Autoimmunity to Peptidyl Arginine Deiminase Type 4 Precedes Clinical Onset of Rheumatoid Arthritis

Abstract

Objective

To determine if antibodies against peptidyl arginine deiminase Type 4 (PAD-4) are present in the pre-clinical phase of rheumatoid arthritis (RA), and to compare their appearance to other pre-clinical autoantibodies.

Methods

Prediagnosis serum samples from 83 subjects with RA were evaluated for presence of anti-PAD-4 antibody, anti-cyclic citrullinated peptide (anti-CCP) antibody, and rheumatoid factor (RF). In addition, a control cohort (N = 83) matched on age, gender, race, number of samples, and duration of serum storage were tested for antibody against PAD-4 to determine its sensitivity and specificity for future RA.

Results

Fifteen of 83 (18.1%) subjects with RA had at least one prediagnosis sample positive for anti-PAD-4. One of 83 (1.2%) control subjects had at least one sample positive, resulting in a sensitivity and specificity of anti-PAD-4 for the future development of RA of 18.1% and 98.8%, respectively. The mean time of first positivity for anti-PAD-4 was ~4.6 years prior to diagnosis. Anti-PAD-4 positivity was associated with anti-CCP positivity (OR 5.13, 95%CI 1.07–24.5, p = 0.04). In subjects with prediagnosis samples positive for both antibodies, anti-CCP positivity predated anti-PAD-4 in 9 of 13 (69%) cases.

Conclusion

Autoantibodies to PAD-4 are present in the pre-clinical phase of RA in a subset of patients and are associated with anti-CCP positivity. Further exploration is needed regarding the timing of appearance and disease-related effects of PAD-4 autoimmunity.

Studies using stored prediagnosis specimens have demonstrated the presence of autoantibodies, cytokines/chemokines, and inflammatory markers years prior to clinical onset and diagnosis of RA (1–5). These findings suggest that there is a pre-clinical period in RA, during which immunologic and inflammatory changes occur that may subsequently lead to symptomatic disease. As such, biomarkers that are present in this pre-clinical period are of great interest and may aid in the understanding of disease pathogenesis.

Autoantibodies against peptidyl arginine deiminase type 4 (PAD-4) have recently been described as a specific biomarker in subjects with clinically apparent RA (6). Peptidyl arginine deiminases (PADs) are a family of enzymes responsible for post-translational modification of the amino acid arginine to citrulline. This process is likely to be of significance in patients with RA given the established association of anti-citrullinated peptide antibodies (ACPAs) with disease presence and severity. Several studies have identified an association between genetic polymorphisms of the PADI4 gene and RA (7–10), although it has not been confirmed across all racial and ethnic groups (11, 12). Subsequent studies showed that PAD-4 may also function as an antigen, generating antibody responses in subjects with RA (13, 14). Recently, researchers demonstrated the presence of specific anti-PAD-4 antibodies in patients with RA, as well as association with disease severity (6, 15, 16). However, the role of PAD-4 in the development of RA has not been fully elucidated. Herein we tested for the presence of anti-PAD-4 antibodies in prediagnosis samples of subjects with RA in order to determine whether these autoantibodies play an early role in disease evolution. In addition, we sought to describe the timing of anti-PAD-4 antibody appearance in the pre-clinical period, its relation to anti-CCP autoimmunity, and potential associations with a more severe RA phenotype.

PATIENTS AND METHODS

Study population

Stored prediagnosis serum samples were utilized from 83 military cases of RA – a cohort previously identified through the Walter Reed Army Medical Center Rheumatology Clinic (4). RA subjects included in this analysis met ≥4 American College of Rheumatology (ACR) classification criteria for RA or were diagnosed by a board-certified rheumatologist (17). Information on gender, race, symptom onset, and age at the time of RA diagnosis was obtained by chart review. The presence or absence of radiographic erosions was determined by a radiologist as part of clinical care. In addition, a control cohort of 83 military subjects without RA was matched to cases on gender, age, race, number of serum samples, and duration of serum storage. The study protocol was approved by the Institutional Review Board at Walter Reed Army Medical Center and the University of Colorado. Further details on the repository and RA cohort are described elsewhere (4).

