Systemic lupus erythematosus (SLE) is an autoimmune systemic disease, and 90% of the cases of SLE occur in women. These patients are at high risk of cardiovascular disease, which often affects women with SLE before menopause (1, 2), the time of life at which women normally are protected from coronary heart disease (3). Premature atherosclerosis was recently demonstrated in patients with SLE (4, 5). In epidemiologic studies, women ages 44–50 years had a 50-fold increased risk of myocardial infarction as compared with controls from the Framingham study (1), and the relative risk for coronary heart disease was 7.5, after adjusting for Framingham risk factors (6). We recently identified both traditional and nontraditional risk factors for cardiovascular disease among female patients with SLE. Briefly, these were markers of inflammation (raised levels of acute-phase reactants and tumor necrosis factor α), dyslipidemia, enhanced low-density lipoprotein (LDL) oxidation, antiphospholipid antibodies (aPL; lupus anticoagulants and antibodies to oxidized LDL [anti-OxLDL]), and high levels of homocysteine (2, 7, 8).
Enhanced titers of antibody to anionic phospholipids, including cardiolipin and lupus anticoagulant, are features of the antiphospholipid syndrome (APS), which is characterized by fetal loss, autoimmune thrombocytopenia, and thrombosis (9). Among patients with SLE, 30–50% have aPL; APS develops in approximately one-third to one-half of these patients (10) and is often referred to as secondary APS. Although this is not included in the definition of APS, some anti-OxLDL can also be regarded as aPL that cross-react with aCL (11, 12).
In APS and SLE, a significant fraction of aCL recognize oxidized phospholipids (OxPL) (12, 13). Furthermore, enhanced lipid peroxidation in patients with APS, as determined by secretion of oxidation products of arachidonic acid in urine, was recently reported (14). An enhanced monocyte expression of tissue factor by oxidative stress in patients with APS, with antioxidant therapy providing beneficial effects, was also reported (15). These findings indicate that lipid peroxidation may be of importance in APS.
According to the modified LDL hypothesis, oxidation of LDL is an important factor in atherogenesis (16). As a result of oxidation, a variety of immunogenic neoepitopes are formed on OxLDL. For example, oxidation of the phosphorylcholine (PC)–containing phospholipids renders them antigenic, and, in fact, such OxPL form ligands on OxLDL recognized by macrophage scavenger receptors, leading to enhanced uptake of OxLDL by macrophages and foam cell formation (17–19). Through this route, macrophages become lipid laden and develop into the characteristic foam cells of the atherosclerotic lesions (18, 20–22). OxLDL is also chemotactic, immune stimulatory, and has toxic properties that promote local inflammatory processes in atherosclerotic lesions (23, 24). Furthermore, OxLDL elicits a humoral immune response, with production of autoantibodies to oxidation-specific epitopes of OxLDL (anti-OxLDL).
EO6 is an IgM monoclonal antibody cloned from apolipoprotein E (Apo E)–deficient mice that was subsequently shown to be equivalent to the classic T15 naturally occurring antibody that specifically binds to the PC moiety of the cell wall polysaccharide of pathogens such as Streptococcus pneumoniae (25). This antibody confers optimal protection to mice against lethal infection with streptococci. EO6 was subsequently shown to recognize the PC headgroup of OxPL, but not the same moiety on native, unoxidized phospholipids. Thus, EO6 binds to OxPL on OxLDL and on apoptotic cells and via this mechanism blocks the uptake of OxLDL and apoptotic cells by macrophage scavenger receptors such as CD36 (19). Therefore, the epitope recognized by EO6 is a key ligand for scavenger receptors (18). In this study, we used EO6 to detect enhanced numbers of OxPL epitopes on Apo B-100 particles (e.g., OxPL/Apo B, a measure of OxLDL that we term OxLDL-EO6) in a large cohort of unselected female patients with SLE. These epitopes were associated with arterial and renal disease manifestations. Furthermore, high-titer aCL were shown to bind primarily to oxidized forms of cardiolipin, further supporting a role for enhanced lipid peroxidation in these patients. The implications of these findings are discussed below.
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- PATIENTS AND METHODS
In this study, we demonstrated that female patients with SLE had more OxLDL-EO6 (i.e., Apo B lipoproteins containing OxPL recognized by monoclonal antibody EO6) compared with controls (19). Arterial and renal manifestations of lupus were associated with higher levels of these oxidized epitopes on LDL.
The epitope recognized by EO6 is the PC headgroup of OxPL, which is present on both OxLDL and minimally modified LDL (e.g., LDL that has undergone early oxidative changes of lipids but, in contrast to OxLDL, changes that are insufficient to cause scavenger receptor recognition) (33). Minimally modified LDL is readily deposited in the artery wall and is of importance for endothelial activation and recruitment of monocytes to the intima. These monocyte–endothelial interactions are inhibited by a platelet-activating factor (PAF) receptor antagonist (34). Recently, it was shown that the same PC epitope is also present on apoptotic cells and pneumococci (18), and that PC is a target not only for scavenger receptors and EO6 antibodies but also for C-reactive protein (35). Thus, the immune response to PC/OxLDL is part of a greater immune network that may have evolved to provide protection against both external invaders such as bacteria and internal debris from dying cells (18).
