Anti-nucleosome antibodies in patients with systemic lupus erythematosus of recent onset. Potential utility as a diagnostic tool and disease activity marker
J. A. Simón,
J. Cabiedes,
E. Ortiz,
J. Alcocer-Varela and
J. Sánchez-Guerrero
Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan, México.
Correspondence to: J. Abraham Simón, Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, Colonia Sección XVI, Tlalpan, México DF CP: 14000. E-mail: abrahamsimon{at}mailcity.com
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Abstract
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Objective. To compare the utility of anti-chromatin antibodies for the diagnosis of systemic lupus erythematosus (SLE) and as markers of disease activity.
Methods. We included 73 consecutive patients (62 female) with SLE (four or more ACR criteria) of recent onset (<1 yr since diagnosis). As control groups we included 130 healthy blood donors and 261 patients with 11 systemic autoimmune diseases (SAD). Disease activity was assessed using the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). A venous blood sample was drawn to measure three anti-chromatin antibodies [anti-nucleosome (anti-NCS), anti-double-stranded DNA (anti-dsDNA) and anti-histones (anti-HST)] by enzyme-linked immunosorbent assay.
Results. The prevalence of anti-chromatin antibodies in SLE patients and healthy controls was 100 and 3% respectively for anti-NCS, 63 and 5% for anti-dsDNA, and 15 and 3% for anti-HST. Anti-NCS had a sensitivity of 100% and specificity of 97% for SLE diagnosis. When SLE and SAD patients were compared [excluding mixed connective tissue disease (MCTD)], the sensitivity of anti-NCS, anti-dsDNA and anti-HST antibodies for SLE diagnosis was 93, 71 and 40% respectively and the specificity was 97, 98 and 98%. Anti-chromatin antibodies were not useful in differentiating between SLE and MCTD patients. Anti-NCS antibodies showed the highest correlation with disease activity (r = 0.45, P < 0.0001), especially in patients negative for anti-dsDNA antibodies (r = 0.58, P = 0.001). Anti-NCS antibodies also showed strong association with renal damage (odds ratio 4.1, 95% confidence interval 1.213.6, P = 0.01).
Conclusion. Anti-NCS antibodies could be a useful tool in the diagnosis and assessment of disease activity in SLE patients, especially in patients who are negative for anti-dsDNA antibodies.
KEY WORDS: Anti-nucleosome antibodies, Anti-chromatin antibodies, Systemic lupus erythematosus, Diagnosis, Disease activity.
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Introduction
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Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown aetiology, characterized by the presence of autoantibodies against cellular antigens, mainly nuclear antigens, such as anti-histones (anti-HST), anti-single-stranded DNA and anti-double-stranded DNA (anti-dsDNA) antibodies [1]. Anti-dsDNA antibodies are considered the main diagnostic tool for SLE and a useful marker of disease activity; however, they are found only in 50% of SLE patients and do not always correlate with disease activity [2, 3]. On the other hand, antinuclear antibodies, the most prevalent antibodies, have low specificity for the diagnosis of SLE, because they are found in most systemic autoimmune diseases (SAD) and even in healthy individuals [4]. Thus, it is important to look for other autoantibodies that may be useful in the diagnosis and assessment of disease activity in SLE patients.
Recently, it was proposed that the nucleosome is the principal antigen in the pathophysiology of SLE, and that anti-nucleosome antibodies (anti-NCS) are associated with organic damage [57]. However, the sensitivity and specificity of anti-NCS antibodies for the diagnosis of SLE in patients with long disease duration have not been shown to be better than those of anti-dsDNA antibodies [8, 9].
In the murine models of lupus, anti-NCS antibodies arise before the development of other anti-chromatin antibodies and they have the highest prevalence from the early stages of life [10]; therefore, we hypothesized that anti-NCS antibodies could be useful in the diagnosis and assessment of disease activity in SLE patients of recent onset.