Autoantibody testing

RF and anti-CCP antibody testing was performed at the University of Colorado Division of Rheumatology Clinical Research Laboratory. RF was measured by nephelometry (RF-Neph) according to manufacturer’s specifications (Dade Behring, Newark, Delaware, USA). The ACR Classification Criteria for RA specifies that a RF level is considered positive if present in < 5% of control subjects (17). Accordingly, we determined a general RF cut-off level for positivity of > 15.2 IU/mL using a 95% cutoff point established from the 83 healthy military control subjects. Antibodies against citrullinated peptides were tested by ELISA using the anti-CCP2 kit (Diastat, Axis-Shield, Dundee, Scotland, UK). Per the manufacturer’s specifications, a positive test was defined as >5 U/mL.

Anti-PAD-4 antibody testing was performed in the Rheumatic Disease Research Core Center at Johns Hopkins University, using an immunoprecipitation method as previously described (6, 18). Briefly, ³⁵S-methionine-labeled human PAD-4 was generated by coupled in vitro transcription/translation (IVTT). Immunoprecipitation was performed in a controlled setting over one hour, at four degrees Celsius and in the absence of calcium. Anti-PAD-4 antibody was detected by fluorography; densitometric scanning allowed for quantitative comparison between patient serum and established positive and negative controls. Positive reference serum was assigned a value of 1.0; patient sera with values > 0.1 were categorized as positive. A semiquantitative scale (0, 1, 2, 3+) based on densitometry of scanned immunoprecipitation autoradiograms was used to assign a value to each serum sample.

Statistical analysis

The prevalence of pre-clinical autoimmunity as well as sensitivity and specificity values of the individual autoantibody tests were calculated. The mean duration of autoantibody positivity in the prediagnosis period (to time of diagnosis) was calculated based on the earliest serum sample that was positive for RA-related autoantibodies. A subset of cases were autoantibody positive in the earliest available serum sample (representing left censorship of data), thus the true duration of antibody positivity may have been underestimated. Survival analysis was not used to adjust for left censorship based on the relatively small number of antibody positive cases in the cohort (4, 19). Differences in the mean duration of antibody positivity were determined by the t-test and adjusted for left censorship (20). The signed-rank test was used to determine if anti-CCP antibody appeared prior to PAD-4 antibody.

Logistic regression was used to analyze the relationship between gender, age, and race with anti-PAD-4 positivity, and to analyze the relationship between anti-CCP and anti-PAD-4 antibodies. Factors associated with radiographic erosions (at or after diagnosis of RA) were determined using logistic regression with the following predictor variables included in the model: PAD-4 and anti-CCP antibody, RF, age, sex, gender, and the interaction term PAD4*CCP. All statistical analyses were performed using SAS 9.2.

RESULTS

The demographic and clinical characteristics of the RA cases are presented in Table 1. Anti-PAD-4 positivity was present in at least one prediagnosis serum sample from 15 RA subjects but only one control, resulting in a sensitivity and specificity for subsequent RA of 18.1% and 98.8% (Table 2). The mean duration of anti-PAD-4 positivity was 4.67 years prior to clinical diagnosis (Table 2). Anti-CCP antibody was positive in 51 cases of RA (61.4%), with a sensitivity and specificity for subsequent RA of 61.4% and 100%, respectively. The mean duration of anti-CCP antibody positivity was 3.49 years prior to diagnosis. RF was positive in 47 cases of RA (56.6%), had a sensitivity and specificity of 56.6% and 86.7%, and was present a mean of 3.8 years prior to diagnosis. In post-diagnosis samples, prevalence increased for RF, anti-CCP and anti-PAD-4 antibodies to 83%, 68%, and 26%, respectively (Table 2).

Table 1.

Demographic and clinical characteristics of RA cohort (N=83)

	Number of subjects (%), unless otherwise specified
Age at diagnosis (years); mean, SD	39.9 (10.0)
Male	49 (59)
Race
White	57 (69)
Black	21 (25)
Other	5 (6)
Erosions
Present	42 (51)
Absent	34 (41)
Unclassified	7 (8)
Preclinical serum samples (N = 243)
Number of samples per case; mean, SD	2.9 (1.2)
Years before diagnosis of first sample; mean, SD	6.6 (3.7)

Adapted from Majka et. al.(4)

Abbreviations: RA: rheumatoid arthritis; SD: standard deviation

Table 2.

