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Systemic lupus erythematosus (SLE)—the prototype of immune complex diseases—is characterised by disturbances of the cellular and humoral immune systems. Antibodies against nuclear components are the hallmark of SLE, but a number of antibodies against proteins, glycoproteins, and carbohydrates have also been reported. Dyslipoproteinaemia and accelerated atherosclerosis that commonly lead to coronary artery disease and other complications are recognised sequelae.1-3 The role of autoimmunity in the aetiology of atherosclerosis has recently been highlighted.1-3Increased levels of antibodies against oxidised low density lipoprotein, lysophosphatidylcholine, and apolipoprotein A1 were found in patients with SLE.4 ,5 As reported earlier, we have shown marked alterations of anticholesterol antibody (ACHA) levels in patients with various atherosclerotic vascular disorders.6In this study ACHA levels of patients with SLE were compared with those of healthy donors.
Sixty eight patients (64 women, four men), aged 39.4 (10.6) (mean (SD)) years, who fulfilled at least four of the diagnostic criteria established by the American Rheumatism Association for SLE7 and 60 healthy donors (55 women, five men), aged 42.6 (8.12), were enrolled into the study. The SLEDAI (SLE Disease Activity Index) score was used to measure disease activity.8Patients were considered to have active lupus if they scored at least 2 on the modified SLEDAI scale (calculated by omitting anti-dsDNA and complement from SLEDAI) and prompt treatment was obviously indicated to control their symptoms.
The level of cholesterol-specific antibodies was measured by a solid phase enzyme immunoassay described earlier.6 Polystyrene plates (Greiner, Frickenhausen, Germany) were coated with 5 μg/well cholesterol dissolved in 100 μl absolute ethanol and incubated at 4°C for 24 hours. After washing with phosphate buffered saline (PBS) and blocking with 0.1% casein (Reanal, Budapest, Hungary) in PBS, the wells were incubated with 100 μl samples of serum diluted 1:800 in PBS containing 0.1% casein. The binding of ACHA was detected by antihuman horseradish peroxidase conjugated γ-chain-specific rabbit antibodies (DAKO, Glostrup, Denmark); and witho-phenylenediamine (Sigma, St Louis, USA) using H2O2 as substrate. Optical density was measured at 492 nm (reference at 620 nm), and the mean of duplicates was calculated. Serial dilutions of purified immunoglobulin were used as standards in all experiments. Data obtained as optical density values were expressed in arbitrary units per millilitre (AU/ml), related to the standard curve. Our previous observations,6in accordance with those of others,9 demonstrated the specificity of ACHA to cholesterol. The inter- and intra-assay variations of this method were 18.7% and 9.5%, respectively.
Differences between the parameters measured in controls and patients with SLE, between patients with active and inactive SLE, and those between patients with and without previous vascular events were calculated with the Mann-Whitney-test. The χ2 test was used to estimate the discriminative power of ACHA between patients with SLE and healthy donors. The correlation of individual parameters with each other and with the SLEDAI score was calculated using Spearman's rank correlation test.
Twelve samples (18%) were classified as having been obtained from patients with active SLE and 56/68 (82%) from patients with inactive disease. Three of the 12 “active” samples were from patients with SLE active in more than one organ system and nine were from patients with disease activity in one organ system only (musculoskeletal (four), central nervous system (two), renal (two), and cutaneous (one)).
The difference between ACHA levels measured in the control group and in patients with SLE was significant (p<0.001) (fig 1). High ACHA levels, in the upper quartile occurred in 29/68 (43%) patients with SLE and 13/60 (22%) controls (p=0.012). ACHA levels (mean (SD)) of patients with active (97 (81) AU/ml) or inactive (64 (38) AU/ml) SLE did not differ significantly (p=0.21) and did not correlate with the SLEDAI score (r s=0.22, p=0.066). No correlation was found between the ACHA level and other parameters (such as anti-dsDNA, CH50, C3, and C4, data not shown). No significant differences were found between patients treated with corticosteroids for inactive disease or those with inactive disease and not receiving corticosteroid treatment (p=0.174). The latter group had not received corticosteroids for at least one year before blood samples were obtained. The difference between the ACHA levels of patients who had or had not experienced vascular events was not significant.
The observed increases of ACHA levels may be related to underlying chronic inflammatory disease. A number of conditions such as dyslipoproteinaemia,1-3 nephrotic syndrome, and changes in cholesterol membrane domains10 might have elicited the increase of ACHA levels. Corticosteroid treatment might also have stimulated ACHA production, though according to our observations this intervention does not alter ACHA levels significantly. A preventive role against atherosclerosis in patients with SLE has been attributed to raised ACHA levels, though these may also exert an atherogenic effect.6 Further studies are necessary to clarify the role of ACHA in the changes of lipid metabolism ascertained in patients with SLE.
This work was supported by grants OTKA No T 029044 (IK) and ETT No 239/2000 (LR).
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