Autoantibodies Against Oxidized Low-Density Lipoprotein in ...

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SUMMARY. The prevalence and clinical significance of anti-oxidized low-density lipoprotein antibodies (anti-ox-LDL) were evaluated in patients with the ...
British Journal of Rheumatology 1997;36:964±968

AUTOANTIBODIES AGAINST OXIDIZED LOW-DENSITY LIPOPROTEIN IN ANTIPHOSPHOLIPID SYNDROME O. AMENGUAL, T. ATSUMI, M. A. KHAMASHTA, F. TINAHONES* and G. R. V. HUGHES Lupus/Arthritis Research Unit, The Rayne Institute, St Thomas' Hospital, London and *Endocrinology Department, Hospital Regional Carlos Haya, MaÂlaga, Spain SUMMARY The prevalence and clinical signi®cance of anti-oxidized low-density lipoprotein antibodies (anti-ox-LDL) were evaluated in patients with the antiphospholipid syndrome (APS). Anti-ox-LDL were measured in the sera of 107 patients with APS (64 primary APS, 43 secondary to systemic lupus erythematosus) by enzyme-linked immunosorbent assay (ELISA) utilizing malondialdehyde (MDA)-modi®ed LDL as antigen. In the same patients, anticardiolipin antibodies (aCL) and anti-b2-glycoprotein I antibodies (anti-b2GPI) were also measured. A positive titre of anti-ox-LDL was detected in 22% of patients, but only in 6% of control subjects (w2 = 12, P = 0.0005). Levels of anti-ox-LDL were higher in patients with arterial thrombosis (n = 58) than in those without (n = 49) (P = 0.0001). Anti-ox-LDL levels correlated weakly with those of aCL (r = 0.196, P = 0.043), but not with those of anti-b2GPI (r = 0.076). Our ®ndings suggest that elevated levels of anti-ox-LDL may represent another potential marker of APS, particularly of patients prone to arterial thrombosis. KEY WORDS: Anticardiolipin antibodies, Anti-b2-glycoprotein I antibodies, Atherosclerosis, Thrombosis, Systemic lupus erythematosus.

(aCL) [12]. The same study suggested cross-reactivity between aCL and anti-ox-LDL. Since aCL is one of the antiphospholipid antibodies associated with the antiphospholipid syndrome (APS), a thrombophilic disorder characterized by arterial and venous thrombosis, recurrent fetal losses and thrombocytopenia [13], we investigated the prevalence and clinical signi®cance of anti-ox-LDL in APS.

LOW-DENSITY lipoprotein (LDL) is a hydrophilic complex of lipids and apoliprotein B100, and represents one of the major cholesterol-carrier lipoproteins in plasma. Epidemiological studies have established that an elevated plasma level of LDL represents one of the most important risk factors for the development of atherosclerosis [1]. In vitro studies have shown that LDL can undergo several chemical modi®cations, such as acetylation and oxidation [2]. The latter process is of great interest because oxidation of LDL may occur in vivo [3±5] and may contribute to the development of atherosclerosis, as suggested by the presence of oxidized-LDL (ox-LDL) particles in the early phase of atherosclerotic plaque formation [6, 7]. Structural changes of LDL may enhance LDL uptake by macrophage scavenger receptors, promoting the transformation of macrophages into foam cells [8], and may favour the recruitment and migration of monocytes and leucocytes within arterial vessels [9]. On the other hand, oxidatively modi®ed LDL is more immunogenic than its native counterpart, eliciting speci®c anti-ox-LDL antibodies (anti-ox-LDL) [2]. The latter have been detected in human sera from a variety of in¯ammatory conditions. Initially reported in patients with chronic periaortitis [10], they were subsequently detected in subjects with carotid atherosclerosis where they represented a marker of progressive disease [11], and they were also found in 80% of patients with systemic lupus erythematosus (SLE) with and without anticardiolipin antibodies

