Previous case series have examined the relationship between anti–Jo-1 antibody levels and myositis disease activity, demonstrating equivocal results. Using enzyme-linked immunosorbent assays (ELISAs) and novel measures of myositis disease activity, the current study was undertaken to systematically reexamine the association between anti–Jo-1 antibody levels and various disease manifestations of myositis.
Serum anti–Jo-1 antibody levels were quantified using 2 independent ELISA methods, while disease activity was retrospectively graded using the Myositis Disease Activity Assessment Tool, which measures disease activity in 7 different organ systems via the Myositis Disease Activity Assessment Visual Analog Scale (VAS) and the Myositis Intention-to-Treat Index (MITAX) components. Spearman's rank correlation coefficients and mixed linear regression analysis were used to identify associations between anti–Jo-1 antibody levels and organ-specific disease activity in cross-sectional and longitudinal analyses, respectively.
Cross-sectional assessment of 81 patients with anti–Jo-1 antibody revealed a modest correlation between the anti–Jo-1 antibody level and the serum creatine kinase (CK) level, as well as muscle and joint disease activity. Correlation coefficients were similar for CK levels (rs = 0.38, P = 0.002), myositis VAS (rs = 0.36, P = 0.002), and arthritis VAS (rs = 0.40, P = 0.001). In multiple regression analyses of 11 patients with serial samples, anti–Jo-1 antibody levels correlated significantly with CK levels (R2 = 0.65, P = 0.0002), myositis VAS (R2 = 0.53, P = 0.0008), arthritis VAS (R2 = 0.53, P = 0.006), pulmonary VAS (R2 = 0.69, P = 0.005), global VAS (R2 = 0.63, P = 0.002), and global MITAX (R2 = 0.64, P = 0.0003).
In this large series of patients with idiopathic inflammatory myopathy, anti–Jo-1 antibody levels correlated modestly with muscle and joint disease, an association confirmed by a custom ELISA using recombinant human Jo-1. More striking associations emerged in a smaller longitudinal subset of patients that link anti–Jo-1 antibody levels to muscle, joint, lung, and global disease activity.
Idiopathic inflammatory myopathy (IIM) represents a group of disorders characterized by immune-mediated destruction and/or dysfunction of muscle (1). The autoimmune process extends beyond muscle disease, with additional clinical features involving the skin, joints, lungs, and circulation (2). These protean manifestations are often difficult to manage, particularly because different features may not be simultaneously present or active in all patients. Further complicating disease management is the imprecise nature of current markers of disease activity. For example, manual muscle testing has poor reproducibility and is not specific for active inflammatory muscle disease versus muscle damage (3). Moreover, while creatine kinase (CK) levels may be helpful in following muscle disease activity, they do not correlate with systemic manifestations such as lung disease. The latter complication can be especially difficult to assess because pulmonary function tests and high-resolution computed tomography cannot easily distinguish infection, active inflammatory disease, or scarring. Overall, these diagnostic and management issues underscore the lack of a clinically useful biomarker for component and global disease activity.
Anti–Jo-1 antibody is a myositis-specific antibody that was first described in 1980 (4). Initially thought to be a marker of inflammatory myopathy alone, anti–Jo-1 antibody is now associated with a distinct clinical entity known as the antisynthetase syndrome, which includes fever, myositis, interstitial lung disease (ILD), nonerosive arthropathy, mechanic's hands, and Raynaud's phenomenon (5). ILD is especially prevalent in the antisynthetase syndrome, occurring in ∼75% of patients with anti–Jo-1 antibody compared to ∼30% of patients with IIM in the absence of antisynthetase antibodies (6–8).
Anti–Jo-1 antibody is one of several antisynthetase antibodies that have been described, each of which has been associated with overlapping features of the antisynthetase syndrome (5, 9). Anti–Jo-1 antibody recognizes different epitopes of histidyl-tRNA synthetase (Jo-1), including the subunit that catalyzes binding between the amino acid histidine and its cognate tRNA in the process of protein synthesis (5, 10, 11). Based on a range of immunologic and immunogenetic data, Jo-1 likely plays a direct role in the induction and maintenance of autoimmunity in the antisynthetase syndrome. For example, the antibody response to histidyl-tRNA synthetase undergoes class switching, spectrotype broadening, and affinity maturation, all of which are indicators of a T cell–dependent, antigen-driven process (10–13). Therefore, although data are lacking that directly implicate anti–Jo-1 antibody in the pathogenesis of the antisynthetase syndrome, this patterned B cell reactivity reflects an underlying T cell response directed against Jo-1 that may drive autoantibody formation and direct tissue damage.