Characteristics of Pre-Clinical Autoantibodies (RF, anti-CCP, and anti-PAD4)

Autoantibody	Positive test before diagnosis N (%)	Sensitivity for subsequent RA (%)	Specificity for subsequent RA (%)	Duration of antibody positivity prior to diagnosis, years Mean (95% CI)	Positive test post diagnosis N (%)***
RF (>15.2 IU/mL)	47 (56.6)	56.6	86.7	3.80 (2.83, 4.76)	39 (83)
Anti-PAD-4	15 (18.1)	18.1	98.8	4.67 (2.54, 6.82)	12 (26)
Anti-CCP	51 (61.4)	61.4	100.0	3.49 (2.59, 4.39)	32 (68)
PAD-4 present*	13 (15.7)			6.16 (4.29, 8.02)
PAD-4 absent**	38 (45.8)			2.58 (1.68, 3.47)

^*= Patients with positive anti-PAD-4 and anti-CCP antibodies in the pre-clinical period (double positive)

^**= Patients with positive anti-CCP but negative anti-PAD-4 antibodies in the pre-clinical period

^***= 47 subjects with available post-diagnosis samples

Abbreviations: RF-neph: rheumatoid factor (by nephelometry); CI: confidence interval

Anti-PAD-4 positivity was significantly associated with anti-CCP positivity: OR 5.13, 95% CI 1.07-24.5. Anti-PAD-4 and anti-CCP positivity (double positive) was seen in 13 of the 83 subjects with RA but no controls; double positivity was 15.7% sensitive and 100% specific for the future development of RA (Table 2). In 9 of 13 ‘double positive’ cases, anti-CCP positivity predated anti-PAD-4, while only one patient developed antibodies to PAD-4 prior to CCP, suggesting that anti-CCP antibody tends to appear prior to anti-PAD-4 in these subjects (p = 0.027, Table 3). Only two subjects with prediagnosis anti-PAD-4 positivity were anti-CCP negative.

Table 3.

Timing of antibody appearance in subjects with both anti-PAD4 and anti-CCP in prediagnosis samples (N = 13)

		p-value
Anti-CCP preceded anti-PAD-4	9/13 cases	0.0273#
Anti-PAD-4 preceded anti-CCP	1/13 cases
First appearance in the same sample	3/13 cases

Abbreviations: PAD-4: peptidyl arginine deiminase-4. CCP: cyclic citrullinated peptide.

^#Signed rank test used for statistical analysis

The mean time between appearance of anti-CCP and clinical diagnosis in 13 double positive subjects was 6.16 years (Table 2), and in subjects without pre-clinical anti-PAD-4 the mean time was 2.58 years (p < 0.0007). The mean anti-CCP antibody titer in all available samples in double positive subjects was 206.3 U/mL, and in subjects without evidence of pre-clinical PAD-4 the mean titer was 82.7 (p = 0.03).

To evaluate the persistence of autoantibodies over time, autoantibody positivity was assessed in subjects who had additional samples collected after their initial positive test. Six of the 14 (43%) anti-PAD-4 positive subjects with multiple samples had reversion from anti-PAD-4 positive to a persistent seronegative status, while eight (57%) remained positive. Six of the 44 (14%) subjects assessed had anti-CCP reversion to a seronegative status, while 38 (86%) remained positive in follow-up samples. Three of the 42 (7%) subjects assessed had RF reversion, while 39 (93%) remained positive. The proportion of subjects with RF reversion was significantly less than anti-PAD-4 (7% vs. 43%; p < 0.01), while the difference between anti-CCP and anti-PAD-4 reversion bordered on significance (14% vs. 43%; p = 0.05). Several cases reverted in the post-diagnosis time period: all three cases of RF reversion, four of six cases of anti-CCP reversion, and two of six cases of anti-PAD-4 reversion. Changes in PAD-4 antibody level between sample collections, based on densitometry and using a semiquantitative scale (0–3+), are outlined in Figure 1. Changes in anti-CCP titer from the same serum samples are plotted alongside anti-PAD-4 for comparison.

Figure 1. Longitudinal Pattern of PAD4 and CCP Antibodies in 15 RA Patients with Pre-Clinical Anti-PAD4.

Testing performed for anti-PAD-4 and anti-CCP antibody in successive samples within each individual. Time between samples varied among subjects and is not reflected in this figure. CCP titers separated in to the following categories: 0 (0 – 5 U/mL), 1 (>5 – 50 U/mL), 2 (>50 – 100 U/mL), 3 (>100 – 150 U/mL), and 4 (>150 U/mL). PAD-4 antibody level determined by semiquantitative scale based upon densitometry (0–3).

