ARTHRITIS & RHEUMATOLOGY Vol. 66, No. 4, April 2014, pp 813–821 DOI 10.1002/art.38307 © 2014, American College of Rheumatology

Rheumatoid Factor as a Potentiator of Anti–Citrullinated Protein Antibody–Mediated Inflammation in Rheumatoid Arthritis Jeremy Sokolove,1 Dannette S. Johnson,2 Lauren J. Lahey,1 Catriona A. Wagner,1 Danye Cheng,1 Geoffrey M. Thiele,3 Kaleb Michaud,3 Harlan Sayles,3 Andreas M. Reimold,4 Liron Caplan,5 Grant W. Cannon,6 Gail Kerr,7 Ted R. Mikuls,3 and William H. Robinson1 Objective. The co-occurrence of rheumatoid factor (RF) and anti–citrullinated protein antibody (ACPA) positivity in rheumatoid arthritis (RA) is well described. However, the mechanisms underlying the potential interaction between these 2 distinct autoantibodies have not been well defined. The aim of this study was to evaluate the epidemiologic and molecular interaction of ACPAs and RF and its association with both disease activity and measures of RA-associated inflammation. Methods. In a cohort of 1,488 US veterans with RA, measures of disease activity and serum levels of cytokines and multiplex ACPAs were compared between the following groups of patients: double-negative (anti– cyclic citrullinated peptide [anti-CCP]ⴚ/RFⴚ), anti-

CCPⴙ/RFⴚ, anti-CCPⴚ/RFⴙ, or double-positive (antiCCPⴙ/RFⴙ). Additional studies were performed using an in vitro immune complex (IC) stimulation assay in which macrophages were incubated with ACPA ICs in the presence or absence of monoclonal IgM-RF, and tumor necrosis factor ␣ production measured as a readout of macrophage activation. Results. Compared with the double-negative subgroup (as well as each single-positive subgroup), the double-positive subgroup exhibited higher disease activity as well as higher levels of C-reactive protein and inflammatory cytokines (all P < 0.001). In vitro stimulation of macrophages by ACPA ICs increased cytokine production, and the addition of monoclonal IgM-RF significantly increased macrophage tumor necrosis factor ␣ production (P ⴝ 0.003 versus ACPA ICs alone). Conclusion. The combined presence of ACPAs and IgM-RF mediates increased proinflammatory cytokine production in vitro and is associated with increased systemic inflammation and disease activity in RA. Our data suggest that IgM-RF enhances the capacity of ACPA ICs to stimulate macrophage cytokine production, thereby providing a mechanistic link by which RF enhances the pathogenicity of ACPA ICs in RA.

Supported by the US Department of Veterans Affairs (research funding to Drs. Sokolove, Mikuls, and Robinson) and the NIH (National Institute of Arthritis and Musculoskeletal and Skin Diseases grants R01-AR-0636763 and U01-AI-101981 to Dr. Robinson). Dr. Sokolove is recipient of an Arthritis Foundation Innovative Research Award. Drs. Mikuls and Robinson are recipient of Rheumatology Research Foundation Within Our Reach Awards. 1 Jeremy Sokolove, MD, Lauren J. Lahey, BS, Catriona A. Wagner, BS, Danye Cheng, MS, William H. Robinson, MD, PhD: VA Palo Alto Health Care System, Palo Alto, California, and Stanford University School of Medicine, Stanford, California; 2Dannette S. Johnson, DO: Jackson VA Medical Center and University of Mississippi, Jackson; 3Geoffrey M. Thiele, PhD, Kaleb Michaud, PhD, Harlan Sayles, MS, Ted R. Mikuls, MD, MSPH: Omaha VA Medical Center and University of Nebraska Medical Center, Omaha; 4Andreas M. Reimold, MD: Dallas VA Medical Center and Texas Southwestern University Medical Center, Dallas; 5Liron Caplan, MD, PhD: Department of Veterans Affairs, Denver and University of Colorado School of Medicine, Denver; 6Grant W. Cannon, MD: George E. Wahlen VA Medical Center and University of Utah, Salt Lake City; 7Gail Kerr, MD, FRCP: Washington DC VA Medical Center and Georgetown University, Washington, DC. Address correspondence to Jeremy Sokolove, MD, or William H. Robinson, MD, PhD, Veteran Affairs Palo Alto Health Care System, Mail Stop 154R, 3801 Miranda Avenue, Palo Alto, CA 94304. E-mail: [email protected] or [email protected]. Submitted for publication July 29, 2013; accepted in revised form December 3, 2013.

Rheumatoid arthritis (RA) is often characterized by the presence of circulating autoantibodies. Rheumatoid factor (RF) was first described more than 50 years ago (1–3) and is detectable in nearly 70% of patients with RA, although its presence is not specific for RA (4,5). Nevertheless, whether RF plays a role in RA pathogenesis has remained unclear. More recently, attention has been given to anti–citrullinated protein antibodies (ACPAs). Anti–cyclic citrullinated peptide (anti-CCP) assays are commonly used to test for ACPAs, which are detectable in ⬃70% of patients with 813

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RA and are highly specific for RA (6). In addition to their use in diagnostic criteria, RF and ACPAs provide important prognostic information. Previous studies have examined the diagnostic utility of RF and ACPAs (7,8), and multiple studies have demonstrated that higher concentrations of both autoantibodies are associated with a more aggressive disease course marked by increased disease activity and reduced rates of remission (9–11). However, there has been little investigation into the mechanisms by which these autoantibodies could interact and/or contribute to RA pathogenesis. Although several in vitro (12–14) and in vivo (15) studies have identified a potential role for ACPAs in disease pathogenesis, the role of RF in RA pathogenesis remains elusive. In this study, we sought to investigate the role of RF as a contributor to the RA inflammatory burden, both independently and in synergy with ACPAs. PATIENTS AND METHODS Patient samples and clinical measures. Study subjects included US veterans enrolled in the Veterans Affairs Rheumatoid Arthritis (VARA) registry, with sites across the US (16). The registry has received Institutional Review Board approval at each site, and the Stanford Institutional Review Board approved the biomarker and in vitro analyses performed using RA samples. All patients satisfied the 1987 American College of Rheumatology classification criteria for RA (17) and provided informed written consent and Health Insurance Portability and Accountability Act authorization. The study group comprised 1,488 veterans with RA (89% male); detailed characteristics of the cohort are shown in Table 1. Banked serum samples were available for a representative population of 1,466 patients and were used for multiplex cytokine and autoantibody analyses. In addition to banked sera, VARA collects clinical data including the baseline and longitudinal 28-joint Disease Activity Score (DAS28) values (18), as well as baseline measurements of ACPAs, obtained using a Diastat secondgeneration CCP-2 antibody enzyme-linked immunosorbent assay (ELISA) (positivity ⱖ5 units/ml; Axis-Shield). VARA also collects RF (positivity ⱖ15 IU/ml) and high-sensitivity C-reactive protein (hsCRP) test results, as determined by nephelometry (Siemens). HsCRP concentrations were not available at followup visits. Although VARA is a multicenter project, standardized autoantibodies are measured at the laboratory of a single investigator (GMT). HLA–DRB1 genotyping was conducted as previously described (19). Patients were categorized as being positive or negative for HLA–DRB1 shared epitope (SE)–containing alleles and HLA–DR3 alleles. Followup measures include tender and swollen joint counts in 28 joints, erythrocyte sedimentation rate (ESR; mm/hour), pain score (range 0–10), Multidimensional Health Assessment Questionnaire (MD-HAQ) score (range 0–3) (20), patient’s and provider’s global assessments (range 0–100), and treatments. A comorbidity count (range 0–9) was calculated