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Patients and methods
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We conducted a cross-sectional study among 73 consecutive patients who attended the lupus clinic at the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ) between August 1999 and November 2001, with SLE of recent onset (<1 yr since diagnosis) according to American College of Rheumatology (ACR) criteria [11]. All patients were evaluated by the same rheumatologist, who assessed disease activity using the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) [12]. A venous blood sample was drawn at the same visit and stored at -70°C to measure immunoglobulin G (IgG)-class anti-chromatin antibodies (anti-NCS, anti-dsDNA and anti-HST). As control groups we included (i) 130 healthy blood donors who tested negative for HIV, hepatitis B and C viruses and VDRL (Venereal Disease Research Laboratory slide test) (56% females, mean age 34 ± 9 yr) and (ii) 261 prevalent patients with 11 SAD, including 50 with mixed connective tissue disease (MCTD), 31 with primary antiphospholipid syndrome, 70 with primary vasculitis (Takayasu arteritis, Wegener's granulomatosis, polyarteritis nodosa, HenochSchönlein purpura, Behçet disease and microscopic polyangiitis), 50 with primary Sjögren syndrome, 40 with inflammatory myopathy and 20 with systemic sclerosis, according to appropriate classification criteria [1320].
Anti-chromatin antibodies were determined by immunoenzymatic assay (EIA), according to the manufacturer's recommendations [Orgentec Diagnostika (Germany) (anti-HST and anti-NCS antibodies) and The Binding Site (UK) (anti-dsDNA antibodies)]. Briefly, plates coated with purified human nucleosomes, histones or calf thymus dsDNA were incubated with 100 µl of 1:100 serum dilutions for 30 min at room temperature. Each plate included a calibration curve of five points, a positive control and a negative control. Plates were washed three times with the wash solutions of the kits. A prediluted anti-human IgG horseradish peroxidase conjugate (100 µl/well) was pipetted into the wells of the microplate and incubated for 30 min (dsDNA plates) or 15 min (histones and nucleosomes plates) at room temperature. After three washes, 100 µl of prediluted substrate was added and incubated for 30 min (dsDNA plates) or 15 min (histones and nucleosomes plates) at room temperature. Colour release was stopped by the addition of 100 µl of prediluted stop solution. Plates were read at 450 nm and a 650 nm reference filter was used. Reproducibility was evaluated using eight positive and negative controls (r = 0.98). Because contamination with Jo-1 in the human nucleosome EIA preparations has been reported (personal communication, Henry Chabre), we tested five anti-Jo-1-positive sera in the three anti-chromatin assays, and all samples were negative to anti-NCS, anti-dsDNA and anti-HST antibodies (data not shown). These results suggest that the preparation used for coating the plates was not contaminated with Jo-1.
The cut-off values of anti-chromatin antibodies were set above the mean ± 2 S.D. of a separate control group of 100 healthy individuals (55, 15 and 100 U/ml for anti-NCS, anti-dsDNA and anti-HST antibodies respectively). Because these values were not useful in differentiating between SAD and SLE patients, we used a ROC (receiver operating characteristic) curve considering the titres found in 73 SLE patients and 261 patients with rheumatic diseases. Antibody determinations were performed by one of the authors (JC), who was blinded to the clinical information. The study was approved by the Institutional Committee of Biomedical Research at the INCMNSZ.
Statistical analysis
To determine the diagnostic utility of anti-chromatin antibodies, we used the sensitivity, specificity, positive predictive value, negative predictive value and likelihood ratio. Continuous variables are expressed as mean ± S.D. Pairs of groups were compared using Student's t test for normally distributed continuous variables or the MannWhitney U test for those without normal distribution. The
2 test or Fisher's exact test was used for categorical variables as needed. For comparisons among three or more groups, an ANOVA (analysis of variance) test or its equivalent non-parametric test was used. As a secondary analysis, the correlation coefficient (Spearman) between anti-chromatin antibodies and disease activity was determined, using a modified SLEDAI in which anti-dsDNA antibodies and complement protein values were excluded to avoid overestimation of the coefficient. Statistical significance was set at P
0.05, two-tailed.
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Results
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We included 73 SLE patients [62 females (85%)] with age 25.6 ± 8.8 yr (range 1561, median 24) and SLE duration 5.5 ± 2.9 months (range 212, median 5). The SLEDAI score at clinical evaluation was 5.3 ± 5.9 (range 025, median 4). The most frequent clinical manifestations, defined according to ACR criteria, are summarized in Table 1.
