In contrast to its influence on the incidence of anti‐CCP positivity, the influence of tobacco exposure on the constitution of the anti‐CCP response is significant in SE‐negative RA. These findings suggest a differential effect of tobacco exposure on the induction as compared with the propagation of the anti‐CCP antibody response.

     

Association of anti–citrullinated vimentin and anti–citrullinated α‐enolase antibodies with subsets of rheumatoid arthritis

Objective

To determine whether the anti–citrullinated vimentin peptide 60–75 (anti–Cit‐vimentin) and the immunodominant anti–citrullinated α‐enolase peptide 1 (anti–CEP‐1) antibodies are associated with subsets of patients with rheumatoid arthritis (RA) independently of the associations between anti–cyclic citrullinated peptide (anti‐CCP) antibodies and clinical features of RA.

Methods

The 3 antibody types were quantified by enzyme‐linked immunosorbent assay (ELISA) in serum samples from 521 patients with RA and 173 healthy controls of Spanish ancestry. Genotypes for HLA–DRB1 alleles and rs2476601 in PTPN22 were available for these patients and controls plus an additional 106 healthy controls. A combined analysis of the 3 antibodies was conducted using stratified contingency tables and logistic regression models.

Results

A differential, particularly strong, and independent association was observed between the presence of anti–Cit‐vimentin antibodies and the presence of shared epitope (SE) alleles, specifically in patients carrying 2 SE alleles, and between the presence of anti– Cit‐vimentin antibodies and the prevalence of joint erosion. Associations were observed between anti–CEP‐1 positivity and the presence of HLA–DRB1 and PTPN22 risk alleles and their additive interaction. These associations were not accounted for by the anti‐CCP status.

Conclusion

Our results indicate that the 2 antibodies against citrullinated peptides analyzed in this study add specific information beyond that obtained with the anti‐CCP status. They define subgroups of patients with RA in which genetic factors have different weight and there is an observed difference in the prevalence of erosions.

     

Regulation of anti–cyclic citrullinated peptide antibodies in rheumatoid arthritis: Contrasting effects of HLA–DR3 and the shared epitope alleles

Objective

To examine the association between HLA–DRB1 alleles and the production of anti–cyclic citrullinated peptide (anti‐CCP) and rheumatoid factor (RF) autoantibodies in patients with rheumatoid arthritis (RA).

Methods

We studied 1,723 Caucasian RA patients enrolled in the North American Rheumatoid Arthritis Consortium (NARAC) family cohort and the Study of New Onset Rheumatoid Arthritis (SONORA) cohort. All patients were tested for anti‐CCP antibodies (by enzyme‐linked immunosorbent assay), RF (by nephelometry), and HLA–DR genotype (by polymerase chain reaction and sequence‐specific oligonucleotide hybridization).

Results

When controlled for the presence of RF, anti‐CCP positivity was strongly associated with the HLA–DRB1 shared epitope (SE). In RF+ patients, the presence of the SE was very significantly associated with anti‐CCP positivity, with an odds ratio (OR) of 5.8 and a 95% confidence interval (95% CI) of 4.1–8.3. This relationship was also seen in RF– patients (OR 3.1 [95% CI 1.8–5.3]). In contrast, RF positivity was not significantly associated with presence of the SE independently of anti‐CCP antibodies. Strikingly, HLA–DRB1*03 was strongly associated with reduced anti‐CCP titers, even after controlling for the presence of the SE and restricting the analysis to anti‐CCP+ patients. HLA–DR3 was also associated with anti‐CCP– RA in our population.

Conclusion

The HLA–DRB1 SE is strongly associated with the production of anti‐CCP antibodies, but not RF. In contrast, HLA–DR3 alleles are associated with anti‐CCP– disease and with lower levels of anti‐CCP antibodies, even when controlling for the SE. These data emphasize the complexity of the genetic effects of the major histocompatibility complex on the RA phenotype.