for each patient, using administrative codes (21). Because we were investigating the association of autoantibodies with followup clinical parameters, patients were excluded if autoantibody or DAS28 data were unavailable, if only a single clinical observation was recorded, or if the total followup duration was ⬍6 months. Multiplex cytokine analysis. Multiplex analysis of cytokines and chemokines in human serum was performed using a Bio-Plex Pro Human Cytokine 17-plex Assay (Bio-Rad) run on a Luminex 200 system according to the manufacturer’s instructions, with the exception that a proprietary Bio-Rad assay dilution buffer was modified to contain reagents demonstrated to reduce the effects of heterophilic antibodies in multiplex immunoassays, as previously described (21). Data processing was performed by using Bio-Plex Manager 5.0 software, and analyte concentrations (picograms per milliliter) were interpolated from standard curves. Multiplex autoantigen arrays. Antibodies targeting 37 putative RA-associated autoantigens were measured using a custom bead-based immunoassay on a Bio-Plex platform, as previously described (22,23). Of the 37 antigens, 30 are citrullinated and 7 are native (native histone 2A, histone 2B, apolipoprotein A-I [Apo A-I], filaggrin 48–65 peptide, vimentin, fibrinogen, and Apo-AI 231–248 peptide). Briefly, serum was diluted and mixed with spectrally distinct florescent beads conjugated with putative RA-associated autoantigens, followed by incubation with anti-human phycoerythrin–labeled antibody and analysis on a Luminex 200 instrument. In vitro ACPA immune complex (IC) stimulation assays. ACPA IC stimulation of human macrophages was performed as previously described (12). Polyclonal rabbit IgG antibodies against fibrinogen (Pierce) or human IgG derived from patients with ACPA-positive RA (RA lgG) was used to generate plate-bound ICs containing citrullinated fibrinogen ICs. RA IgG derived from 3 pooled plasma samples that were shown by ELISA to contain high levels of anti–citrullinated fibrinogen antibodies was purified by affinity chromatography on protein G columns, according to the instructions of the manufacturer (Pierce). Monoclonal IgM-RF derived from a lymphoblastoid cell line generated from a patient with RA (24) was a generous gift from Drs. Michael Steinitz and Reuven Laskov (Hebrew University, Jerusalem, Israel). IgM-RF was isolated from cell culture supernatant by ammonium sulfate precipitation, washed, and dialyzed against phosphate buffered saline (PBS). The resultant product was shown to contain primarily IgM, with no contamination by human IgG. This antibody has been demonstrated to bind human and rabbit IgG but not IgM and, when bound to ICs, fails to bind complement (25). Additional monoclonal IgM antibodies with in vitro RF activity that were isolated from patients with mixed cryoglobulinemia were generously provided by Dr. Mariana Newkirk (McGill University, Montreal, Quebec, Canada). The purified RA IgG and monoclonal RF were separately concentrated by centrifugation over a 100-kd molecular weight filter column with buffer exchange to PBS (Amicon Ultra; Millipore) and were depleted of endotoxin by filtration through a polymyxin B column (Detoxi-Gel; Pierce). RA IgG and IgM-RF concentrations were estimated according to optical density at 280 nm, aliquoted, and stored at ⫺80°C. For generation of citrullinated fibrinogen ICs, flat-bottomed

INTERACTION BETWEEN RF AND ACPAs IN RA

815

Table 1. Characteristics of the patients with RA at the time of study enrollment*

Characteristic Sociodemographics and comorbidity Age, mean ⫾ SD years Male sex Race/ethnicity White African American Other High school education or higher Comorbidity count, mean ⫾ SD RA factors Ever smoking† HLA–DRB1 SE positive† 2 HLA–DRB1 alleles HLA–DR3† Age at diagnosis, mean ⫾ SD years† Disease duration, mean ⫾ SD years‡ Prednisone treatment Methotrexate treatment‡ Biologic agent treatment Nodules†

Concordant seronegative (n ⫽ 206)

Anti-CCP⫹/RF⫺ (n ⫽ 102)

Anti-CCP⫺/RF⫹ (n ⫽ 134)

Concordant seropositive (n ⫽ 1,046)

64.5 ⫾ 12.2 89.3

61.2 ⫾ 11.9 88.2

63.3 ⫾ 11.9 91.8

63.0 ⫾ 11.2 91.1

79.6 15.1 5.3 86.7 2.6 ⫾ 1.6

75.5 16.7 7.8 86.7 2.2 ⫾ 1.5

76.9 15.7 7.7 85.6 2.4 ⫾ 1.5

76.7 16.7 6.6 84.0 2.3 ⫾ 1.6

73.2 54.6 8.7 25.6 55.9 ⫾ 15.0 8.7 ⫾ 10.1 35.0 45.4 16.9 12.1

73.4 77.8 23.2 14.1 50.7 ⫾ 13.7 10.5 ⫾ 10.7 40.7 64.8 24.2 24.5

73.9 54.4 6.5 32.6 53.8 ⫾ 13.7 9.5 ⫾ 10.7 40.7 51.2 23.6 26.1

82.7 76.5 24.7 12.8 51.8 ⫾ 13.6 11.2 ⫾ 11.4 45.2 49.7 21.1 33.8

* Except where indicated otherwise, values are the percent. RA ⫽ rheumatoid arthritis; anti-CCP ⫽ anti–cyclic citrullinated peptide; RF ⫽ rheumatoid factor; SE ⫽ shared epitope. † Overall P ⬍ 0.001, by analysis of variance. ‡ Overall P ⬍ 0.01, by analysis of variance.

96-well culture plates were coated overnight at 4°C with 50 ␮l of citrullinated fibrinogen (20 ␮g/ml), washed in PBS containing 0.05% Tween 20, and incubated for 2 hours at 4°C with 100 ␮l of polyclonal rabbit antibodies against fibrinogen (50 ␮g/ml), 100 ␮l of anti–citrullinated fibrinogen–positive IgG (10 mg/ml), or 100 ␮l of anti–citrullinated fibrinogen–positive IgG preincubated with monoclonal IgM-RF (stock concentration 5 mg/ml used at 1:20 or 1:100 dilution); PBS alone was used as a control. Wells were again washed in PBS containing 0.05% Tween 20, and macrophages (50,000/well) in 200 ␮l of RPMI containing 5% fetal calf serum were then added to the wells and incubated for 16 hours, at which time levels of tumor necrosis factor ␣ (TNF␣) in culture supernatant were measured by ELISA (PeproTech). All in vitro cell stimulations were performed in triplicate and in at least 2 separate experiments. Levels of TNF␣ production from in vitro macrophagestimulation assays were compared using Student’s unpaired t-test. Statistical analysis. Patients were categorized into the following groups: double-negative (anti-CCP⫺/RF⫺; n ⫽ 206), anti-CCP⫹/RF⫺ (n ⫽ 102), anti-CCP⫺/RF⫹ (n ⫽ 134), and double-positive (anti-CCP⫹/RF⫹; n ⫽ 1,046). Comparisons of patient characteristics were examined according to autoantibody subgroup, using chi-square tests or analysis of variance (ANOVA). Unadjusted comparisons of the 8 continuous disease activity measures assessed at enrollment were examined using one-way ANOVA with Tukey’s post hoc test to compare each of the 4 subgroups. Levels of each cytokine as well as hsCRP were compared between the double-negative, anti-CCP⫹/RF⫺, anti-CCP⫺/RF⫹, and double-positive subgroups, using the Kruskal-Wallis test with Dunn’s post hoc test.

The ESR was log-transformed prior to analysis to render a more normal distribution. Given the skewed distributions, joint counts were dichotomized into 0 tender/swollen joints and ⱖ1 tender/swollen joints, with comparisons examining the probability of having a joint count of ⬎0. Continuous joint counts were reported for descriptive purposes. We examined whether the associations observed between autoantibody status and disease activity at enrollment were independent of other covariates, and whether these associations were apparent over an extended period of observation. For multivariable analyses, double-negative cases served as the referent population. Generalized linear mixed models were used to evaluate multivariable associations of autoantibody group with disease activity assessed during followup. The generalized linear mixed models adjusted for the random effects from sites and correlations between the outcomes from the same patient over time via a compound symmetry correlation structure. Analyses were completed using Stata version 12, SAS version 9.3, and GraphPad Prism version 5. Additional comparisons were performed on multiplex cytokines as well as multiplex ACPAs, using SAM (Significance Analysis of Microarrays) version 3.08 (24). Output was sorted based on false discovery rates (FDRs) in order to identify the antigens with the greatest differences in autoantibody reactivity between serologic subgroups. The use of FDRs obviates the need to adjust for multiple comparisons. Hierarchical clustering was performed using Cluster 3.0 to arrange the SAM results according to similarities among autoantibody specificities, and the results were displayed using Java TreeView (version 1.1.3).