Anti-chromatin antibodies
The prevalence of anti-NCS, anti-dsDNA and anti-HST antibodies in SLE patients was 100, 63 and 15% respectively; whereas in healthy controls it was 3, 5 and 3% for anti-NCS, anti-ds DNA and anti-HST antibodies respectively. Titres of anti-chromatin antibodies in SLE patients were 244 ± 88 U/ml (range 57481 U/ml) for anti-NCS, 335 ± 425 U/ml (range 01540 U/ml) for anti-dsDNA and 48 ± 58 U/ml (range 0237 U/ml) for anti-HST. Comparing SLE patients with healthy controls, anti-NCS, anti-dsDNA and anti-HST antibodies had a sensitivity of 100, 63 and 15% respectively, specificity of 97, 95 and 97%, positive predictive value of 95, 90 and 73%, negative predictive value of 100, 93 and 67%, and likelihood ratio values of
, 12.6 [95% confidence interval (CI), 5.524.5] and 5.0 (95% CI, 2.614.8) for the diagnosis of SLE (Table 2).
Utility of anti-chromatin antibodies in differentiating SAD from SLE
Anti-chromatin antibodies were highly prevalent in MCTD patients, whereas in the other SAD studied they were found in <7% of patients (Table 3). Comparing SLE patients and patients with SAD (excluding MCTD), the anti-NCS, anti-dsDNA and anti-HST antibodies had a sensitivity of 93, 71 and 40% respectively and specificity of 97, 98 and 98% for SLE diagnosis. When MCTD patients were included in the analysis, the sensitivity was identical, but the specificity decreased to 85, 92 and 96% for anti-NCS, anti-dsDNA and anti-HST antibodies respectively.
Among MCTD patients, the prevalence of anti-NCS antibodies was 70% (Table 3). The sensitivity and specificity of anti-NCS antibodies for identifying SLE from MCTD patients was 93 and 30% respectively. Therefore, anti-NCS antibodies were not useful for this task.
Correlation between anti-chromatin antibodies and SLE activity
All anti-chromatin antibodies correlated with SLE activity (Fig. 1). The correlation coefficient was stronger for anti-NCS antibodies (r = 0.45, P < 0.0001) than for anti-HST (r = 0.28, P = 0.01) and anti-dsDNA antibodies (r = 0.25, P = 0.03).

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FIG. 1. (a) The correlation coefficient of the anti-HST antibodies was 0.28, P=0.01, (b) anti-dsDNA 0.25, P=0.03 and (c) the anti-NCS 0.45, P<0.0001.
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To determine the utility of anti-NCS antibodies as a marker of disease activity in patients negative for anti-dsDNA antibodies, we analysed a subgroup of 27 patients who tested negative for anti-dsDNA antibodies. We found that the correlation of anti-NCS with disease activity was stronger in this subgroup than in the whole group of SLE patients (r = 0.58, P = 0.001) (Fig. 2).

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FIG. 2. The correlation coefficient between anti-NCS antibodies and disease activity, in anti-dsDNA negative SLE patients was 0.58 (P=0.001).
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Association between anti-NCS antibodies and SLE manifestations
To establish the association between anti-chromatin antibodies and SLE manifestations, we converted the antibody titres to a dichotomous variable, defining high and low titres as values above and below the median respectively. High titres of anti-NCS antibodies were associated with proteinuria [odds ratio (OR) 4.1, 95% CI 1.213.6, P = 0.01], haematuria (OR 4.2, 95% CI 1.115.4, P = 0.03), malar rash (OR 4.7, 95% CI 1.119.1, P = 0.03), arthritis (OR 6.5, 95% CI 1.820.6, P = 0.003) and oral ulcers (OR 10.0, 95% CI 2.442.5, P = 0.001). High titres of anti-dsDNA antibodies were associated with oral ulcers (OR 5.4, 95% CI 1.127.3, P = 0.02) and high titres of anti-HST antibodies were associated with pleuritis (OR 2.1, 95% CI 1.62.7, P = 0.05) (Table 4).
Global effect of anti-chromatin antibodies on SLE activity
In order to evaluate the impact of anti-chromatin antibodies on SLE activity, we studied a multiple linear regression model using disease activity as the dependent variable and the titres of anti-NCS, anti-HST and anti-dsDNA antibodies as independent variables. Considered together, anti-chromatin antibodies explained almost 40% of SLE activity (r2 = 0.364, P < 0.001) (Fig. 3).

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FIG. 3. The global impact of all anti-chromatin antibodies on SLE activity is shown in this multivariate model. Altogether anti-chromatin antibodies explained 36% of the disease activity.