PATIENTS AND METHODS Patients A total of 107 patients were included in the study [94 female and 13 male; mean age 41 yr (range 22± 66)]. Of these, 64 patients had primary APS (60%) and 43 had APS secondary to SLE (40%). Clinical features of the patients are reported in Table I. All patients ful®lled the proposed criteria for the APS [14]. One hundred and four sex- and age-matched healthy controls were also included. LDL isolation Human LDL was isolated from pooled plasma of healthy fasting adults by density gradient ultracentrifugation with BrK (Beckman L8-70 ultracentrifuge, rotor VTI 65) at 65 000 r.p.m. for 35 min, followed by a second ultracentrifugation with BrK at 49 000 r.p.m. for 18 h. The LDL layer was then dialysed for 30 h against phosphate-bu€ered saline (PBS) (0.14 M NaCl/0.01 M phosphate bu€er). Puri®ed LDL showed a single band on 1% agarose gel electrophoresis in borate bu€er. Modi®cation of LDL Malondialdehyde (MDA) was freshly generated from malonaldehyde bis dimethylacetal by acid hydrolysis as described by Palinski et al. [15]. MDA-

Submitted 31 December 1996; revised version accepted 10 March 1997. Correspondence to: M. A. Khamashta, Lupus Research Unit, The Rayne Institute, St Thomas' Hospital, London SE1 7EH.

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AMENGUAL ET AL.: ANTI-ox-LDL ANTIBODIES IN APS

TABLE I Patient characteristics

Primary APS Secondary APS to SLE aCL Lupus anticoagulant Thrombosis Arterial Venous Arterial + Venous Recurrent miscarriages Thrombocytopenia

No.

Percentage

64 43 75 78/95 91 58 55 23 38/94 22

60 40 70 82 84 54 51 21 40 21

APS, antiphospholipid syndrome; SLE, systemic lupus erythematosus; aCL, anticardiolipin antibodies

LDL was prepared by incubating LDL (100 ml/mg) for 3 h at 378C with 0.5 M MDA. The reaction was stopped by adjusting the pH to 7.4 with NaOH. After conjugation, MDA-LDL was dialysed extensively against PBS. Anti-ox-LDL ELISA Half of a microtitre plate (Immulon 4; Dynatech Laboratories Inc., VA, USA) was coated with LDL and the other half with MDA-LDL, both at 5 mg/ml in PBS containing 2 mM ethylenediaminetetra-acetic acid tetrasodium salt (EDTA) (BDH Chemicals Ltd, Poole) and 20 mM butylated hydroxytoluene (Sigma Chemical Co., St Louis, MO, USA), and incubated at 378C for 2 h and then overnight at 48C. Plates were washed four times with PBS containing 0.05% Tween 20 (Sigma) (PBS-Tween) and wells were blocked with 150 ml of 0.5% gelatin (BDH) for 1 h at 378C. After washing, 50 ml of serum diluted with PBS-Tween containing 1% bovine serum albumin (BSA) (Sigma) at 1:100 were added in duplicate. Plates were incubated for 2 h at room temperature, and washed four times. Fifty microlitres per well of the appropriate dilution of alkaline phosphatase-conjugated goat anti-human IgG (Sigma) in PBS-Tween containing 1% BSA were added and incubated for 1 h at room temperature. After four washes, 100 ml/ well of 1 mg/ml p-nitrophenylphosphate disodium (Sigma) in 1 M diethanolamine bu€er (pH 9.8) were added. Following colour development, optical density at 405 nm (OD 405) was measured by a Titertek Multiskan MC apparatus (Flow Laboratories, Herts). Results were expressed as OD, and binding to oxLDL was calculated by subtracting OD 405 obtained by LDL-coated wells from that by MDA-LDLcoated wells. A normal range was established using 104 controls with a cut-o€ of 172 OD units being 2 S.D. above the mean. Anti-b2-glycoprotein I antibodies (anti-b2GPI) ELISA Anti-b2GPI were detected by ELISA as previously described [16]. Brie¯y, irradiated microtitre plates (Sumilon Bakelite type C, Tokyo, Japan) were coated