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RESULTS ACPAs and RF in the VARA cohort. The characteristics of the 1,488 patients are shown in Table 1. The mean ⫾ SD followup was 3.6 ⫾ 2.8 years, with 16,822 encounters and 5,284 patient-years of observation. A majority of the patients (⬃90%) were male, with a mean age of ⬃63 years. Most of the patients (70%) were anti-CCP⫹/RF⫹, 14% were anti-CCP⫺/RF⫺, 9% were anti-CCP⫺/RF⫹, and 6.9% were anti-CCP⫹/RF⫺. There were significant differences across the autoantibody groups for age at diagnosis, disease duration, methotrexate treatment, and presence of nodules. Compared with the other patients, those who were antiCCP⫺/RF⫺ were older at the time of diagnosis, had a shorter disease duration, were less likely to be receiving methotrexate, and had a lower prevalence of nodules. Ever smoking was more common among antiCCP⫹/RF⫹ patients (83%) compared with patients in the other subgroups (73–74%; P ⬍ 0.001). HLA–DRB1 SE positivity was higher in groups characterized by anti-CCP positivity (P ⬍ 0.001), irrespective of RF status. HLA–DR3 positivity was less common among patients positive for anti-CCP antibodies (P ⬍ 0.001 across groups). The average anti–CCP-2 titer was nearly identical in the CCP⫹/RF⫺ and CCP⫹/RF⫹ groups (275.85 AU versus 273.64 AU), and similarly, the average RF titer in the CCP⫺/RF⫹ group was nearly identical to that in the CCP⫹/RF⫹ group (329.03 IU versus 328.12 IU). Relationship between the concurrent presence of ACPAs and RF and increased RA disease activity. The double-positive group exhibited significantly higher

levels of RA disease activity, with a mean ⫾ SD baseline DAS28 of 4.2 ⫾ 1.6 compared with 3.7 ⫾ 1.6 in the double-negative group, 3.4 ⫾ 1.6 in the anti-CCP⫹/RF⫺ group, and 3.9 ⫾ 1.7 in the anti-CCP⫺/RF⫹ group (overall P ⬍ 0.001, by ANOVA) (Table 2). After adjusting for multiple comparisons across the 4 groups, the ESR, and hsCRP and DAS28 values at the time of enrollment were higher in the double-positive group compared with the other groups. Post-test comparisons between each pair of groups demonstrated significantly higher ESRs and CRP levels among the double-positive group compared with the double-negative, anti-CCP⫹/ RF⫺, and anti-CCP⫺/RF⫹ groups, and the DAS28 was significantly higher in the double-positive group compared with the double-negative and anti-CCP⫹/RF⫺ subgroups, with a nonsignificant trend compared with the anti-CCP⫺/RF⫹ subgroup. Similarly, in multivariable models, concurrent anti-CCP–positive and RF-positive autoantibody status was associated with longitudinal clinical and laboratory measures of disease activity, including the swollen joint count, ESR, and DAS28 when compared with the double-negative or anti-CCP⫹/RF⫺ subgroups, while only a similar trend was noted when compared with the anti-CCP⫺/RF⫹ subgroup (Table 3). Concurrent presence of ACPAs and RF is predictive of RA-associated inflammation. Compared with the double-negative group, the double-positive group exhibited significantly higher ESRs and hsCRP levels as well as higher levels of multiple circulating inflammatory cytokines, including TNF␣, interleukin-1␤ (IL-1␤), IL-6,

Table 2. Measures of disease activity in patients with rheumatoid arthritis at the time of study enrollment, according to autoantibody status*

Measure

Concordant seronegative (n ⫽ 206)

Anti-CCP⫹/RF⫺ (n ⫽ 102)

Anti-CCP⫺/RF⫹ (n ⫽ 134)

Concordant seropositive (n ⫽ 1,046)

Pain score (range 0–10) MD-HAQ (range 0–3) Tender 28 joint count Swollen 28 joint count Patient’s global assessment (range 0–100) Provider’s global assessment (range 0–10) Log-transformed ESR, mm/hour† CRP, mg/liter‡ DAS28§

5.1 ⫾ 2.8 0.94 ⫾ 0.63 6.0 ⫾ 7.5 4.1 ⫾ 5.4 45.1 ⫾ 27.0 39.9 ⫾ 24.5 19.4 ⫾ 19.6 0.92 ⫾ 1.78 3.7 ⫾ 1.6

4.3 ⫾ 2.7 0.81 ⫾ 0.62 4.5 ⫾ 7.0 4.0 ⫾ 5.7 35.8 ⫾ 23.2 29.1 ⫾ 19.8 21.2 ⫾ 21.9 0.92 ⫾ 0.74 3.4 ⫾ 1.6

4.8 ⫾ 2.7 0.96 ⫾ 0.58 5.5 ⫾ 6.7 5.3 ⫾ 6.6 43.4 ⫾ 26.1 32.0 ⫾ 25.2 24.8 ⫾ 24.7 1.20 ⫾ 1.78 3.9 ⫾ 1.7

4.8 ⫾ 2.8 0.98 ⫾ 0.60 6.3 ⫾ 7.3 5.8 ⫾ 6.4 43.8 ⫾ 25.8 37.8 ⫾ 23.3 30.2 ⫾ 24.0 1.36 ⫾ 2.13 4.2 ⫾ 1.6

* Joint counts were dichotomized as 0 versus ⱖ1. Statistical significance, as determined by analysis of variance, was defined as P ⬍ 0.00625. Values are the mean ⫾ SD. Anti-CCP ⫽ anti–cyclic citrullinated peptide; RF ⫽ rheumatoid factor; MD-HAQ ⫽ Multidimensional Health Assessment Questionnaire; ESR ⫽ erythrocyte sedimentation rate; CRP ⫽ C-reactive protein; DAS28 ⫽ Disease Activity Score in 28 joints. † P ⬍ 0.001, overall and concordant seropositive versus concordant seronegative; P ⫽ 0.007, concordant seropositive versus anti-CCP⫺/RF⫹; P ⫽ 0.001, concordant seropositive versus anti-CCP⫹/RF⫺. ‡ P ⬍ 0.001, overall, concordant seropositive versus concordant seronegative and versus anti-CCP⫹/RF⫺; P ⫽ 0.01, concordant seropositive versus anti-CCP⫺/RF⫹. § P ⬍ 0.001 overall; P ⫽ 0.001, concordant seropositive versus concordant seronegative and versus anti-CCP⫹/RF⫺.

INTERACTION BETWEEN RF AND ACPAs IN RA

817

Table 3. Multivariable associations of autoantibody status with measures of disease activity in patients with RA during followup* Discordant Anti-CCP⫹/RF⫺ Variable

Anti-CCP⫺/RF⫹

Concordant, anti-CCP⫹/RF⫹

␤ coefficient

P

␤ coefficient

P

␤ coefficient

P

⫺0.489 ⫺0.161 ⫺0.251 0.100 ⫺6.088 ⫺3.804 0.444 ⫺0.003

0.094 0.034 0.181 0.562 0.015 0.069 ⬍0.001 0.987

⫺0.008 ⫺0.000 0.326 0.619 ⫺0.212 2.597 0.410 0.387

0.972 0.996 0.042 ⬍0.001 0.925 0.191 ⬍0.001 0.005

⫺0.288 ⫺0.015 0.052 0.507 ⫺1.588 2.418 0.652 0.418

0.097 0.725 0.624 ⬍0.001 0.315 0.069 ⬍0.001 ⬍0.001

Pain score (range 0⫺10) MD-HAQ (range 0⫺3) Tender joint count (0 vs. ⬎0) Swollen joint count (0 vs. ⬎0)† Patient’s global assessment (range 0⫺100 mm) Provider’s global assessment (range 0⫺100 mm) Log-transformed ESR, mm/hour‡ DAS28§

* The models were adjusted for age, sex, disease duration, comorbidity score, race/ethnicity, and treatment, including methotrexate, prednisone, and biologic agents. The concordant seronegative group served as the referent population. RA ⫽ rheumatoid arthritis; anti-CCP ⫽ anti–cyclic citrullinated peptide; RF ⫽ rheumatoid factor; MD-HAQ ⫽ Multidimensional Health Assessment Questionnaire; ESR ⫽ erythrocyte sedimentation rate; DAS28 ⫽ Disease Activity Score in 28 joints. † P ⫽ 0.426, anti-CCP⫹/RF⫹ versus anti-CCP⫺/RF⫹; P ⫽ 0.008, anti-CCP⫹/RF⫹ versus anti-CCP⫹/RF⫺. ‡ P ⫽ 0.005, concordant seropositive versus anti-CCP⫺/RF⫹; P ⫽ 0.036, concordant seropositive versus anti-CCP⫹/RF⫺. § P ⫽ 0.786, concordant seropositive versus anti-CCP⫺/RF⫹; P ⫽ 0.002, concordant seropositive versus anti-CCP⫹/RF⫺.