× Anti-PAD-4 Antibody (0–3)

Anti-CCP Antibody (0–4)

No association was seen between age, gender, or race and the presence of anti-PAD-4 antibody (Table 4). In multivariate analysis, after accounting for PAD-4 autoantibody presence, an independent association was identified between anti-CCP antibody positivity in the prediagnosis period and subsequent erosions (OR 12.1, 95% CI 2.34–62.9). There was a non-significant association between anti-PAD-4 and subsequent erosive disease (OR 1.71, 95% CI 0.45–6.54, p = 0.43). Sub-group analysis of subjects with persistent anti-PAD-4 positivity over time (N = 8) demonstrated similar results (OR 2.11, 95% CI 0.41–13.9, p = 0.48).

Table 4.

Anti-PAD-4 antibody association with patient demographics⁺

Variable	OR, 95% CI	P-value
Age*	0.99 (0.93 – 1.06)	0.80
Female gender	1.77 (0.30– 10.4)	0.59
African-American race**	1.06 (0.09 – 12.1)	0.86
Other race**	1.37 (0.33 – 5.76)	0.66

⁺Analysis based on logistic regression

^*analyzed as a continuous variable

^**analyzed using non-Hispanic whites as the reference category; ‘Other Race’ categories: American Indian, Asian, Biracial, Hispanic

DISCUSSION

We have found that autoantibodies against the PAD-4 enzyme are present in the prediagnosis period and are specific for the future development of RA. Anti-PAD-4 antibodies were evident as early as four years prior to clinical diagnosis, similar to findings reported in other studies for pre-clinical anti-CCP and RF (1, 3, 4, 21, 22). In addition, the presence of anti-PAD-4 antibody was significantly associated with anti-CCP antibody. Patients with both anti-PAD-4 and anti-CCP present in the pre-clinical period appeared to have a longer time interval between the appearance of anti-CCP and clinical disease.

To detect antibody against the PAD-4 enzyme, we used a novel approach using immunoprecipitation with in vitro transcribed/translated (IVTT) PAD-4. While others have used ELISAs to detect anti-PAD-4 antibody (15, 16, 23, 24), we have found a significant false-positive rate (~10%) in RA using this method compared to immunoprecipitation. One may question whether or not the anti-PAD-4 antibodies detected in the above experiments were directed against citrullinated targets on the PAD-4 enzyme. A recent study by Andrade and colleagues has described the presence of antibodies in a subset of patients with RA against both citrullinated and non-citrullinated epitopes on the PAD-4 enzyme (Arthritis & Rheumatism, in press). As such, the conditions of our study were chosen to minimize the risk of IVTT PAD-4 autocitrullination, which could lead to identification of antibodies against citrullinated residues. Calcium is required as a cofactor for proper functioning of the PAD-4 enzyme (25); therefore, the assay in our study was performed at four degrees Celsius in the absence of calcium. Under similar conditions, Andrade and colleagues demonstrated an inability of PAD-4 to autocitrullinate, providing further evidence that the antibodies identified in our study are directed against non-citrullinated residues.

The prevalence of anti-PAD-4 antibody detected in our study deserves additional comment. Data from prior studies has determined the prevalence of anti-PAD-4 antibody in established RA at 35–45% (6, 15, 16, 24), in contrast to the prevalence in the pre and post-diagnosis time period noted in our study (18% and 26%, respectively). However, the duration of disease in these cohorts ranged from 7 to 13.5 years, in contrast to the mean disease duration in our cohort of 3.3 years at the time of last post-diagnosis sample. In addition, a prior study by Harris et. al. discovered a decreased prevalence of anti-PAD-4 antibody in early RA compared to established disease (6). Together, these findings suggest that the prevalence of anti-PAD-4, while low in the pre-diagnosis period, may increase over time. The prevalence of anti-PAD-4, anti-CCP and RF all increased in our cohort between the pre-diagnosis and early post-diagnosis time period, supporting a hypothesis of amplification of multiple autoimmune pathways. This latter data also supports prior descriptions of increasing anti-CCP prevalence in the pre-diagnosis time period as patients draw closer to the time of RA symptoms (1).

Another area of particular interest is the description of autoantibody reversion over time. Reversion to a seronegative status occurred for each autoantibody tested, although the percentage of subjects with anti-PAD-4 reversion was higher than either anti-CCP or RF. Most instances of RF and anti-CCP reversion occurred in the post-diagnosis period, as noted in the results section. Studies in established RA have shown declining RF and anti-CCP antibody titers in patients with good clinical response to ongoing therapy (26, 27). The loss of seropositivity in subjects during the post-diagnosis period may reflect improved disease control upon initiation of immunosuppressive therapy; however, supporting data regarding disease activity, medication use, and other factors after the time of diagnosis are not available.

