Association of biological markers of activity of systemic lupus erythematosus with levels of anti-oxidized low-density lipoprotein antibodies
J. M. Gómez-Zumaquero,
F. J. Tinahones,
E. De Ramón1,
M. Camps1,
L. Garrido and
F. J. Soriguer
Endocrinology and Nutrition Service, 1Systemic Autoimmune Diseases Unit, Internal Medicine Service, Carlos Haya University Hospital, Malaga, Spain.
Correspondence to J. M. Gómez-Zumaquero. E-mail: jmgz{at}ole.com
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Abstract
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Objectives. To study the levels of anti-oxidized low-density lipoprotein (LDL) antibodies in patients with systemic lupus erythematosus (SLE) at two different points during the disease, and evaluate their relation with markers of SLE activity in serial blood samples. To investigate the correlations at two points in time between anti-oxidized LDL antibodies and anti-ß2-glycoprotein-I antibodies, leucocytes, immunoglobulin G, anti-deoxyribonucleic acid, complement 3, complement 4 and the disease activity index.
Methods. A total of 49 patients with SLE according to ACR criteria were studied at two points, 3 to 4 months apart, Time 1 and Time 2.
Results. There were ostensible changes in levels of anti-oxidized LDL antibodies between Times 1 and 2, which correlated significantly with disease activity markers. The association between levels of anti-oxidized LDL antibodies and complement system activation remained after multiple regression analysis with stepwise adjustment.
Conclusions. Antibody levels against oxidized LDL vary with time and are closely related to the degree of SLE activity. There is an association between levels of autoantibodies to oxidized LDL and complement system activation.
KEY WORDS: Systemic lupus erythematosus, Oxidized LDL antibodies, Activity markers.
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Introduction
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Several studies have related the presence of anti-low-density lipoprotein (LDL) antibodies in humans with atherosclerosis, so that they are predictors of progression of atherosclerotic lesions in the carotid artery [1] and the development of cerebral infarction [2]. The clinical value of the measurement of these autoantibodies is at present under debate, since there is no association in diabetic patients with microvascular involvement [3] or between levels of anti-oxidized LDL antibodies and cholesterol [4], or with the degree of oxidation in serum [5].
A greater presence of anti-oxidized LDL antibodies has been found in patients with systemic lupus erythematosus (SLE) [6], there being a close relation between the presence of anti-cardiolipin (aCL) antibodies (Ig isotype) and anti-oxidized LDL antibodies [7]. However, the importance of anti-oxidized LDL antibodies in patients with SLE remains controversial. Some authors have found a close relation with arterial thrombosis [8, 9], whereas others have failed to find this association [10]. The variation of these anti-oxidized LDL antibodies during the course of the disease is unknown, as is their relation with known biological markers of SLE disease activity. We have attempted to determine the possible variation in levels of anti-oxidized LDL antibodies over time and their association with other biological indicators of SLE activity.
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Material and methods
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Patients
A total of 49 patients with SLE according to ACR criteria were studied at two points in time, 3 to 4 months apart, and referred to as Time 1 and Time 2.
The SLE activity was measured with the Lupus Activity Index (LAI), a valid and reliable index of disease activity which considers the physician's overall assessment, clinical and analytical variables, and treatment aspects during the previous 2 weeks providing a final score from 0 to 3. It has been shown to be sensitive to changes in disease activity over time [11]. A blood test was taken, and levels of complement C3 and C4 were determined by nephelometry (Beckman: kit-100, ref. 449530), levels of immunoglobulin G (IgG) by nephelometry (Beckman: kit-100, ref. 449400), and anti-native deoxyribonucleic acid (DNA) antibodies by Crithidia luciliae assay.
LDL isolation
LDL was isolated from pooled plasma of healthy fasting human donors by density gradient ultracentrifugation. The LDL was then dialysed against phosphate-buffered saline (PBS) (4°C for 30 h) (0.14 M NaCl/0.01 M phosphate buffer).
LDL oxidation
Oxidized LDL was prepared by incubating the LDL for 3 h at 37°C with 0.5 M malonyldialdehyde (MDA) at a constant ratio of 100 ml/mg of LDL. The reaction was stopped by adjusting the pH to 7.4 with 1 M NaOH. After conjugation, MDALDL was extensively dialysed against PBS.
Anti-oxidized LDL antibodies
Microtitre plates for determination of anti-oxidized LDL antibodies were coated with either native or MDALDL, both at 10 mg/ml in PBS. The plates were incubated for 2 h at 37°C and overnight at 4°C. After washing four times with PBS, plates were blocked with 1% bovine serum albumin (BSA)/PBS for 2 h at room temperature. Serum samples were diluted 1:100 in 1% BSA/PBS and incubated for 3 h at room temperature. After washing, an alkaline phosphatase-conjugated anti-human IgG (Sigma Immuno Chemicals) was diluted 1:1000 in 1% BSA/PBS and added. It was then left for 3 h at room temperature. 1 mg/ml p-nitrophenyl phosphate (Sigma) in 500 mM carbonate buffer containing 1 mM MgCl2 (pH 9.8) was used as substrate. The reaction was stopped with 1 M NaOH after 60 min. The absorbance was read at 405 nm. Binding of antibodies to oxidized LDL was calculated by subtracting the binding of native LDL from binding to MDALDL. Optic density values above the mean plus 2 S.D. of the values of a group of 65 healthy young persons aged 2024 yr were considered positive.