***

80 60 40 20

A.A.

D

***

150

100

C

C P+ /R F+

C P/R F+

/R F-

C

C P+

ns

80

*

100

50

F

ns

60

M-CSF

*

150

IL-17A

40

50 20

50

/R F+ C P+ C

C P/R F+ C

/R FC P+ C

C P/R F-

C P+ /R F+ C

C P/R F+ C

C P+ /R FC

C P/R F-

0

C

/R F+ C P+ C

C P/R F+ C

C P+

/R F-

0

C

C

C P/R F-

0

C

IL-12p70

200

*

100

0

E ***

250

150

C P/R F-

C P/R F+ C

C

C P/R FC

C P+ /R F+ C

C

C P/R F+

/R FC P+ C

C P/R FC

C P+ /R F-

0

0

***

200

C

100

C

100

IL-6 (pg/ml)

IL-1β (pg/ml)

TNF-α (pg/ml)

ns

200

B

C P+ /R F+

***

300

C

A

17 potentially independent cytokines would require a P value of 0.0029 for significance, and this was achieved for all of the cytokines shown in Figure 1. Additionally, all cytokine levels were compared between the double-negative group, the anti-CCP⫺/ RF⫹ group, and a set of double-positive patients

C

IL-12p70, and IL-17A (Figure 1) (all P ⬍ 0.001 by ANOVA). Although it is likely that many cytokines, including those most up-regulated in the double-positive group, are highly related and thus not subject to a high risk of Type I error in multiplex cytokine analysis, stringent implementation of Bonferroni’s correction for

Figure 1. Higher serum levels of rheumatoid arthritis (RA)–associated cytokines in RA patients who were double-positive for anti–cyclic citrullinated peptide (anti-CCP) and rheumatoid factor (RF). Patients were categorized as being double-negative (anti-CCP⫺/RF⫺; n ⫽ 204), anti-CCP⫹/RF⫺ (n ⫽ 96), anti-CCP⫺/RF⫹ (n ⫽ 135), or double-positive (anti-CCP⫹/RF⫹; n ⫽ 1,031). Between-group comparisons were performed using the Kruskal-Wallis test with Tukey’s multiple comparison post hoc test. ⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 versus anti-CCP⫺/RF⫺. TNF␣ ⫽ tumor necrosis factor ␣; NS ⫽ not significant; IL-1␤ ⫽ interleukin-1␤; M-CSF ⫽ macrophage colony-stimulating factor.

matched for age, sex, and disease duration. Three-way group comparisons were performed using SAM version 3.08 (23), and output was sorted based on FDRs. Notably, the use of FDRs obviates the need to adjust for multiple comparisons. Supplementary Figure 1 (available on the Arthritis & Rheumatology web site at http:// onlinelibrary.wiley.com/doi/10.1002/art.38307/abstract) demonstrates significant up-regulation of 10 of 17 cytokines in the double-positive group compared with the double-negative group and both the anti-CCP⫹/RF⫺ and anti-CCP⫺/RF⫹ groups. A smaller but, in many cases, statistically significant elevation of cytokine levels (as well as some clinical measures of disease activity) was noted in the antiCCP⫺/RF⫹ group compared with the double-negative and anti-CCP⫹/RF⫺ groups. We hypothesized that such elevations in the anti-CCP⫺/RF⫹ group may reflect the presence in some patients of physiologic levels of ACPAs not detected by or below the range of the commercial anti–CCP-2 ELISA. To support this hypothesis, we used a multiplex antigen array to assess ACPA subspecificities that might be present in the anti-CCP⫺/ RF⫹ group. Figure 2 shows that the levels of 11 of 30 ACPAs were elevated in the anti-CCP⫺/RF⫹ group compared with the double-negative group. Thus, we hypothesized that low levels of ACPAs that are not DV093_CCP_1_RF_1