     

Secretion of anti–citrulline‐containing peptide antibody by B lymphocytes in rheumatoid arthritis

Objective

To understand the regulation of anti–citrulline‐containing peptide antibody (anti‐CCP) production in rheumatoid arthritis (RA), production of anti‐CCP by B cells derived from peripheral blood (PB), bone marrow (BM), and synovial fluid (SF) was examined.

Methods

Purified PB and SF B cells were isolated by negative selection and then cultured in the absence or presence of L–CD40 ligand cells and interleukin‐10 or anti‐CD3–activated T cells. Total IgM and IgM–anti‐CCP were detected after 14 days of culture by enzyme‐linked immunosorbent assay. Enzyme‐linked immunospot assays were performed to analyze the frequency of cells that spontaneously produced IgM–anti‐CCP in BM and SF B cells.

Results

IgM–anti‐CCP autoantibodies were induced in PB B cells from healthy controls and RA patients following coculture with activated T cells or application of the CD40 activation system, whereas no production could be detected when PB B cells were cultured in the absence of a stimulus. SF and BM B cells from anti‐CCP–seropositive RA patients, but not anti‐CCP–seronegative patients, actively produced IgM–anti‐CCP without stimulation. The frequency of spontaneous production of IgM–anti‐CCP among the IgM‐secreting cells ranged from 2.2% to 25%.

Conclusion

These results indicate the presence of B cell precursors for anti‐CCP autoantibodies that are able to produce antibodies upon stimulation in the PB B cell repertoire of healthy controls and patients with RA. In contrast, B cells that actively secreted anti‐CCP were specifically present in the BM and SF compartment of anti‐CCP–seropositive RA patients. The local presence of anti‐CCP–secreting cells in the inflamed joints provides evidence for an antigen‐driven maturation of CCP‐specific B cells at the site of inflammation in RA.

     

Isotype distribution of ANTI–CYCLIC citrullinated peptide antibodies in undifferentiated arthritis and rheumatoid arthritis reflects an ongoing immune response

Objective

The evolution of the rheumatoid arthritis (RA)–specific anti–cyclic citrullinated peptide (anti‐CCP) antibody response, as measured by the isotypes of anti‐CCP, has not been described. This study was undertaken to determine anti‐CCP isotype usage in patients with undifferentiated arthritis (UA), patients with recent‐onset RA, and patients with RA of long duration.

Methods

IgA, IgM, and IgG subclasses of anti‐CCP were measured by enzyme‐linked immunosorbent assay in serum samples that were obtained from IgG anti‐CCP antibody–positive patients with UA (n = 110) and IgG anti‐CCP antibody–positive patients with RA (n = 152) early after the onset of arthritis. Patients with UA in whom RA developed within 1 year (UA→RA) were compared with patients with UA in whom RA did not develop within 1 year (UA→UA). In addition, baseline serum samples obtained from a subset of patients with RA (n = 64) were compared with sera obtained from the same patients a median of 7 years later.

Results

IgM anti‐CCP was present in early samples from both patients with UA and patients with RA and in followup samples from patients with RA. Several IgG anti‐CCP antibody–positive patients who did not have IgM anti‐CCP early after disease onset did display IgM anti‐CCP later in the course of the arthritis. A diverse pattern of isotype usage was detected in early samples, with a trend toward lower frequencies of all isotypes of anti‐CCP in patients with UA compared with patients with RA and in UA→UA patients compared with UA→RA patients. Levels of all isotypes except IgG1 had decreased after 7 years.

Conclusion

These data indicate development of the anti‐CCP isotype repertoire into full usage early in the course of arthritis. The sustained presence of IgM anti‐CCP indicates ongoing recruitment of new B cells into the anti‐CCP response, reflecting a continuous (re)activation of the RA‐specific anti‐CCP response during the course of anti‐CCP–positive arthritis.

     

Association of rheumatoid arthritis treatment response and disease duration with declines in serum levels of IgM rheumatoid factor and anti–cyclic citrullinated peptide antibody

Objective

To examine the association of treatment response and disease duration with changes in rheumatoid factor (RF) and anti–cyclic citrullinated peptide (anti‐CCP) antibody levels among patients with rheumatoid arthritis (RA).