In turn, the likely role of Jo-1 in the immunopathogenesis of the antisynthetase syndrome suggests that anti–Jo-1 antibody levels may correlate with disease activity. A limited number of previous studies that address this question have been hampered by factors such as small sample size, imprecise indices of disease activity, and variable and/or unreliable methods of quantifying anti–Jo-1 antibody levels (12, 14, 15). Furthermore, none of the earlier studies was designed to specifically examine the correlation between antibody levels and disease activity. In contrast, the current analysis directly addresses the association between enzyme-linked immunosorbent assay (ELISA)–determined anti–Jo-1 antibody levels and disease activity as measured by the Myositis Disease Activity Assessment Tool (MDAAT) (16), a partially validated index for measuring disease activity in IIM.
PATIENTS AND METHODS
Serum was collected from patients with suspected autoimmune disease during inpatient and outpatient encounters at the University of Pittsburgh Medical Center from 1975 through 2006. Serum obtained at the time of clinical evaluation was tested for the presence of anti–Jo-1 antibody using immunodiffusion. Patients were included in the current study if they provided written consent and tested positive for anti–Jo-1 antibody by this method, but were excluded if adequate clinical or laboratory data were unavailable.
Clinical indices of disease activity.
Disease activity was measured through chart review and retrospective application of the 2005 MDAAT. The MDAAT consists of the Myositis Disease Activity Assessment Visual Analog Scale (MYOACT) and the Myositis Intention-to-Treat Index (MITAX) components, each of which contains specific guidelines for physician scoring of disease activity. While the MYOACT measures myositis-associated disease activity during the previous 4 weeks in 7 organ systems (constitutional, cutaneous, joint, gastrointestinal, pulmonary, cardiac, and muscle) using a continuous 10-cm visual analog scale (VAS), the MITAX incorporates the physician's intention to treat active disease present during the preceding month in the same 7 organ systems using a 4-point ordinal scale (0 = disease not present, 1 = disease improving, 2 = disease unchanged, 3 = disease worsening, 4 = new disease manifestations).
Anti–Jo-1 antibody levels were determined in stored serum samples with a commercially available ELISA kit (Inova Diagnostics, San Diego, CA) that uses calf thymus extract. Samples were prepared according to the manufacturer's instructions. To confirm the results from this system, a custom ELISA was independently devised using full-length recombinant human Jo-1 (rHuJo-1)/maltose binding protein fusion protein as the substrate. Briefly, 96-well microtiter plates (Nunc, Rochester, NY) were coated with rHuJo-1 (0.1 μg/ml) in carbonate buffer (50 mM NaHCO3/Na2CO3, pH 9.6) and incubated overnight at 4°C. Plates were then washed 4 times with phosphate buffered saline (PBS) containing 0.05% Tween 20. After blocking wells with PBS containing 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO) at room temperature for 2 hours, appropriately diluted serum samples (1:10,000) were added to triplicate wells for 2 hours.
Following repeated washes with PBS–Tween 20 and a 60-minute incubation with a 1:10,000 dilution of horseradish peroxidase–conjugated goat anti-human IgG (stock = 0.4 mg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), the enzymatic reaction was visualized using tetramethylbenzidine (Sigma-Aldrich) and subsequently terminated with 1N H2SO4. Color development was measured at 450 nm using a Wallac 1420 multilabel counter (PerkinElmer, Wellesley, MA). Quantification of the rHuJo-1–based ELISA and assignment of relative unit values were based on comparisons of readings at an optical density of 450 nm with those obtained from serial dilutions of a control anti–Jo-1 antibody serum sample obtained from the Centers for Disease Control and Prevention (Atlanta, GA). The specificity of this ELISA for human Jo-1 was confirmed through pilot experiments that showed no serum reactivity to plates coated with the maltose binding protein fusion partner alone.
The mean ± SD anti–Jo-1 antibody level was determined using both the commercial Jo-1 and the rHuJo-1 ELISAs. Spearman's rank correlation coefficients were calculated to quantify relationships between disease activity and anti–Jo-1 antibody levels at a single point in time. In patients for whom serial samples were available, mixed linear regression analysis for repeated measures was performed to determine correlations between anti–Jo-1 antibody levels and disease activity. All statistical analyses were performed using SAS software (SAS Institute, Cary, NC). P values less than 0.05 were considered significant.
Patient selection and demographics.