DA092_CCP_1_RF_1

DC097_CCP_1_RF_1

SL299_CCP_1_RF_1

DA195_CCP_1_RF_1

DC090_CCP_1_RF_1

SL293_CCP_1_RF_1

SL196_CCP_1_RF_1

OM090_CCP_1_RF_1

SL095_CCP_1_RF_1

SL194_CCP_1_RF_1

SL297_CCP_1_RF_1

DA295_CCP_1_RF_1

DA298_CCP_1_RF_1

SL291_CCP_1_RF_1

OM099_CCP_1_RF_1

MS080_CCP_1_RF_1

SL283_CCP_1_RF_1

DC080_CCP_1_RF_1

DA383_CCP_1_RF_1

DA482_CCP_1_RF_1

DA185_CCP_1_RF_1

MS089_CCP_1_RF_1

SL182_CCP_1_RF_1

MS082_CCP_1_RF_1

SL086_CCP_1_RF_1

SL183_CCP_1_RF_1

DA485_CCP_1_RF_1

OM085_CCP_1_RF_1

OM184_CCP_1_RF_1

DC085_CCP_1_RF_1

DA487_CCP_1_RF_1

DA585_CCP_1_RF_1

SL188_CCP_1_RF_1

DA087_CCP_1_RF_1

DA287_CCP_1_RF_1

DV080_CCP_1_RF_1

SL286_CCP_1_RF_1

SL280_CCP_1_RF_1

DC088_CCP_1_RF_1

DA580_CCP_1_RF_1

DA074_CCP_1_RF_1

DA275_CCP_1_RF_1

SL278_CCP_1_RF_1

SL175_CCP_1_RF_1

DA171_CCP_1_RF_1

DA176_CCP_1_RF_1

DA374_CCP_1_RF_1

DA073_CCP_1_RF_1

SL272_CCP_1_RF_1

OM072_CCP_1_RF_1

OM177_CCP_1_RF_1

SL276_CCP_1_RF_1

OM173_CCP_1_RF_1

DA476_CCP_1_RF_1

DA376_CCP_1_RF_1

OM077_CCP_1_RF_1

DA078_CCP_1_RF_1

DV073_CCP_1_RF_1

DA070_CCP_1_RF_1

OM172_CCP_1_RF_1

DA468_CCP_1_RF_1

SL162_CCP_1_RF_1

DA469_CCP_1_RF_1

DA360_CCP_1_RF_1

SL063_CCP_1_RF_1

DV069_CCP_1_RF_1

DA567_CCP_1_RF_1

DA569_CCP_1_RF_1

DA160_CCP_1_RF_1

DA568_CCP_1_RF_1

DA068_CCP_1_RF_1

DV063_CCP_1_RF_1

DA563_CCP_1_RF_1

DA161_CCP_1_RF_1

DC062_CCP_1_RF_1

DC150_CCP_1_RF_1

DA452_CCP_1_RF_1

DA555_CCP_1_RF_1

DA355_CCP_1_RF_1

DA558_CCP_1_RF_1

DA357_CCP_1_RF_1

OM157_CCP_1_RF_1

DA556_CCP_1_RF_1

DA450_CCP_1_RF_1

DA258_CCP_1_RF_1

DC057_CCP_1_RF_1

DA554_CCP_1_RF_1

DA254_CCP_1_RF_1

DA256_CCP_1_RF_1

DA053_CCP_1_RF_1

OM053_CCP_1_RF_1

DA154_CCP_1_RF_1

DA052_CCP_1_RF_1

DA352_CCP_1_RF_1

DC157_CCP_1_RF_1

SL154_CCP_1_RF_1

DA456_CCP_1_RF_1

SL148_CCP_1_RF_1

DA549_CCP_1_RF_1

MS043_CCP_1_RF_1

OM043_CCP_1_RF_1

DC144_CCP_1_RF_1

OM140_CCP_1_RF_1

DC043_CCP_1_RF_1

DA143_CCP_1_RF_1

DA343_CCP_1_RF_1

DC046_CCP_1_RF_1

DA041_CCP_1_RF_1

DA344_CCP_1_RF_1

DA149_CCP_1_RF_1

SL242_CCP_1_RF_1

DA340_CCP_1_RF_1

DC147_CCP_1_RF_1

MS045_CCP_1_RF_1

SL046_CCP_1_RF_1

DC148_CCP_1_RF_1

DC042_CCP_1_RF_1

DA541_CCP_1_RF_1

DA140_CCP_1_RF_1

DA139_CCP_1_RF_1

DA031_CCP_1_RF_1

SL131_CCP_1_RF_1

DA034_CCP_1_RF_1

OR030_CCP_1_RF_1

SL130_CCP_1_RF_1

DA331_CCP_1_RF_1

SL030_CCP_1_RF_1

OR034_CCP_1_RF_1

DV130_CCP_1_RF_1

SL231_CCP_1_RF_1

CCP-, RF+ DA333_CCP_1_RF_1

DA039_CCP_1_RF_1

DC038_CCP_1_RF_1

MS034_CCP_1_RF_1

DA520_CCP_1_RF_1

SL129_CCP_1_RF_1

OR026_CCP_1_RF_1

SL128_CCP_1_RF_1

SL120_CCP_1_RF_1

MS124_CCP_1_RF_1

DC122_CCP_1_RF_1

DA026_CCP_1_RF_1

MS027_CCP_1_RF_1

DA329_CCP_1_RF_1

SL025_CCP_1_RF_1

DA322_CCP_1_RF_1

DA021_CCP_1_RF_1

DV026_CCP_1_RF_1

DA023_CCP_1_RF_1

SL126_CCP_1_RF_1

DA025_CCP_1_RF_1

DA127_CCP_1_RF_1

MS026_CCP_1_RF_1

DV027_CCP_1_RF_1

SL213_CCP_1_RF_1

OM112_CCP_1_RF_1

OM118_CCP_1_RF_1

OM019_CCP_1_RF_1

DA119_CCP_1_RF_1

MS013_CCP_1_RF_1

OR017_CCP_1_RF_1

IA016_CCP_1_RF_1

SL211_CCP_1_RF_1

DA112_CCP_1_RF_1

MS112_CCP_1_RF_1

DA513_CCP_1_RF_1

DV018_CCP_1_RF_1

OM116_CCP_1_RF_1

SL114_CCP_1_RF_1

SL115_CCP_1_RF_1

DA414_CCP_1_RF_1

OM017_CCP_1_RF_1

OR012_CCP_1_RF_1

OM111_CCP_1_RF_1

DA519_CCP_1_RF_1

DA316_CCP_1_RF_1

DC119_CCP_1_RF_1

SL206_CCP_1_RF_1

DA207_CCP_1_RF_1

IA009_CCP_1_RF_1

OM105_CCP_1_RF_1

SL105_CCP_1_RF_1

BY002_CCP_1_RF_1

MS100_CCP_1_RF_1

SL301_CCP_1_RF_1

DA302_CCP_1_RF_1

OM009_CCP_1_RF_1

DC001_CCP_1_RF_1

MS103_CCP_1_RF_1

DV008_CCP_1_RF_1

DA008_CCP_1_RF_1

DV107_CCP_1_RF_1

BY006_CCP_1_RF_1

DA402_CCP_1_RF_1

DV104_CCP_1_RF_1

DA201_CCP_1_RF_1

DA403_CCP_1_RF_1

DV100_CCP_1_RF_1

OM003_CCP_1_RF_1

OM101_CCP_1_RF_1

DC005_CCP_1_RF_1

DA206_CCP_1_RF_1

SL193_CCP_0_RF_1

SL093_CCP_0_RF_1

OM095_CCP_0_RF_1

MS095_CCP_0_RF_1

DV096_CCP_0_RF_1

DV095_CCP_0_RF_1

DC096_CCP_0_RF_1

DA296_CCP_0_RF_1

DA294_CCP_0_RF_1

DA290_CCP_0_RF_1

DA194_CCP_0_RF_1

DA190_CCP_0_RF_1

DA094_CCP_0_RF_1

SL285_CCP_0_RF_1

SL185_CCP_0_RF_1

SL088_CCP_0_RF_1

SL085_CCP_0_RF_1

SL080_CCP_0_RF_1

DA587_CCP_0_RF_1

DA382_CCP_0_RF_1

DA380_CCP_0_RF_1

DA285_CCP_0_RF_1

DA187_CCP_0_RF_1

SL172_CCP_0_RF_1

SL171_CCP_0_RF_1

SL071_CCP_0_RF_1

SL070_CCP_0_RF_1

OM079_CCP_0_RF_1

OM070_CCP_0_RF_1

MS076_CCP_0_RF_1

DV078_CCP_0_RF_1

DA477_CCP_0_RF_1

DA379_CCP_0_RF_1

DA172_CCP_0_RF_1

SL176_CCP_0_RF_1

SL261_CCP_0_RF_1

SL160_CCP_0_RF_1

OM069_CCP_0_RF_1

OM068_CCP_0_RF_1

DV065_CCP_0_RF_1

DC162_CCP_0_RF_1

DC066_CCP_0_RF_1

DA464_CCP_0_RF_1

DA362_CCP_0_RF_1

SL167_CCP_0_RF_1

SL250_CCP_0_RF_1

SL158_CCP_0_RF_1

MS057_CCP_0_RF_1

MS055_CCP_0_RF_1

DC159_CCP_0_RF_1

DA454_CCP_0_RF_1

DA358_CCP_0_RF_1

DA250_CCP_0_RF_1

DA155_CCP_0_RF_1

DA051_CCP_0_RF_1

OM148_CCP_0_RF_1

OM144_CCP_0_RF_1

OM142_CCP_0_RF_1

OM048_CCP_0_RF_1

OM045_CCP_0_RF_1

MS049_CCP_0_RF_1

MS047_CCP_0_RF_1

DV042_CCP_0_RF_1

DC049_CCP_0_RF_1

DA542_CCP_0_RF_1

DA446_CCP_0_RF_1

DA341_CCP_0_RF_1

DA247_CCP_0_RF_1

SL238_CCP_0_RF_1

SL232_CCP_0_RF_1

SL230_CCP_0_RF_1

OM132_CCP_0_RF_1

OM035_CCP_0_RF_1

OM031_CCP_0_RF_1

MS038_CCP_0_RF_1

MS035_CCP_0_RF_1

DV133_CCP_0_RF_1

DV035_CCP_0_RF_1

DC034_CCP_0_RF_1

DC032_CCP_0_RF_1

DA239_CCP_0_RF_1

DA238_CCP_0_RF_1

DA134_CCP_0_RF_1

DA035_CCP_0_RF_1

DA137_CCP_0_RF_1

SL021_CCP_0_RF_1

OM127_CCP_0_RF_1

OM126_CCP_0_RF_1

OM120_CCP_0_RF_1

DV129_CCP_0_RF_1

DV128_CCP_0_RF_1

DV123_CCP_0_RF_1

DV022_CCP_0_RF_1

DC029_CCP_0_RF_1

DC021_CCP_0_RF_1

CCP-, RFDA525_CCP_0_RF_1

DA522_CCP_0_RF_1

DA323_CCP_0_RF_1

DA320_CCP_0_RF_1

DA224_CCP_0_RF_1

DA128_CCP_0_RF_1

OM113_CCP_0_RF_1