Methods

The study sample included 66 RA patients who completed double‐blind, randomized clinical protocols and for whom baseline and followup serum samples were available. Anti‐CCP and RF levels were measured using commercially available assay kits. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were used to describe the association of response and disease duration with declines in antibody levels.

Results

Patients had a mean ± SD age of 49.9 ± 12.0 years and were predominantly female (n = 51; 77%). The mean ± SD duration between the times at which the baseline and followup serum samples were obtained was 13.7 ± 8.6 months. Among the 64 subjects with positive antibody at baseline, 33 (52%) experienced a ≥25% reduction in the anti‐CCP antibody level during the course of treatment, and 35 patients (55%) had a ≥25% reduction in RF. After adjustment for the baseline anti‐CCP antibody level, only a shorter disease duration (≤12 months) was significantly associated with a decline in the level of anti‐CCP antibody (OR 3.0, 95% CI 1.0–8.8), and no association with treatment response was observed. Conversely, treatment response was the only significant determinant of a decrease in RF levels (OR 3.6, 95% CI 1.2–10.4).

Conclusion

Shorter disease duration predicts greater declines in anti‐CCP antibody levels with treatment in RA. Although treatment response is a robust determinant of a decrease in RF, it does not appear to be associated with declines in the anti‐CCP antibody level.

     

Interaction between smoking,the shared epitope,and anti–cyclic citrullinated peptide: A mixed picture in three large North American rheumatoid arthritis cohorts

Objective

Recently, Swedish members of the Epidemiological Investigation of Rheumatoid Arthritis (EIRA) provided evidence that smoking may trigger RA‐specific immune reactions to citrullinated protein in carriers of HLA–DR shared epitope alleles. In an effort to confirm this interaction between smoking and shared epitope alleles, we performed a case‐only analysis of 3 North American RA cohorts.

Methods

A total of 2,476 white patients with RA were studied, 1,105 from the North American Rheumatoid Arthritis Consortium (NARAC) family collection, 753 from the National Inception Cohort of Rheumatoid Arthritis Patients (Inception Cohort), and 618 from the Study of New Onset Rheumatoid Arthritis (SONORA). All patients were HLA–DRB1 typed, and tested for anti–cyclic citrullinated peptide (anti‐CCP) and rheumatoid factor. Information about smoking history was obtained by questionnaire.

Results

A significant association was found between smoking and the presence of anti‐CCP in the NARAC and the Inception Cohort, but not in the SONORA. The shared epitope alleles consistently correlated with anti‐CCP in all 3 populations. Using multiple logistic regression analyses, shared epitope alleles were still the most significant risk factor for anti‐CCP positivity. Weak evidence of gene–environment interaction between smoking and shared epitope alleles for anti‐CCP formation was found only in the NARAC.

Conclusion

Unlike the EIRA data, we could not confirm a major gene–environment interaction for anti‐CCP formation between shared epitope alleles and smoking in 3 North American RA cohorts. Our data indicate a need for further studies to address the full range of environmental factors other than smoking that may be associated with citrullination and RA.

     

Antibodies to several citrullinated antigens are enriched in the joints of rheumatoid arthritis patients

Objective

High titers of specific anti–citrullinated protein antibodies (ACPAs) are frequently present in the serum of rheumatoid arthritis (RA) patients, but their presence in synovial fluid is less well characterized. The purpose of this study was to compare the levels of antibody to 4 well‐defined citrullinated candidate RA autoantigens in serum and synovial fluid and to determine whether antibodies to one citrullinated antigen are dominant over another. Furthermore, we studied their relationships with mutated citrullinated vimentin (MCV), a newly identified RA‐specific serum assay, and the classic cyclic citrullinated peptide (CCP) in the synovial fluid of well‐defined HLA–DR groups.

