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Originally published In Press as doi:10.1194/jlr.M200329-JLR200 on January 16, 2003

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Journal of Lipid Research, Vol. 44, 716-726, April 2003
Copyright © 2003 by Lipid Research, Inc.

Circulating oxidized LDL forms complexes with ß2-glycoprotein I

: implication as an atherogenic autoantigen

Kazuko Kobayashi*, Makoto Kishi*,§, Tatsuya Atsumi**, Maria L. Bertolaccini{dagger}{dagger}, Hirofumi Makino{dagger}, Nobuo Sakairi§§, Itaru Yamamoto§, Tatsuji Yasuda*, Munther A. Khamashta{dagger}{dagger}, Graham R. V. Hughes{dagger}{dagger}, Takao Koike**, Dennis R. Voelker*** and Eiji Matsuura1,*

* Department of Cell Chemistry, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan
{dagger} Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan
§ Department of Immunochemistry, Faculty of Pharmaceutical Science, Okayama University, Okayama 700-8530, Japan
** Department of Medicine II, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan
{dagger}{dagger} Lupus Research Unit, The Rayne Institute, St. Thomas' Hospital London, SE1 7EH, UK
§§ Division of Bioscience, Graduate School of Environment Earth Science, Hokkaido University, Sapporo 060-0810, Japan
*** Program in Cell Biology, Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206

Published, JLR Papers in Press, January 16, 2003. DOI 10.1194/jlr.M200329-JLR200

1 To whom correspondence should be addressed. e-mail: eijimatu{at}md.okayama-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ß2-glycoprotein I (ß2-GPI) is a major antigen for antiphospholipid antibodies (Abs, aPL) present in patients with antiphospholipid syndrome (APS). We recently reported (J. Lipid Res., 42: 697, 2001; J. Lipid Res., 43: 1486, 2002) that ß2-GPI specifically binds to Cu2+-oxidized LDL (oxLDL) and that the ß2-GPI ligands are {omega}-carboxylated 7-ketocholesteryl esters. In the present study, we demonstrate that oxLDL forms stable and nondissociable complexes with ß2-GPI in serum, and that high serum levels of the complexes are associated with arterial thrombosis in APS. A conjugated ketone function at the 7-position of cholesterol as well as the {omega}-carboxyl function of the ß2-GPI ligands was necessary for ß2-GPI binding. The ligand-mediated noncovalent interaction of ß2-GPI and oxLDL undergoes a temperature- and time-dependent conversion to much more stable but readily dissociable complexes in vitro at neutral pH. In contrast, stable and nondissociable ß2-GPI-oxLDL complexes were frequently detected in sera from patients with APS and/or systemic lupus erythematodes. Both the presence of ß2-GPI-oxLDL complexes and IgG Abs recognizing these complexes were strongly associated with arterial thrombosis. Further, these same Abs correlated with IgG immune complexes containing ß2-GPI or LDL.

Thus, the ß2-GPI-oxLDL complexes acting as an autoantigen are closely associated with autoimmune-mediated atherogenesis.

Abbreviations: Ab, antibody; APS, antiphospholipid syndrome; ß2-GPI, ß2-glycoprotein I; oxLDL, oxidized LDL; PL, phospholipid

Supplementary key words antiphospholipid syndrome • arterial thrombosis • autoantibody


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative modification of LDL is a physiologically relevant mechanism for atherogenesis. Experimental evidence clearly demonstrates that oxidized LDL (oxLDL) exists in vivo in the artery wall and contributes to the initiation and progression of atherosclerotic lesions (1–3). When LDL undergoes oxidation, "biologically active" lipids are generated. The process involves oxidative breakdown of either free polyunsaturated fatty acids or those esterified at the sn-2 position of phospholipids (PLs) to form fatty-acid hydroperoxides. The resulting fatty-acid hydroperoxides decompose to form highly reactive products containing an aldehyde (or ketone) function (4–10). Such active functions can form Schiff-base adducts with lysine residues of the apolipoprotein B (apoB) moiety of LDL (11) and primary amine-containing PLs, such as phosphatidylserine and phosphatidylethanolamine.