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with 50 ml of 4 mg/ml of puri®ed human b2GPI in PBS at 48C overnight. Wells were blocked with 3% gelatin for 1 h at 378C. After three washes with PBSTween, 50 ml of serum diluted in PBS containing 1% BSA at 1:50 were added in duplicate. Plates were incubated for 1 h at room temperature, followed by alkaline phosphatase-conjugated goat anti-human IgG and substrate. Statistical analysis All statistical analysis was performed by Statview II (Apple Macintosh software). Comparisons were determined by w2 test or Mann±Whitney non-parametric test. RESULTS Positivity for anti-ox-LDL was found in 22% (24/ 107) of APS patients, but in only 6% (6/104) of controls (w2 = 12, P = 0.0005). Likewise, levels of antiox-LDL were higher in patients than in controls (P = 0.0001) (Fig. 1). In the patient group, a positive level of anti-ox-LDL was detected in 17% (11/64) of primary APS patients and in 30% (13/43) of patients whose APS was secondary to SLE. By correlating levels of anti-ox-LDL with clinical manifestations of APS (arterial/venous thrombosis, miscarriages and thrombocytopenia), it was found that patients with a history of arterial thrombosis had higher levels of anti-ox-LDL than patients without (P = 0.0001) (Fig. 2), but no correlation was found between levels of anti-ox-LDL and a history of venous thrombosis or miscarriages (data not shown). A positive titre of aCL IgG and anti-b2GPI IgG was found in 70% (75/107) and 49% (52/107) of patients, respectively. Positivity for anti-ox-LDL was more frequent in patients with aCL IgG (31%, 23/75) than in those without aCL IgG (3%, 1/32) (w2 = 9.7, P = 0.0018), but no di€erence in positivity for anti-ox-LDL was

FIG. 1.Ð Levels of IgG anti-ox-LDL (optical density values) in patients with APS (n = 107) and in healthy controls (n = 104). Levels of anti-ox-LDL were higher in APS patients than in controls.

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FIG. 2.Ð Presence of anti-ox-LDL in APS patients with or without arterial thrombosis. APS patients with arterial thrombosis showed higher levels of anti-ox-LDL than those without.

noted between patients with (29%, 5/52) or without anti-b2GPI (16%, 9/55). Anti-ox-LDL IgG levels correlated weakly with the titres of aCL IgG (r = 0.196, P = 0.043), but not with those of anti-b2GPI (r = 0.076) (Fig. 3). DISCUSSION The present study reveals that anti-ox-LDL antibodies are often found in patients with APS, both primary and secondary. Levels of anti-ox-LDL were found to be higher in patients with a history of arterial thrombosis compared to those without. It has been hypothesized that ox-LDL may combine with anti-ox-LDL, leading to the formation of an immune complex, the uptake of which by Fc receptors on macrophages occurs in synergy but faster than by the scavenger pathway [2, 17]. An in vitro study showed that uptake of radiolabelled ox-LDL