Methods

Paired serum and synovial fluid samples from 290 RA patients and serum samples from 100 age‐ and sex‐matched healthy controls were analyzed for the presence of anti‐MCV and anti‐CCP antibodies and for reactivity to citrullinated fibrinogen, α‐enolase, type II collagen, and vimentin. A total of 219 of the 290 patients were genotyped for the HLA–DR shared epitope alleles.

Results

Significantly higher proportions of antibodies against all RA‐associated citrullinated antigens were found in synovial fluid as compared with serum. This was also true for the MCV and CCP responses but not for non–RA‐associated anti–tetanus toxoid antibodies. As expected, we found a high correlation between citrullinated vimentin and MCV responses. All synovial fluid ACPAs were predominantly associated with HLA–DRB1*04 alleles and were confined to the CCP+/MCV+ subset of patients.

Conclusion

MCV and CCP positivity represent a similar subset of RA patients, whereas ACPAs with different fine specificities fall into subgroups of anti‐CCP+/anti‐MCV+ patients. The levels of all specific ACPAs were elevated in synovial fluid, suggesting that there is local antibody production and/or retention of ACPAs at the site of inflammation governed by RA‐predisposing genes.

     

Quantitative heritability of anti–citrullinated protein antibody–positive and anti–citrullinated protein antibody–negative rheumatoid arthritis

Objective

The majority of genetic risk factors for rheumatoid arthritis (RA) are associated with anti–citrullinated protein antibody (ACPA)–positive RA, while far fewer genetic risk factors have been identified for ACPA‐negative RA. This study was undertaken to quantify the contribution of genetic risk factors in general, and of the predisposing HLA–DRB1 shared epitope (SE) alleles in particular, to the ACPA‐positive and ACPA‐negative subsets of RA, by computing their heritability and assessing the contribution of the HLA SE alleles.

Methods

One hundred forty‐eight RA twin pairs, in which at least 1 twin of each pair had RA, were tested for ACPAs and typed for HLA–DRB1 genotypes. Heritability was assessed in a logistic regression model including a bivariate, normally distributed random effect, representing the contribution of unobserved genetic factors to RA susceptibility, with the correlation of the random effects fixed according to twin zygosity. The contribution of the HLA SE alleles to genetic variance was assessed using a similar model, except that estimates were based on genotype‐specific population prevalences.

Results

The heritability of RA among the twin pairs was 66% (95% confidence interval [95% CI] 44–75%). For ACPA‐positive RA, the heritability was 68% (95% CI 55–79%), and for ACPA‐negative RA it was 66% (95% CI 21–82%). Presence of the HLA SE alleles explained 18% (95% CI 16–19%) of the genetic variance of ACPA‐positive RA but only 2.4% (95% CI 1.6–10%) of the genetic variance of ACPA‐negative RA.

Conclusion

The heritability of ACPA‐positive RA is comparable with that of ACPA‐negative RA. These data indicate that genetic predisposition plays an important role in the pathogenesis of ACPA‐negative RA, for which most individual genetic risk factors remain to be identified.

     

Association of Anti–Cyclic citrullinated peptide antibody levels with PADI4 haplotypes in early rheumatoid arthritis and with shared epitope alleles in very late rheumatoid arthritis

Objective

Anti–cyclic citrullinated peptide (anti‐CCP) antibodies are rheumatoid arthritis (RA)–specific serologic markers. RA susceptibility has been associated with HLA–DRB1 shared epitope (SE) alleles and single‐nucleotide polymorphism (SNP) haplotypes in the peptidyl arginine deiminase 4 gene (PADI4). This study was undertaken to determine whether anti‐CCP levels are associated with PADI4 haplotypes and/or SE alleles in Korean patients with RA.

Methods

Three nonsynonymous SNPs in PADI4 (padi4_89, padi4_90, and padi4_92) and SE alleles were genotyped, and serum anti‐CCP levels were measured, in 311 patients with nonerosive or erosive RA. The relationships between anti‐CCP levels and PADI4 haplotypes and/or SE alleles were analyzed statistically.