Statistical analysis
The hypothesis contrast between the means of the continuous variables was analysed by the MannWhitney test. The tendency between variables was measured by Spearman's correlation coefficient. In all cases the rejection level for a null hypothesis was an
= 0.05 for two tails. A multiple regression test was made, considering the levels of C4 and C3 as dependent variables, with the independent variables consisting of levels of anti-oxidized LDL antibodies, IgG aCL antibodies, ß2-glycoprotein-I (ß2-GPI) antibodies and anti-native DNA antibodies, total IgG and LAI score.
The study was authorized by the Hospital Ethics Committee.
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Results
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At Time 1, 38 patients were positive for anti-oxidized LDL antibodies and 11 negative, and at Time 2, 31 patients were positive and 18 negative. Of the 38 who were initially positive at Time 1, 27 remained positive at Time 2. Of the 11 patients who were negative at Time 1, 7 remained negative. Thus, the antibody titres of 15 patients changed in a period of less than 6 months.
Those patients with positive levels of anti-oxidized LDL antibodies at both time points had significantly higher mean values of anti-native DNA, IgG and disease activity index and significantly lower levels of C4 (Table 1).
The levels of anti-oxidized LDL antibodies correlated positively with the levels of IgG, anti-native DNA antibodies and the disease activity index and negatively with the levels of C3 and C4 at one or other of the two time points (Tables 2 and 3 and Fig. 1).
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TABLE 2. Spearman correlation coefficients between the levels of anti-oxidized LDL antibodies (anti-LDLox) and the analytical variables at Time 1
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TABLE 3. Spearman correlation coefficients between the levels of anti-oxidized LDL antibodies (anti-LDLox) and the analytical variables at Time 2
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FIG. 1. Scatter plots of C4 and IgG versus anti-oxidized LDL antibodies at Times 1 and 2, with the coefficients of regression.
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Multiple regression analysis showed that the complement C3 and C4 levels were directly related with the level of anti-oxidized LDL antibodies at both Times 1 and 2, though there was no correlation between C3 and anti-oxidized LDL at Time 1. At Time 2 the disease activity index and levels of anti-oxidized LDL antibodies accounted for 45% of the variability of the levels of C3 (Table 4), with levels of IgG antibodies, anti-native DNA antibodies and anti-oxidized LDL antibodies accounting for 52% of the variability of the levels of C4 (Table 5).
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TABLE 4. Multiple linear regression analysis. Dependent variable: C3 levels. Independent variables: disease activity index, anti-oxidized LDL antibodies, IgG aCL antibodies, anti-native DNA antibodies, IgG, ß2-glycoprotein-I antibodies. The analysis was carried out at both Times 1 and 2
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TABLE 5. Multiple linear regression analysis. Dependent variable: C4 levels. Independent variables: disease activity index, anti-oxidized LDL antibodies, IgG aCL antibodies, anti-native DNA antibodies, IgG, ß2-glycoprotein-I antibodies. The analysis was carried out at both Times 1 and 2
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Discussion
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Our results show that 30% of the patients with SLE have marked changes in titres of anti-oxidized LDL antibodies, and that this variability is related to the analytical variables related to disease activity. As far as we are aware, no studies have yet been undertaken demonstrating these associations between SLE activity and anti-oxidized LDL antibodies. The close relation between the levels of these antibodies and the levels of total IgG, together with the great individual variability, suggest that the high levels of anti-oxidized LDL antibodies reflect the autoimmune activity of the disease.
Oxidatively modified LDLs have been implicated in the pathogenesis of atherosclerosis and are found in human vascular lesions. Furthermore, there is increasing evidence that complement activation may also play a role in atherogenesis. For some years it has been speculated that the complement system may be related to LDL uptake by macrophages [12]. It has also been pointed out that oxidized LDL could be an agent activating the complement system in atherosclerotic lesions [13]. Others, however, have found that oxidation does not confer relevant complement-activating properties on LDL, suggesting that the lesion complement activator is not directly related to oxidized LDL [14].
Another factor involved in the relation between LDL and the complement system during atherosclerosis is the presence of antibodies to modified LDL. Since the first report of anti-oxidized LDL antibodies, it has been known that they contained immunoglobulins of the three major isotypes, with a predominance of IgG subclasses 1 and 3. These IgG isotypes are known to interact with Fc receptor (FcR) gamma-I and to activate the complement system, and are thus potentially able to activate macrophages and cause foam cell formation [15]. Furthermore, it has been shown that monoclonal antibodies directed against oxidized LDL, including epitopes for the MDAlysin adduct, bind to apoptotic cells. This inhibits phagocytosis of these cells by macrophages [16].
Our study reports for the first time in a clinical model an inverse relation between levels of anti-oxidized LDL antibodies and complement, so that these antibodies may be responsible for activation of the complement system. Several questions, however, still remain; some consider that it is not the oxidized LDL which is related to activation of the complement system, but rather the involvement of enzymatically modified LDL [17, 18]. Moreover, there are numerous forms of modified LDL which may have an immunogenic capacity, and it has recently been shown there is no cross-reaction between anti-oxidized LDL antibodies and glycosylated LDL [19].
Our study shows that the levels of anti-oxidized LDL antibodies in a clinical model of an autoimmune disease associated with atherosclerosis vary, and that they are related to disease activity. We have shown for the first time in a clinical model a direct relation between activation of the complement system and levels of anti-oxidized LDL antibodies. Further studies to examine the importance of continuous monitoring of anti-oxidized LDL antibodies in these patients and their relation with clinical symptoms may prove interesting.
The authors have declared no conflicts of interest.
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Acknowledgments
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J. M. Gómez-Zumaquero is the holder of a grant from the FIS (96/5327). The authors are grateful to Ian Johnstone for help with the English language editing of the manuscript.
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Submitted 23 December 2002;
Accepted 12 November 2003