by a monocyte/macrophage-like cell line was more rapid in the presence of anti-ox-LDL than the uptake of ox-LDL alone [2]. On the other hand, it may be that the mechanism described contributes to the elimination of excess ox-LDL produced in some disease states, and that anti-ox-LDL in serum may represent a marker of inappropriate ox-LDL generation [18]. Antibodies against ox-LDL have been detected in a variety of diseases, including SLE [12, 19±23]. Although the role of anti-ox-LDL remains unclear, there are several pieces of evidence supporting their involvement in the development of atherosclerosis: (a) the correlation between anti-ox-LDL and disease progression in carotid atherosclerosis [11]; (b) the predictive value of anti-ox-LDL for myocardial infarction [18]; (c) the presence of ox-LDL by immunostaining in atherosclerotic plaques of human aortas [7]; (d) the in vitro e€ects of ox-LDL on monocyte/ macrophages [2]. Clari®cation of the latter issue is of practical importance because it contributes signi®cantly towards the morbidity and mortality of a€ected patients. In our study with APS patients, we did not ®nd a convincing relationship between the titres of aCL and anti-ox-LDL, in agreement with earlier data from Vaarala et al. [12], eventually implying that anti-ox-LDL may represent a di€erent autoantibody in the APS scenario. There are some reports suggesting cross-reactivity between aCL and anti-ox-LDL. Initially, this was reported by Vaarala et al. [12], in SLE patients, showing that aCL activity was inhibited by ox-LDL in the ELISA system. Recent studies proved the cross-reaction using murine monoclonal antibodies [24]. Monoclonal anti-ox-LDL raised from apo E-de®cient mice bound to di€erent immunogenic structures generated by the oxidation of LDL, and some of them bound strongly to cardiolipin as it was progressively oxidized [25]. On the other hand, monoclonal aCL established from NZW/BXSBF1 mice (both b2GPI dependent and independent) bound to ox-LDL [26]. In another study, puri®ed IgG from

FIG. 3.Ð Correlation of the levels of anti-ox-LDL with those of (A) aCL and (B) anti-b2GPI in sera from patients with APS.

AMENGUAL ET AL.: ANTI-ox-LDL ANTIBODIES IN APS

SLE patients containing aCL was fractionated according to their anity for cardiolipin and oxLDL binding was found in some fractions, implying that at least some aCL may have binding activity to ox-LDL [23]. However, b2GPI-dependent murine monoclonal aCL were reported to bind ox-LDL only in the presence of b2GPI, showing that monoclonal aCL recognize a cryptic epitope on b2GPI which bound to lipids contained in ox-LDL molecules [27]. Thus, some but not all aCL may cross-react with ox-LDL. A number of mechanisms have been hypothesized in the pathophysiology of thrombosis in APS [28] and the atherosclerosis process could be implicated as one of them [29±31]. Oxidative modi®cation of LDL may occur as a result of free radical generation in the blood stream, particularly on the arterial side where primed neutrophils may release reactive oxygen species [29]. In keeping with this concept, we feel that our data regarding higher anti-ox-LDL levels in APS patients with a history of arterial thrombosis may represent a novel marker for arterial disease in APS. We used MDA-LDL as an antigen in ELISA. MDA is a highly reactive dialdehyde generated during arachidonic acid catabolism in thrombocytes and it is also produced from lipid peroxidation during phagocytosis of monocytes. Thus, MDA-LDL may be a `physiological' ox-LDL and it is known that MDA-LDL favours the binding of anti-ox-LDL as well as Cu2+-oxidized LDL [15]. Our anti-ox-LDL ELISA was carried out in the absence of b2GPI in order to reduce the heterogeneity of antibodies detected by the assay. In that way, we reduced the chances that our assay could detect b2GPI-dependent aCL (= anti-b2GPI). In fact, the levels of anti-oxLDL did not correlate with those of anti-b2GPI and the correlation with aCL was extremely weak. Therefore, anti-ox-LDL may represent a distinct subset of antibodies co-existing with b2GPI-dependent aCL (speci®city directed towards the epitope on b2GPI) and with conventional `aCL' detected by standard aCL assay [16]. In conclusion, our data from a large number of APS patients suggest that high titres of anti-ox-LDL may be a potential marker of arterial disease in APS and anti-ox-LDL might contribute to a better characterization of this syndrome. ACKNOWLEDGEMENTS This work was supported by grants from Fondo de InvestigacioÂn de la Seguridad Social of Spain (FIS 95/5011, FIS 96/5093), the Daiwa Anglo-Japanese Foundation and Lupus UK. The authors wish to thank Dr P. R. J. Ames (Haematology Department, St Thomas' Hospital, London) for his helpful suggestions and comments on this manuscript. REFERENCES 1. Brown MS, Goldstein JL. The hyperlipoproteinemias and other disorders of lipid metabolism. In: Isselbacher

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