Results

Anti‐CCP levels were significantly higher in patients carrying the PADI4 RA risk haplotype than in patients who did not have the risk haplotype, among anti‐CCP–positive patients with RA with a disease duration of ≤34 months (P = 0.041), but not among patients with a longer disease duration or among those who had erosive RA versus nonerosive RA. In contrast, the levels were significantly higher in SE carriers than in noncarriers among patients with RA with a disease duration of ≥141 months (P = 0.0037) and among those who had erosive RA (P = 0.000098), but not among patients who had a shorter disease duration or those who had nonerosive RA.

Conclusion

The PADI4 RA risk haplotype is associated with increased anti‐CCP levels in RA patients with disease of short duration, and PADI4 may play a role in early RA. In contrast, SE alleles are associated with increased anti‐CCP levels in RA patients with very longstanding disease and in patients with erosive RA, suggesting that SE alleles play a role in very late RA.

     

Refining the complex rheumatoid arthritis phenotype based on specificity of the HLA–DRB1 shared epitope for antibodies to citrullinated proteins

Objective

The main genetic risk factor for rheumatoid arthritis (RA), the HLA region, has been known for 25 years. Previous research has demonstrated, within the RA population, an association between HLA–DRB1 alleles carrying the shared epitope (SE) and antibodies directed against cyclic citrullinated peptides (anti‐CCP antibodies). We undertook this study to make the first comparison of SE allele frequencies in the healthy population with those in RA patients who do or do not harbor anti‐CCP antibodies.

Methods

HLA–DRB1 typing was performed in 408 RA patients from the Leiden Early Arthritis Clinic (the Leiden EAC; a Dutch population‐based inception cohort in which disease course was followed up over time), in 423 healthy Dutch controls, and in 720 affected members of 341 US multiplex (sibpair) families of Caucasian origin from the North American RA Consortium (NARAC) with well‐established disease and fulfilling the American College of Rheumatology classification criteria for RA. The presence of anti‐CCP antibodies was determined by enzyme‐linked immunosorbent assay.

Results

For the Leiden EAC, the odds ratio (OR) describing the association of 2 copies of the SE allele with anti‐CCP positivity (using no copies of the SE allele in the healthy control group as the referent) was 11.79 (P < 0.0001), while the OR for 1 SE allele was 4.37 (P < 0.0001). No association with the SE was observed in the Dutch anti‐CCP–negative RA patients. For the NARAC families, linkage and association analysis revealed the SE to be associated only with anti‐CCP–positive disease and not with anti‐CCP–negative disease. Stratified analyses indicated that anti‐CCP antibodies primarily mediated association of the SE with joint damage or disease persistence.

Conclusion

HLA–DRB1 alleles encoding the SE are specific for disease characterized by antibodies to citrullinated peptides, indicating that these alleles do not associate with RA as such, but rather with a particular phenotype.

     

Alterations of the synovial T cell repertoire in anti–citrullinated protein antibody–positive rheumatoid arthritis

Objective

The association of HLA–DRB1 alleles with anti–citrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) suggests the potential involvement of T lymphocytes in ACPA‐seropositive disease. The purpose of this study was to investigate this hypothesis by systematic histologic and molecular analyses of synovial T cells in ACPA+ versus ACPA– RA patients.

Methods

Synovial biopsy samples were obtained from 158 RA patients. Inflammation was determined histologically and immunohistochemically. RNA was extracted from peripheral blood mononuclear cells and synovial tissues obtained from 11 ACPA+ RA patients, 7 ACPA– RA patients, and 10 spondylarthritis (SpA) patients (arthritis controls). T lymphocyte clonality was studied by combined quantitative and qualitative T cell receptor CDR3 length distribution (LD) analysis and direct sequencing analysis.