OM012_CCP_0_RF_1

MS019_CCP_0_RF_1

MS017_CCP_0_RF_1

MS015_CCP_0_RF_1

IA019_CCP_0_RF_1

IA018_CCP_0_RF_1

DV016_CCP_0_RF_1

DC018_CCP_0_RF_1

DC013_CCP_0_RF_1

DA410_CCP_0_RF_1

DA216_CCP_0_RF_1

DA110_CCP_0_RF_1

BY010_CCP_0_RF_1

SL306_CCP_0_RF_1

SL304_CCP_0_RF_1

SL104_CCP_0_RF_1

SL006_CCP_0_RF_1

OM104_CCP_0_RF_1

OM102_CCP_0_RF_1

OM007_CCP_0_RF_1

MS001_CCP_0_RF_1

DV103_CCP_0_RF_1

DA508_CCP_0_RF_1

DA502_CCP_0_RF_1

DA309_CCP_0_RF_1

DA307_CCP_0_RF_1

DA205_CCP_0_RF_1

DA204_CCP_0_RF_1

DA203_CCP_0_RF_1

DA106_CCP_0_RF_1

DA103_CCP_0_RF_1

DA101_CCP_0_RF_1

BY008_CCP_0_RF_1

SL195_CCP_0_RF_0

DA196_CCP_0_RF_0

DC092_CCP_0_RF_0

SL090_CCP_0_RF_0

DA395_CCP_0_RF_0

DV099_CCP_0_RF_0

DA392_CCP_0_RF_0

SL295_CCP_0_RF_0

SL099_CCP_0_RF_0

DA595_CCP_0_RF_0

SL098_CCP_0_RF_0

DA397_CCP_0_RF_0

DA491_CCP_0_RF_0

DA599_CCP_0_RF_0

DC095_CCP_0_RF_0

DA393_CCP_0_RF_0

SL281_CCP_0_RF_0

SL282_CCP_0_RF_0

OM183_CCP_0_RF_0

OM084_CCP_0_RF_0

DA286_CCP_0_RF_0

SL082_CCP_0_RF_0

DA081_CCP_0_RF_0

DA581_CCP_0_RF_0

SL184_CCP_0_RF_0

DV083_CCP_0_RF_0

DA480_CCP_0_RF_0

DA489_CCP_0_RF_0

DA085_CCP_0_RF_0

OM087_CCP_0_RF_0

DC086_CCP_0_RF_0

OM170_CCP_0_RF_0

DA371_CCP_0_RF_0

OM174_CCP_0_RF_0

OM178_CCP_0_RF_0

DA473_CCP_0_RF_0

DA470_CCP_0_RF_0

SL079_CCP_0_RF_0

DA573_CCP_0_RF_0

MS071_CCP_0_RF_0

SL274_CCP_0_RF_0

DC070_CCP_0_RF_0

DA270_CCP_0_RF_0

DA472_CCP_0_RF_0

DA571_CCP_0_RF_0

DV076_CCP_0_RF_0

DV070_CCP_0_RF_0

DA178_CCP_0_RF_0

DA378_CCP_0_RF_0

DC074_CCP_0_RF_0

DC076_CCP_0_RF_0

OM179_CCP_0_RF_0

DA478_CCP_0_RF_0

DA475_CCP_0_RF_0

DC078_CCP_0_RF_0

DA479_CCP_0_RF_0

DC060_CCP_0_RF_0

DA262_CCP_0_RF_0

DA369_CCP_0_RF_0

SL061_CCP_0_RF_0

OM161_CCP_0_RF_0

SL168_CCP_0_RF_0

SL066_CCP_0_RF_0

SL267_CCP_0_RF_0

DA269_CCP_0_RF_0

SL260_CCP_0_RF_0

DA562_CCP_0_RF_0

DA368_CCP_0_RF_0

OM063_CCP_0_RF_0

MS060_CCP_0_RF_0

DC160_CCP_0_RF_0

OM162_CCP_0_RF_0

DA367_CCP_0_RF_0

OM163_CCP_0_RF_0

SL269_CCP_0_RF_0

SL252_CCP_0_RF_0

DV059_CCP_0_RF_0

DA251_CCP_0_RF_0

MS059_CCP_0_RF_0

SL052_CCP_0_RF_0

SL153_CCP_0_RF_0

MS056_CCP_0_RF_0

DA259_CCP_0_RF_0

OM055_CCP_0_RF_0

MS052_CCP_0_RF_0

SL254_CCP_0_RF_0

DA257_CCP_0_RF_0

DA353_CCP_0_RF_0

DA158_CCP_0_RF_0

OM150_CCP_0_RF_0

SL140_CCP_0_RF_0

DA545_CCP_0_RF_0

SL149_CCP_0_RF_0

OR042_CCP_0_RF_0

OM047_CCP_0_RF_0

SL145_CCP_0_RF_0

DA246_CCP_0_RF_0

OM042_CCP_0_RF_0

DA440_CCP_0_RF_0

SL042_CCP_0_RF_0

OM146_CCP_0_RF_0

SL146_CCP_0_RF_0

DA543_CCP_0_RF_0

SL141_CCP_0_RF_0

DC140_CCP_0_RF_0

MS048_CCP_0_RF_0

SL045_CCP_0_RF_0

DA540_CCP_0_RF_0

SL248_CCP_0_RF_0

DC035_CCP_0_RF_0

DA530_CCP_0_RF_0

DC039_CCP_0_RF_0

SL034_CCP_0_RF_0

DA132_CCP_0_RF_0

OR032_CCP_0_RF_0

OM139_CCP_0_RF_0

DA433_CCP_0_RF_0

DV135_CCP_0_RF_0

DA133_CCP_0_RF_0

OM137_CCP_0_RF_0

DV139_CCP_0_RF_0

SL135_CCP_0_RF_0

DA338_CCP_0_RF_0

MS033_CCP_0_RF_0

DV039_CCP_0_RF_0

SL237_CCP_0_RF_0

DC134_CCP_0_RF_0

DA237_CCP_0_RF_0

MS039_CCP_0_RF_0

DA334_CCP_0_RF_0

DA234_CCP_0_RF_0

DC131_CCP_0_RF_0

DC130_CCP_0_RF_0

DA339_CCP_0_RF_0

OR029_CCP_0_RF_0

DC127_CCP_0_RF_0

MS024_CCP_0_RF_0

DC024_CCP_0_RF_0

DA129_CCP_0_RF_0

SL124_CCP_0_RF_0

DA227_CCP_0_RF_0

IA022_CCP_0_RF_0

DA029_CCP_0_RF_0

DC027_CCP_0_RF_0

DA325_CCP_0_RF_0

DA428_CCP_0_RF_0

DA121_CCP_0_RF_0

MS122_CCP_0_RF_0

DA327_CCP_0_RF_0

DV023_CCP_0_RF_0

DA124_CCP_0_RF_0

DA228_CCP_0_RF_0

DA429_CCP_0_RF_0

SL024_CCP_0_RF_0

MS021_CCP_0_RF_0

OM128_CCP_0_RF_0

DA120_CCP_0_RF_0

DA526_CCP_0_RF_0

DC023_CCP_0_RF_0

DC125_CCP_0_RF_0

MS014_CCP_0_RF_0

DA514_CCP_0_RF_0

DC117_CCP_0_RF_0

DC015_CCP_0_RF_0

DA416_CCP_0_RF_0

DA212_CCP_0_RF_0

DV114_CCP_0_RF_0

DV112_CCP_0_RF_0

SL117_CCP_0_RF_0

OM114_CCP_0_RF_0

DV113_CCP_0_RF_0

DC014_CCP_0_RF_0

DV111_CCP_0_RF_0

DA512_CCP_0_RF_0

SL110_CCP_0_RF_0

SL116_CCP_0_RF_0

BY015_CCP_0_RF_0

OM115_CCP_0_RF_0

OM119_CCP_0_RF_0

MS114_CCP_0_RF_0

IA012_CCP_0_RF_0

IA011_CCP_0_RF_0

DV110_CCP_0_RF_0

IA014_CCP_0_RF_0

DC007_CCP_0_RF_0

MS003_CCP_0_RF_0

SL109_CCP_0_RF_0

SL308_CCP_0_RF_0

DV001_CCP_0_RF_0

DA007_CCP_0_RF_0

SL004_CCP_0_RF_0

DV108_CCP_0_RF_0

DV004_CCP_0_RF_0

DA500_CCP_0_RF_0

OR004_CCP_0_RF_0

SL205_CCP_0_RF_0

DA303_CCP_0_RF_0

OM006_CCP_0_RF_0

BY005_CCP_0_RF_0

DA505_CCP_0_RF_0

IA002_CCP_0_RF_0

OR003_CCP_0_RF_0

MS009_CCP_0_RF_0

818 SOKOLOVE ET AL

CCP+, RF+ q < 0.1% 4x 2x Average 1/2 x 1/4 x

H2B/A 62-81 Cit Cyclic

Vimentin Cit

H2A/a-2 1-20 Cit

H2A/a 1-20 Cit sm2 Cyclic Enolase 1A 5-21 Cit Clusterin 221-240 Cit Cyclic Vim 58-77 Cit3 Cyclic sm1

Clusterin 231-250 Cit sm1 cyclic

ApoE 277-296 Cit2 sm2 cyclic

Fil 48-65 Cit Cyclic Fil 48-65 Cit2 V1 Cyclic H2B Cit

H2A Cit CCP

Biglycan 247-266 Cit sm1 cyclic FibA 556-575 Cit smCyclic

FibA 616-635 Cit3 smCyclic

Figure 2. Heatmap showing elevated levels of anti–citrullinated protein antibodies in the anti-CCP⫺/RF⫹ group compared with the doublenegative group. Levels of autoantibodies against 37 putative targets of the RA immune response were compared between the anti-CCP⫹/RF⫺ group (n ⫽ 96) and the anti-CCP⫺/RF⫹ group (n ⫽ 135) using a multiplex antigen microarray. SAM software was used to sort output based on false discovery rates in order to identify antigens with the greatest differences in autoantibody reactivity between serologic subgroups. Labels on the color key are the fluorescence intensity relative to the average values in the evaluated cohort. Cit ⫽ citrullinated; Vim ⫽ vimentin; ApoE ⫽ apolipoprotein E; Fil ⫽ filaggrin; FibA ⫽ fibrinogen A (see Figure 1 for other definitions).