Also of interest is the presence of a significant association between anti-PAD-4 and anti-CCP antibodies, a finding similar to cohorts with established RA (6, 16, 28). Epitope spreading may be a potential hypothesis to explain this association. According to this model, anti-CCP antibody develops first due to novel antigen structures generated by PAD activity. The PAD-4 enzyme might then become an antigen itself through association with its substrates. A similar mechanism has been proposed in the development of celiac disease, where positive correlation has been found between autoantibodies directed against tissue transglutaminase and its substrate, gliadin (28). However, this hypothesis would not clearly explain the two cases in our cohort which developed PAD-4 antibodies in the absence of anti-CCP. These two cases suggest that either the PAD-4 enzyme can serve as an auto-antigen independently of its interaction with citrullinated residues, or that ACPAs that precede anti-PAD-4 antibody are present that are not detected by the commercial anti-CCP2 assay. Antigen formation through PAD-4 automodification is another interesting possibility, especially given the recent observation that PAD-4 can be autocitrullinated, and that this may modify the enzyme’s tertiary structure (Andrade et. al., accepted for publication).

We noted a prolonged latency between the appearance of anti-CCP antibodies and clinical disease in patients with anti-PAD-4 positivity. On average, this time period was over three years longer in double positive subjects compared to those without anti-PAD-4. The mechanism responsible for this finding is unknown, but these data generate hypotheses regarding the influence of PAD-4 antibody on subsequent PAD enzyme function. A gain or loss-of-function in response to antibody binding could explain several features relevant to PAD-4 autoimmunity in RA. For example, a loss-of-function following antibody binding could result in decreased levels of protein citrullination. This could result in a longer period of pre-clinical autoimmunity during which time additional genetic or environmental factors are needed for transition to clinical disease. In contrast, antibody binding could potentially lead to altered substrate specificities, generating additional citrullinated epitopes such as vimentin, fibrinogen, and other peptide targets. This could lead to increased levels of autoimmune activation and account for the association between PAD-4 antibody and decreased functional status, increased disease activity, and advanced radiographic progression (15, 16, 24). A recent study by Auger et. al. has provided some insight in to this area by demonstrating that autoantibodies to PAD-4 can inhibit PAD-4 mediated citrullination (23). These hypotheses remain an area where additional research is necessary.

This study has generated important initial data on the presence of anti-PAD-4 antibodies in early RA development; however, there are several limitations of the analysis. The RA cohort examined here consists of military personnel, resulting in an increased proportion of male subjects compared to the general RA population. However, the age at diagnosis and prevalence of RF, anti-CCP and radiographic erosions are similar to cohorts previously described in the literature (29–31). Control subjects were selected from military personnel as well and may represent a younger and healthier cohort compared to the general non-RA population. This could affect our specificity analysis as older subjects and those with co-existent disease may potentially have a higher prevalence of RA-related autoimmunity. Additionally, analysis describing the timing of antibody appearance in relation to diagnosis is limited in subjects with positive antibodies in the first available serum sample. In these subjects we were unable to determine the interval of conversion from seronegative to seropositive status based on left censorship of the data. Statistical adjustment was performed for analyses which included this data, as mentioned in the Methods section. Our study is also limited by the relatively small number of subjects in our RA cohort, resulting in only 15 subjects with anti-PAD-4 antibody in the pre-clinical period. As a result, we were limited in our ability to identify an association with clinical endpoints such as erosive disease, particularly in the subset of double positive subjects. Furthermore, the prevalence of erosions in our study may be an underestimate, as cases without available radiographs were considered negative and some subjects may have radiographs from the time of diagnosis. This will be an important area for further study, as evidence suggests the co-existence of both anti-CCP and anti-PAD-4 is associated with more severe disease (6, 15).

In conclusion, this study has identified anti-PAD-4 antibodies in the pre-clinical period of RA development which appear to be specific for subsequent development of clinically apparent disease. Further research is needed to investigate the relative timing of appearance of multiple autoantibodies in pre-clinical RA, the physiologic implications of autoantibody binding to the PAD-4 enzyme, and potential influence on disease development and severity.