Results

ACPA+ and ACPA– RA patients were similar at both the clinical and histologic levels. At the molecular level, however, patients with ACPA+ synovitis displayed a marked elevation of qualitative CDR3 LD alterations as compared with those with ACPA– synovitis and with the SpA controls. These differences in CDR3 LD were not observed in the peripheral blood, indicating a selective recruitment and/or local expansion of T cells in the synovial compartment. The CDR3 LD alterations reflected true monoclonal or oligoclonal expansions, as confirmed by direct sequencing of the T cell receptor. The CDR3 LD alterations in RA synovium did not correlate with B cell clonal expansions but were inversely associated with synovial lymphoid neogenesis.

Conclusion

The T cell repertoire is specifically restricted in RA patients with ACPA+ synovitis. Whereas the origin and role of these clonal alterations remain to be determined, our data suggest the preferential involvement of T lymphocytes in ACPA‐seropositive RA.

     

Benefit of early treatment in inflammatory polyarthritis patients with anti–cyclic citrullinated peptide antibodies versus those without antibodies

Objective

To compare the clinical utility of anti–cyclic citrullinated peptide (anti‐CCP) antibodies and rheumatoid factor (RF) testing in predicting both functional outcome and response to treatment in early inflammatory polyarthritis (IP) patients.

Methods

A total of 916 IP subjects from a primary care incidence registry (1990–1994) had anti‐CCP antibody and RF status determined at baseline. Mean change in Health Assessment Questionnaire (HAQ) score between baseline and 5 years was compared by antibody status. The effect of treatment with disease‐modifying antirheumatic drugs and/or steroids over 5 years, early (<6 months of symptom onset) versus late initiation, and duration of treatment were also compared by anti‐CCP antibody status. The analysis was adjusted for treatment decisions and censoring over the followup, using marginal structural models.

Results

Anti‐CCP antibody–positive patients (n = 268) had more severe disease both at presentation and 5 years of followup, and this was independent of RF. On adjustment, anti‐CCP antibody–negative patients treated early experienced a significant improvement in functional disability compared with anti‐CCP antibody–negative patients who were never treated (?0.31; 95% confidence interval [95% CI] ?0.53, ?0.08), and experienced additional benefit for each additional month of early treatment. Anti‐CCP antibody–positive patients treated early did not have a significant improvement in HAQ score compared with those not treated (?0.14; 95% CI ?0.52, 0.24).

Conclusion

In this first observational study to examine the influence of anti‐CCP antibody status on treatment response, anti‐CCP antibody–positive IP patients showed less benefit from treatment, particularly early treatment, than anti‐CCP antibody–negative patients. This provides support for the inclusion of anti‐CCP antibodies as well as RF in the classification criteria for rheumatoid arthritis and for stratification by anti‐CCP antibody status in clinical trials.

     

Anti–cyclic citrullinated peptide antibodies from rheumatoid arthritis patients activate complement via both the classical and alternative pathways

Objective

It has been suggested that anti–citrullinated protein antibodies (ACPAs) play an important role in the pathogenesis of rheumatoid arthritis (RA). To exert their pathologic effects, ACPAs must recruit immune effector mechanisms such as activation of the complement system. Mouse models of RA have shown that, surprisingly, arthritogenic antibodies activate the alternative pathway of complement rather than the expected classical pathway. This study was undertaken to investigate whether human anti–cyclic citrullinated peptide (anti‐CCP) antibodies activate the complement system in vitro and, if so, which pathways of complement activation are used.

Methods

We set up novel assays to analyze complement activation by anti‐CCP antibodies, using cyclic citrullinated peptide–coated plates, specific buffers, and normal and complement‐deficient sera as a source of complement.

Results

Anti‐CCP antibodies activated complement in a dose‐dependent manner via the classical pathway of complement, and, surprisingly, via the alternative pathway of complement. The lectin pathway was not activated by anti‐CCP antibodies. Complement activation proceeded in vitro up to the formation of the membrane attack complex, indicating that all activation steps, including the release of C5a, took place.

Conclusion

Our findings indicate that anti‐CCP antibodies activate the complement system in vitro via the classical and alternative pathways but not via the lectin pathway. These findings are relevant for the design of interventions aimed at inhibition of complement‐mediated damage in RA.