strongly represented by the anti–CCP-2 assay may synergize with RF to induce RA-associated inflammation. Augmentation of the stimulatory capacity of ACPA ICs by monoclonal RF. We previously demonstrated the ability of ACPA ICs to stimulate macrophage cytokine production via costimulation of Fc␥ receptor and the innate immune receptor Toll-like receptor 4 (12). To evaluate the effect of RF on ACPAs, we preincubated ACPA-containing RA IgG with monoclonal IgM-RF before formation of ACPA ICs. As previously described, in vitro stimulation of monocyte-derived macrophages by ACPA ICs demonstrated increased cytokine production (P ⫽ 0.002 versus citrullinated fibrinogen only), and the addition of monoclonal IgM-RF resulted in a further significant increase in macrophage TNF␣ production as compared with ACPA ICs alone (P ⫽ 0.003) (Figure 3A). Notably, we additionally tested 2 monoclonal IgM antibodies with in vitro RF activity, which were isolated from patients with mixed cryoglobulinemia. However, the addition of these IgM antibodies at 20 ␮g/ml, 100 ␮g/ml, and 200 ␮g/ml failed to augment macrophage TNF secretion in response to ACPA ICs (data not shown). Given the potential inclusion of IgG-RF (or IgM-RF) in our ACPA-positive IgG preparation, we further investigated the ability of monoclonal IgM-RF to

INTERACTION BETWEEN RF AND ACPAs IN RA

A TNF-α (pg/ml)

800

819

IgM-RF to augment anti–citrullinated fibrinogen IC– induced macrophage activation, as evidenced by increased cytokine production (Figure 3B).

P=0.003

600 P=0.002

DISCUSSION

400 200

1: 10 0

1: 10 0 cF b

+

R F

F

cF bIC

cF b

cF bIC +R

co at ed

0

B P=0.005

800 600 P=0.03

400 200

cF cF bbIC IC +R F 1: cF 10 b0 IC +R F 1: cF 20 b+ R cF F b+ 1: R 10 F 0 1: 20 al on e

cF b

co at

ed

0

Figure 3. IgM-RF augments macrophage activation by anti– citrullinated protein antibody (ACPA) immune complexes (ICs) in vitro. Monocyte-derived macrophages were added to plates precoated with human citrullinated fibrinogen (cFb) ICs (A) or with citrullinated fibrinogen ICs formed using polyclonal rabbit IgG antibodies against fibrinogen (B), in the presence or absence of monoclonal IgM-RF. Macrophage stimulation was assessed by measuring the level of TNF␣ in harvested cell culture supernatants. Results are representative of experiments performed at least twice. Bars show the mean ⫾ SEM of triplicate cultures. See Figure 1 for other definitions.

enhance the stimulatory activity of anti–citrullinated fibrinogen ICs, this time formed with a polyclonal rabbit antibody against fibrinogen previously demonstrated to bind native and citrullinated fibrinogen (12) and which could be targeted by our monoclonal IgM-RF by ELISA and Western blotting (data not shown). In results analogous to those of previous studies, we observed the ability of anti–citrullinated fibrinogen ICs to stimulate macrophage cytokine production and, as with human RA–derived IgG preparations, the ability of monoclonal

Although several previous studies have demonstrated increased disease activity in the presence of either anti-CCP or RF, to our knowledge, this study is the first to identify a synergistic role for ACPAs and RF in mediating RA-associated inflammation and disease activity. We demonstrated that baseline autoantibody status was associated with select measures of disease activity, both at presentation and over time. Specifically, joint swelling and higher baseline DAS28 values were more commonly observed in patients with RA who were positive for both anti-CCP and RF and, to a lesser extent in those positive for RF regardless of anti-CCP status. Interestingly, ESRs were similarly increased in all autoantibody-positive groups (both double-positive and single-positive) compared with seronegative patients, even after accounting for the numerically higher frequency of RA-related treatments used in seropositive cases; however, values were further increased in the double-positive group compared with each singlepositive group. The observed increases in disease activity and the levels of clinical markers of inflammation were paralleled by a nearly identical pattern of elevation among several inflammatory cytokines previously associated with RA pathophysiology (26), including those that we and other investigators have previously demonstrated to be produced in response to macrophage stimulation by ACPA ICs (12,13). The increased inflammation and disease activity observed in the double-positive group is supportive of a potential role for these RA-associated autoantibodies in mediating the pathogenesis of RA. However, because association does not prove causality, we used an in vitro model of ACPA IC–mediated inflammation to identify a novel mechanism by which the interaction of ACPAs and RF may contribute to the pathophysiology of RA inflammation and disease activity. We thus demonstrate a unifying mechanism by which the 2 overtly distinct autoantibody types that characterize RA can interact to promote RA disease pathogenesis. RF was first identified by the ability of RA serum to agglutinate IgG-coated sheep red blood cells, was subsequently defined as an immunoglobulin targeting the Fc portion of IgG (3), and is now considered to be characteristic of the presence of RA and has been associated with increased disease severity (9,11). How-

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ever, the pathogenic role of RF has been questioned and, to date, poorly defined. In 1961, Ragan stated that “the significance of rheumatoid factor in the pathogenesis of rheumatoid arthritis remains conjectural” (3). More than 50 years later, little progress has been made in our understanding. Previous studies have suggested the ability of RF to accelerate experimental models of IC-mediated vascular damage (27), and although there is evidence for the ability of RF to fix complement (28,29), other studies suggest that RF may in fact prevent complement activation by IgG ICs, thus leading to the speculation that the effects of RF are in fact mediated by attachment to the Fc region of the IgG molecule rather than complement activation (30). As such, this study is the first to suggest a mechanistic role of RF in RA disease propagation. Additionally, it has long been known that RF is present in the setting of non-RA immune activation, including most notably, viral and bacterial infection. Prior studies have identified the ability of RFexpressing B cells to recognize ICs (but not monomeric IgG) and to present the retained antigen to T cells (31), thus enhancing the immune response to foreign antigen. Therefore, the generation of RF may have a teleological purpose in its potential ability to stabilize protective ICs. We propose that soluble RF may have developed to provide a protective evolutionary advantage against infectious pathogens. Because the immune response associated with RA and similar autoimmune conditions is also associated with the presence of RF, we hypothesize that inflammatory disease pathogenesis has coopted the beneficial role of IC stabilization, thus enhancing the pathogenic capacity of disease-associated autoantibodies and resultant ICs. The current study has several limitations. Given the large proportion of older patients examined (who are reflective of current VA beneficiaries), caution should be used before applying these results to a broader RA patient population. Also, given reports highlighting the pathogenic role that ACPAs might play in propagating RA-related joint damage (14), further analyses examining the relationship of autoantibody status with clinical and radiographic outcomes in RA are important. Despite use of methods optimized to minimize the effects of heterophilic antibodies, our laboratory studies do not eliminate the potential of heterophilic antibodies such as RF to induce erroneously elevated signal in sandwich immunoassays (32,33). Thus, the risk remains that the observed elevation of cytokine levels in the double-positive group is in part a result of such bias. However, the relative lack of signal in the RF single-

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positive group, as well as supporting clinical and ESR data, provide evidence against a significant contribution of such confounding in this study. Finally, the complexity and heterogeneity of IgM-RF remain great. Although most monoclonal RF characterized to date target the C␥2–C␥3 interface in the Fc region on the IgG molecule (34), the variations in exact binding sites as well as the role of various posttranslational modifications (35,36) to affect RF binding, as well as the typically polyclonal nature of RF in vivo, may limit the generalizability of our IC data generated with a single monoclonal IgM-RF. In summary, our results showed that a significantly increased level of disease activity as well as increased levels of systemic inflammation markers were associated with the concordant presence of RF and ACPAs in a multivariable analysis accounting for a variety of other potentially associated clinical variables. Here, we demonstrate a mechanism by which the IgMRF–ACPA interaction may directly contribute to RA disease pathogenesis. These results not only provide useful information for predicting the severity of RA but also, by identifying a mechanistic interaction between ACPAs and IgM-RF, advance our understanding of the role of RA-associated autoantibodies in mediating the pathogenesis of RA. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Sokolove had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Sokolove, Johnson, Michaud, Reimold, Kerr, Mikuls, Robinson. Acquisition of data. Sokolove, Johnson, Lahey, Wagner, Cheng, Thiele, Reimold, Caplan, Cannon, Kerr, Mikuls, Robinson. Analysis and interpretation of data. Lahey, Wagner, Thiele, Sayles, Caplan, Cannon, Kerr, Mikuls, Robinson.