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Discussion
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SAD, including SLE, share several clinical and biological features that make their differential diagnosis difficult, especially in the early stages. Traditional tests used in the diagnosis of SLE, such as antinuclear antibodies, have low specificity because they identify antibodies against nuclear antigens generically [4]. Because the nucleosomes have been considered the principal antigen in the pathophysiology of SLE [5, 6, 2123], we investigated the prevalence of anti-NCS antibodies in SLE patients with recent onset.
In this study, anti-NCS antibodies were detected in all SLE patients with recent onset. They showed a high correlation with disease activity, especially among SLE patients who tested negative for anti-dsDNA antibodies, and were strongly associated with renal damage. All the SLE patients we studied had anti-NCS antibodies, whereas anti-dsDNA antibodies were present in only 63%. Previous reports [8, 9], in which SLE patients with longer disease duration (mean 8 yr) were studied, did not show a higher prevalence of anti-NCS antibodies than anti-dsDNA antibodies. We cannot explain the discrepancy in the results between these previous studies and our study. Whether these differences depend on the effect of disease activity or SLE treatment on the antibody titres needs to be determined.
The high sensitivity and specificity of anti-NCS antibodies for SLE diagnosis in our study is consistent with findings in murine models of lupus, in which it has been demonstrated that the development of anti-NCS antibodies occurs before other anti-chromatin antibodies [10]. Our results show that the prevalence of anti-NCS antibodies is higher than that of anti-dsDNA antibodies in the initial stages. This is similar to what has been reported in murine models of lupus [10].
Considered together, anti-chromatin antibodies explained close to 40% of SLE activity. Anti-NCS antibodies themselves explained almost 35% of disease activity. These results suggest that anti-NCS antibodies, besides being potentially useful in SLE diagnosis, could be a biological marker of disease activity.
The presence of anti-NCS antibodies in all SLE patients who were anti-dsDNA-negative and the stronger correlation with disease activity needs to be highlighted, because in this group there is no reliable diagnostic test and no marker of disease activity.
In our study, only anti-NCS antibodies were associated with renal damage, which suggests a possible role in the pathogenesis of lupus nephritis [24, 25]. A positive association of anti-NCS antibodies with renal damage has been demonstrated previously, both in murine models of lupus and in SLE [5, 6, 24, 25]. This association seems to depend on a complex interaction between charges associated with the quaternary structure of the nucleosomes and epitope targets in renal tissue. That is, the histones that constitute part of the nucleosomes have a cationic charge, whereas the glomerular basement membrane has an anionic charge, which permits an interaction between them.
As also reported by Amoura et al. [8] and Bruns et al. [9], in our study the presence of anti-NCS antibodies was restricted to patients with SLE and MCTD diagnoses. This suggests that anti-NCS antibodies could be useful not only in differentiating individuals with SLE from healthy individuals, but also in differentiating SLE from other forms of SAD. However, the high prevalence of anti-NCS antibodies in MCTD restricted their utility in the differential diagnosis of SLE. Nevertheless, we cannot definitively rule out their usefulness in differentiating SLE and MCTD patients at early stages of disease evolution, because the MCTD patients studied did not have disease of recent onset. However, this result is interesting in view of the fact that MCTD and SLE share many clinical and biological characteristics.
We must acknowledge some limitations of this study. We did not test cross-reaction among anti-chromatin antibodies. However, in two previous reports it was demonstrated that this cross-reaction did not affect the interpretation of the test [8, 9]. Furthermore, we studied a group of SLE patients who were positive only for anti-NCS antibodies and negative for other anti-chromatin antibodies, which is clear evidence of the existence of a group of antibodies that recognize only the quaternary structure of the nucleosomes [26].
Although a strong correlation was found between anti-NCS antibodies and SLEDAI scores, given the cross-sectional nature of the study we were not able to determine whether the titres of anti-NCS antibodies are sensitive to change with disease activity over time.
Despite these limitations, we may conclude that anti-NCS antibodies could be a useful tool for the diagnosis of SLE, especially at early stages of the disease, and a useful marker of disease activity, particularly in patients who are negative for anti-dsDNA antibodies.
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Acknowledgments
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The authors thank Drs Zahir Amoura, Jean-Charles Piette and Henry Chabre for sharing their expertise in the field of nucleosomes and anti-nucleosome antibodies; and Dr Luis Felipe Flores-Suárez for his collaboration regarding the sera of patients with primary vasculitis. This study was supported by grant 34750-N from Consejo Nacional de Ciencia y Tecnología.
The authors have declared no conflicts of interest.
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Submitted 10 June 2003;
Accepted 31 July 2003