Ninety-four patients with anti–Jo-1 antibody (detected by immunodiffusion) from the University of Pittsburgh database were evaluated for this study, but 13 individuals were excluded from this analysis due to incomplete clinical information or lack of informed consent. Of the 81 patients who satisfied the inclusion requirements, ≥3 serum samples were available from 11 patients. Based on the criteria established by Bohan and Peter (17), 55 patients (68%) had definite or probable polymyositis, 16 (20%) had definite or probable dermatomyositis, and 10 (12%) had undifferentiated connective tissue disease or overlap syndromes. Among the patients with overlap diagnoses, 6 had myositis and systemic sclerosis (SSc), 1 had myositis and systemic lupus erythematosus (SLE), and 1 had combined features of myositis, SSc, and SLE. Additional serologic abnormalities in the overlap group included the presence of anti–double-stranded DNA (anti-dsDNA) antibody (n = 1), anti–PM-Scl antibody (n = 1), and anticentromere antibody (n = 3).
As summarized in Table 1, the mean age of these patients at their first University of Pittsburgh visit was 47.7 years. The cohort was predominantly female (73%) and white (91%), which reflects racial and ethnic differences in disease distribution as well as regional demographics. At the time of initial evaluation, the mean duration of symptoms (not restricted to myositis) was 3.8 years. Organ system involvement and other extramuscular features that characterized this patient group are summarized in Table 1.
Table 1. Demographic and clinical features of the study patients
Age at initial evaluation, mean ± SD (range) years
47.7 ± 12.5 (21.6–75.3)
Duration of symptoms at initial evaluation, mean ± SD (range) years
3.8 ± 5.1 (0.09–24.5)
Clinical feature, no./no. assessed (%)
Interstitial lung disease
ELISA assessment of anti–Jo-1 antibody titer and correlation with measures of clinical disease activity.
To determine potential associations between anti–Jo-1 antibody levels and disease activity in different organ systems, anti–Jo-1 antibody titers were initially measured using a commercially available ELISA kit. A review of the anti–Jo-1 antibody titer distribution indicated heavy skewing toward the high end of the scale, potentially confounding correlations with clinical indices of disease activity (Table 2). Yet, comparing antibody levels in the initial serum samples from all 81 patients with measures of clinical disease activity yielded statistically significant correlations between anti–Jo-1 antibody titers and serum CK levels, muscle as well as articular disease activity (recorded using both the MITAX and the MYOACT/VAS), and a composite index of global disease activity (global MITAX) (Figure 1 and Table 3). Of note, excluding patients with overlap diagnoses had no effect on the findings of this analysis (results not shown).
Table 2. Results of a commercial ELISA for anti–Jo-1 antibody*
No. of samples (n = 106)
Units represent conversion derived from a standard curve of values at an optical density of 450 nm. ELISA = enzyme-linked immunosorbent assay.
Negative (<20 units)
Weakly positive (20–39 units)
Moderately positive (40–80 units)
Strongly positive (>80 units)
Table 3. Cross-sectional correlations with Jo-1 ELISA results*
Commercial Jo-1 ELISA
Custom rHuJo-1 ELISA
Assessments were made in all 81 study patients. ELISA = enzyme-linked immunosorbent assay; rHuJo-1 = recombinant human Jo-1; CK = creatine kinase; ULN = upper limit of normal; VAS = visual analog scale; MITAX = Myositis Intention-to-Treat Index.
CK (× the ULN)
Although the overall distribution of anti–Jo-1 antibody titers determined with the commercial ELISA (substrate antigen derived from calf thymus extract) appeared similar to that obtained with the alternative ELISA using rHuJo-1, the relatively modest correlation coefficient between these 2 systems (r = 0.59; P < 0.0001) suggests antibody recognition of species-specific Jo-1 epitopes that could be a key parameter influencing correlations with measures of myositis disease activity. In support of this conclusion, Table 3 shows greater correlation coefficients between the rHuJo-1 ELISA values and the MDAAT components such as global MITAX and global VAS.
Longitudinal correlations between anti–Jo-1 antibody titer and clinical disease activity.
In our cohort, 11 patients had 3–6 serum samples collected at different time points in their disease course. The interval between the first and the last sample ranged from 1.25 to 16 years. Separate analysis of these serial patient samples through mixed linear regression (after accounting for interpatient variability) showed significant correlations between the anti–Jo-1 antibody level and the following clinical parameters: muscle, joint, lung, and global disease activity (Table 4). Paralleling the results with the cross-sectional data set, use of the rHuJo-1 ELISA generated stronger associations with parameters of disease activity than did the commercial ELISA using calf thymus extract (Table 4). Further review of these serial data revealed that anti–Jo-1 antibody levels assessed by both ELISA methods became negative during periods of disease inactivity in 3 patients. As shown by data from 2 such patients (Figure 2), this temporal correlation further links anti–Jo-1 antibody titers with muscular as well as extramuscular features of myositis disease activity.