REFERENCES 1. Rose HM, Ragan C, Pearce E, Lipman MO. Differential agglutination of normal and sensitized sheep erythrocytes by sera of patients with rheumatoid arthritis. Proc Soc Exp Biol Med 1948; 68:1–6. 2. Franklin EC, Holman HR, Muller-Eberhard HJ, Kunkel HG. An unusual protein component of high molecular weight in the serum of certain patients with rheumatoid arthritis. J Exp Med 1957;105: 425–38. 3. Ragan C. The history of the rheumatoid factor. Arthritis Rheum 1961;4:571–3. 4. Nell VP, Machold KP, Stamm TA, Eberl G, Heinzl H, Uffmann M, et al. Autoantibody profiling as early diagnostic and prognostic tool for rheumatoid arthritis. Ann Rheum Dis 2005;64:1731–6. 5. Nishimura K, Sugiyama D, Kogata Y, Tsuji G, Nakazawa T,

INTERACTION BETWEEN RF AND ACPAs IN RA

6.

7.

8.

9.

10. 11. 12.

13.

14.

15.

16.

17.

18.

19.

20.

Kawano S, et al. Meta-analysis: diagnostic accuracy of anti-cyclic citrullinated peptide antibody and rheumatoid factor for rheumatoid arthritis. Ann Intern Med 2007;146:797–808. Schellekens GA, Visser H, de Jong BA, van den Hoogen FH, Hazes JM, Breedveld FC, et al. The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis Rheum 2000;43:155–63. Greiner A, Plischke H, Kellner H, Gruber R. Association of anti-cyclic citrullinated peptide antibodies, anti-citrullin antibodies, and IgM and IgA rheumatoid factors with serological parameters of disease activity in rheumatoid arthritis. Ann N Y Acad Sci 2005;1050:295–303. Silveira IG, Burlingame RW, von Muhlen CA, Bender AL, Staub HL. Anti-CCP antibodies have more diagnostic impact than rheumatoid factor (RF) in a population tested for RF. Clin Rheumatol 2007;26:1883–9. Miriovsky BJ, Michaud K, Thiele GM, O’Dell J, Cannon GW, Kerr G, et al. Anti-CCP antibody and rheumatoid factor concentrations predict greater disease burden in U.S. veterans with rheumatoid arthritis. Ann Rheum Dis 2010;69:1292–7. Lee DM, Schur PH. Clinical utility of the anti-CCP assay in patients with rheumatic diseases. Ann Rheum Dis 2003;62:870–4. Hueber W, Utz PJ, Robinson WH. Autoantibodies in early arthritis: advances in diagnosis and prognostication. Clin Exp Rheumatol 2003;21:S59–64. Sokolove J, Zhao X, Chandra PE, Robinson WH. Immune complexes containing citrullinated fibrinogen costimulate macrophages via Toll-like receptor 4 and Fc␥ receptor. Arthritis Rheum 2011;63:53–62. Clavel C, Nogueira L, Laurent L, Iobagiu C, Vincent C, Sebbag M, et al. Induction of macrophage secretion of tumor necrosis factor ␣ through Fc␥ receptor IIa engagement by rheumatoid arthritis–specific autoantibodies to citrullinated proteins complexed with fibrinogen. Arthritis Rheum 2008;58:678–88. Harre U, Georgess D, Bang H, Bozec A, Axmann R, Ossipova E, et al. Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin. J Clin Invest 2012; 122:1791–802. Kuhn KA, Kulik L, Tomooka B, Braschler KJ, Arend WP, Robinson WH, et al. Antibodies against citrullinated proteins enhance tissue injury in experimental autoimmune arthritis. J Clin Invest 2006;116:961–73. Mikuls TR, Kazi S, Cipher D, Hooker R, Kerr GS, Richards JS, et al. The association of race and ethnicity with disease expression in male US veterans with rheumatoid arthritis. J Rheumatol 2007;34:1480–4. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24. Prevoo ML, van ’t Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB, van Riel PL. Modified disease activity scores that include twenty-eight–joint counts: development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum 1995;38:44–8. Mikuls TR, Gould KA, Bynote KK, Yu F, Levan TD, Thiele GM, et al. Anticitrullinated protein antibody (ACPA) in rheumatoid arthritis: influence of an interaction between HLA-DRB1 shared epitope and a deletion polymorphism in glutathione s-transferase in a cross-sectional study. Arthritis Res Ther 2010;12:R213. Pincus T, Swearingen C, Wolfe F. Toward a multidimensional Health Assessment Questionnaire (MDHAQ): assessment of ad-

821

21.

22.

23.

24. 25. 26. 27. 28. 29.

30.

31. 32.

33.

34.

35.

36.

vanced activities of daily living and psychological status in the patient-friendly Health Assessment Questionnaire format. Arthritis Rheum 1999;42:2220–30. Mikuls TR, Padala PR, Sayles HR, Yu F, Michaud K, Caplan L, et al. Prospective study of posttraumatic stress disorder and disease activity outcomes in US veterans with rheumatoid arthritis. Arthritis Care Res (Hoboken) 2013;65:227–34. Sokolove J, Bromberg R, Deane KD, Lahey LJ, Derber LA, Chandra PE, et al. Autoantibody epitope spreading in the preclinical phase predicts progression to rheumatoid arthritis. PloS One 2012;7:e35296. Sokolove J, Lindstrom TM, Robinson WH. Development and deployment of antigen arrays for investigation of B-cell fine specificity in autoimmune disease. Front Biosci (Elite Ed) 2012;4: 320–30. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 2001;98:5116–21. Steinitz M, Tamir S. Human monoclonal autoimmune antibody produced in vitro: rheumatoid factor generated by Epstein-Barr virus-transformed cell line. Eur J Immunol 1982;12:126–33. McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. N Engl J Med 2011;365:2205–19. Baum J, Stastny P, Ziff M. Effects of the rheumatoid factor and antigen-antibody complexes on the vessels of the rat mesentery. J Immunol 1964;93:985–92. Bianco NE, Dobkin LW, Schur PH. Immunological properties of isolated IgG and IgM anti-␥-globulins (rheumatoid factors). Clin Exp Immunol 1974;17:91–101. Sabharwal UK, Vaughan JH, Fong S, Bennett PH, Carson DA, Curd JG. Activation of the classical pathway of complement by rheumatoid factors: assessment by radioimmunoassay for C4. Arthritis Rheum 1982;25:161–7. Balestrieri G, Tincani A, Migliorini P, Ferri C, Cattaneo R, Bombardieri S. Inhibitory effect of IgM rheumatoid factor on immune complex solubilization capacity and inhibition of immune precipitation. Arthritis Rheum 1984;27:1130–6. Roosnek E, Lanzavecchia A. Efficient and selective presentation of antigen-antibody complexes by rheumatoid factor B cells. J Exp Med 1991;173:487–9. Hueber W, Tomooka BH, Zhao X, Kidd BA, Drijfhout JW, Fries JF, et al. Proteomic analysis of secreted proteins in early rheumatoid arthritis: anti-citrulline autoreactivity is associated with up regulation of proinflammatory cytokines. Ann Rheum Dis 2007; 66:712–9. Todd DJ, Knowlton N, Amato M, Frank MB, Schur PH, Izmailova ES, et al. Erroneous augmentation of multiplex assay measurements in patients with rheumatoid arthritis due to heterophilic binding by serum rheumatoid factor. Arthritis Rheum 2011;63: 894–903. Artandi SE, Calame KL, Morrison SL, Bonagura VR. Monoclonal IgM rheumatoid factors bind IgG at a discontinuous epitope comprised of amino acid loops from heavy-chain constant-region domains 2 and 3. Proc Natl Acad Sci U S A 1992;89:94–8. Bonagura VR, Artandi SE, Davidson A, Randen I, Agostino N, Thompson K, et al. Mapping studies reveal unique epitopes on IgG recognized by rheumatoid arthritis-derived monoclonal rheumatoid factors. J Immunol 1993;151:3840–52. Newkirk MM, Goldbach-Mansky R, Lee J, Hoxworth J, McCoy A, Yarboro C, et al. Advanced glycation end-product (AGE)-damaged IgG and IgM autoantibodies to IgG-AGE in patients with early synovitis. Arthritis Res Ther 2003;5:R82–90.