Our study provides evidence to support the hypothesis that there is enhanced lipid oxidation in SLE. Iuliano et al also showed that patients with SLE had enhanced urinary excretion of isoprostanes, consistent with enhanced lipid peroxidation (14). In keeping with this hypothesis is a recent study that demonstrated depressed activity of the antioxidant enzyme paraoxonase in the circulation of patients with SLE (36). These findings are also supported by studies in hamsters, in which both infection and inflammation induced LDL oxidation as expressed by another LDL epitope, lysophosphatidylcholine (37). We have previously shown that patients with SLE have antibodies to lysophosphatidylcholine (38).
Another finding reported here is that an enhanced content of OxLDL-EO6 (OxPL/Apo B) was associated with arterial disease. The present findings confirm and extend our earlier results, indicating that OxLDL is an important factor contributing to the very high risk of cardiovascular disease in patients with SLE (2).
LDL cholesterol is also a well-known risk factor for cardiovascular disease, and in this study LDL levels were associated with arterial disease. Because OxLDL-EO6, as detected here, is a measure of OxPL/Apo B, it is independent of LDL particle number. Our measure of OxLDL thus represents a risk factor that is independent of LDL levels alone. High levels of LDL and enhanced LDL oxidation may thus be regarded as separate risk factors for cardiovascular disease in patients with SLE. In recent studies, Lp(a) has been demonstrated to be closely related to the EO6 measurement of OxLDL (39). Consistent with this are previous reports indicating that the level of Lp(a) is elevated in SLE (40).
OxLDL was also associated with renal disease in SLE. It is well recognized that renal disease and renal failure are strong risk factors for cardiovascular disease. OxLDL (as determined by another technique) has also been implicated in renal disease in the general population (41), and OxLDL was demonstrated in sclerotic and mesangial regions of biopsy specimens obtained from patients with chronic renal disease (42). Furthermore, expression of macrophage scavenger receptors can be induced in human mesangial cells (43). Whether enhanced LDL oxidation has a role in the development of renal manifestations in SLE or occurs secondary to nephritis cannot be determined from this study.
It is not known where the epitopes recognized by EO6 are generated. In the circulation there are strong antioxidant defenses, and in animal experiments OxLDL is rapidly taken up by the liver (44). It is therefore highly unlikely that any significant degree of oxidation of LDL occurs within the circulation. In general, LDL is believed to become oxidized after entering the intima, where it is retained by a network of proteoglycans, which further facilitates oxidative and enzymatic modifications (45). LDL may also be oxidized in other tissues, including the liver (46). Previous studies have indicated that the oxidation epitopes demonstrated here are more likely to be derived from oxidation of phospholipids in tissues. In turn, this suggests that the oxidized phospholipids found on plasma Apo B-100 particles reflect lipid peroxidation that occurs in atherosclerotic lesions or other sites of inflammation.
We can also confirm earlier observations that autoantibodies related to epitopes of OxLDL and oxidized cardiolipin are enhanced in SLE (11, 38, 47). In this study, arterial and renal disease manifestations were weakly associated with anti-OxLDL of the IgM subclass. We previously observed an association between IgG anti-OxLDL antibodies and arterial disease in a smaller cohort of older patients with SLE (2). In 2 other studies, anti-OxLDL and arterial disease were not associated (48, 49). These differences between studies may be related to both the selection of patients and the methods. Although in this study anti-OxLDL titers were generally increased, the elevated titers were not associated with the disease manifestations studied.
As reported previously, in patients with APS, many aCL recognize OxPL (12, 13), and in the present report we have demonstrated that in SLE plasma with high levels of aCL, antibody binding to cardiolipin was out-competed by oxidized cardiolipin but not by reduced cardiolipin. This clearly shows that many aCL in plasma with high titers of OxPL from patients with SLE recognize oxidized cardiolipin. Because aPL are pathogenic in animal studies (50) and also predispose to arterial and venous thromboses (51, 52), the possibility that potent antioxidant therapy may be valuable in this patient group should be considered (14). Furthermore, the epitope recognized by EO6 promotes monocyte–endothelial interactions, which can be inhibited by PAF antagonists. This is consistent with a recent study in which PAF antagonists ameliorated atherosclerosis in experimental animals (53). Interference with PAF-related inflammatory effects may thus also be of therapeutic potential in these patients.
In conclusion, enhanced lipid peroxidation may play an important role in SLE, especially with respect to the premature atherosclerotic process observed among these patients. If further evidence can be provided to support this hypothesis, potent antioxidant therapy may be of value for patients with SLE.