Table 4. Longitudinal correlations with Jo-1 ELISA results*
Taken together, these data demonstrate a moderate correlation between anti–Jo-1 antibody titers and clinical indicators of myositis disease activity that include elevation of CK levels, muscle dysfunction, and articular involvement. The strength of these associations is comparable to that between anti-dsDNA and renal disease in SLE (18), lending clinical and biologic significance to our findings. Importantly, several of the statistical correlations in this analysis are associated with increased rs values when using a custom-designed ELISA based on rHuJo-1—a critical factor given variations in immunodominant epitopes between calf and human Jo-1 sequences that are reflected by the relatively modest correlation coefficient between the 2 detection methods (rs = 0.59). Ultimately, the strength of association with measures of clinical disease activity may be enhanced through correlations with antibodies that recognize specific epitopes that are truly related to, or reflective of, pathogenic immune responses. This type of analysis may also establish connections between anti–Jo-1 antibody titers and features of extramuscular disease activity not captured by the current ELISA systems.
In contrast to this cross-sectional analysis, examination of associations between anti–Jo-1 antibody levels and parameters of clinical disease activity at different time points (in individual patients) extends the relationship to other manifestations of the antisynthetase syndrome, including ILD. Although conclusions based on these statistical associations must be viewed with some caution given the limited numbers of serial specimens, the findings are provocative and suggest that following anti–Jo-1 antibody titers over time may be clinically useful considering the limitations in assessing active disease in extramuscular tissues such as lung. From a practical standpoint, therefore, interpreting serial anti–Jo-1 antibody levels may be analogous to the analysis of serum uric acid in gout, being most useful in longitudinal patient assessment.
Coupled with previous smaller case series that examined the relationship between anti–Jo-1 antibody levels and muscle involvement in idiopathic inflammatory myopathy (12, 14, 15), the power of this much larger analysis provides compelling evidence that the antibody titer parallels myositis disease activity. The lone study questioning this association reported serial assessment of antibody levels in only a single patient, which hampered definitive conclusions (15).
Overall, the analysis presented here extends the findings of these earlier studies by demonstrating novel associations between anti–Jo-1 antibody levels and specific extramuscular manifestations such as articular and lung disease activity, particularly in longitudinal assessment. Although the retrospective application of this instrument is an inherent limitation that must be addressed in future prospective studies, comparative analysis of clinical evaluations in 34 patients (encompassing 237 disease subcategories) performed both retrospectively (by KBS) and prospectively (by CVO) shows an 85% concordance rate for the MITAX component of the MDAAT, with no significant alteration of statistical associations (data not shown). By extension, therefore, the results of this study provide additional validation of the MDAAT.
Perhaps more important than the implications for anti–Jo-1 antibody as a biomarker of clinical disease activity, this work lends potential insight regarding the immunopathogenesis of the antisynthetase syndrome. While histopathologic and immunohistochemical studies support T cell–mediated tissue damage rather than humoral mechanisms for myocytotoxicity (19–21), the association of class-switched anti–Jo-1 antibody with muscular as well as extramuscular disease activity suggests that underlying Jo-1–specific T cells fuel both anti–Jo-1 antibody formation and cell-mediated immune responses. In fact, we have previously demonstrated the existence of Jo-1–specific T cells in anti–Jo-1 antibody–positive patients (22). Such T cells also exist in the repertoire of nondiseased control subjects, but the absence of anti–Jo-1 antibody in these individuals indicates that Jo-1–specific T cells have not been activated in vivo.
Regardless of the precise mechanism, the relationship between anti–Jo-1 antibody levels and different clinical aspects of the antisynthetase syndrome supports a pathogenic link between Jo-1 antigen and inflammation in tissues as disparate as muscle and lung. Ultimately, however, establishing this etiologic relationship more firmly and elucidating factors that govern organ specificity will require additional in vitro and in vivo models based on Jo-1–specific T cells.
Dr. Stone 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 design. Stone, Oddis, Ascherman.
Acquisition of data. Stone, Fertig, Katsumata, Lucas.
Analysis and interpretation of data. Stone, Oddis, Katsumata, Vogt, Ascherman.