Several reports indicate that auto-antibodies (Abs) against oxidatively generated neoepitopes of LDL are present in patients or animals with atherosclerosis. Anti-oxLDL Abs are elevated in patients with early-onset peripheral vascular disease, severe carotid atherosclerosis, and angiographically verified coronary artery disease (12–17). In addition, a monoclonal auto-Ab (EO6) from an apoE-deficient mouse recognizes an adduct formed with oxidized phosphatidylcholine, i.e., 1-palmitoyl-2-(5'-oxo) valeroyl-sn-glycero-3-phosphorylcholine and lysine, and its ß-hydroxyaldehyde (aldol) condensates (18–20).

The autoimmune disorder antiphospholipid syndrome (APS) is characterized by the presence of a group of heterogeneous antiphospholipid antibodies (Abs, aPL), such as anticardiolipin Abs (aCL) and lupus anticoagulants (LAs), and by the occurrence of thromboembolic complications in the arterial and/or venous vasculatures (21, 22). In 1990, it was first reported that a plasma cofactor [ß2-glycoprotein I (ß2-GPI)] complexed with negatively charged PLs such as cardiolipin (CL) was an antigenic target for aCL (23–25). ß2-GPI is a 50 kDa protein present in plasma at a concentration of ~200 µg/ml. It binds to negatively charged molecules, including PLs (26) and heparin (27), and to plasma membranes of activated platelets and apoptotic cells on which phosphatidylserine is exposed (28, 29). However, the exact mechanism of the interaction between ß2-GPI and anti-ß2-GPI Abs remains uncertain (30–37).

ß2-GPI is a member of the short consensus repeats of the complement control protein superfamily, and its fifth domain contains a binding region for negatively charged PLs. X-ray crystal analysis (38) showed that the PL binding is provided by a patch consisting of 14 residues of positively charged amino acids and by a flexible loop between S311-K317 in domain V. Recent analysis with domain V mutant proteins confirmed interactions of the flexible loop with hydrophobic ligands (39, 40).

Several lines of evidence suggest that the interaction between aPL and malonedialdehyde-modified LDL (MDA-LDL) may be important in relation to the pathogensis of atherosclerosis and/or atherothrombosis in APS (41–43). We previously reported that ß2-GPI bound directly to Cu2+-oxLDL, and that the complex of oxLDL and ß2-GPI was subsequently recognized by a mouse monoclonal anti-ß2-GPI IgG auto-Ab (WB-CAL-1) established from NZW x BXSB F1 (WB F1) male mouse as a model of APS (44). Uptake of oxLDL by mouse macrophages is significantly increased by phagocytosis of an immune complex consisting of ß2-GPI, oxLDL, and WB-CAL-1 Ab (44). The major ligand responsible for the ß2-GPI binding to oxLDL is 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) (45). It was further demonstrated that oxLDL recognition by ß2-GPI and an anti-ß2-GPI Ab, such as WB-CAL-1 Ab and a human monoclonal IgM auto-Ab (EY2C9) derived from an APS patient, requires an {omega}-carboxyl function introduced by Cu2+-oxidation of an unsaturated acyl chain moiety in cholesteryl esters (46). All these observations imply that auto-Abs against ß2-GPI induced in APS patients may be "atherogenic."

In the present study, we demonstrate that oxLDL forms stable but readily dissociable complexes with ß2-GPI after an initial noncovalent interaction in vitro. In contrast, stable and nondissociable ß2-GPI-oxLDL complexes are detected in sera of patients with APS and/or systemic lupus erythematodes (SLE) and are etiologically important. Further, the ß2-GPI-oxLDL complexes exist as an IgG immune complex in those patients. Clinical analysis indicates that the serum ß2-GPI-oxLDL complexes are associated with arterial thrombosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
This study utilized materials from British Caucasian patients with APS and/or SLE [n = 127, 41.0 ± 11.9 years (mean ± SD); range: 16–67 years] who were examined at Lupus Clinic of St. Thomas' Hospital, London, UK (Table 1). All APS patients were positive for ß2-GPI-dependent aCL (IgG) and/or LA on two or more occasions at least 6 weeks apart. Clinical records were carefully reviewed retrospectively. One hundred sixteen APS and/or SLE patients were female. Of them, 82 patients fulfilled the new preliminary criteria for APS (47) and seven patients had a history of thrombocytopenia alone. Arterial events comprised stroke, myocardial infarction, and peripheral artery occlusion, confirmed by computed tomography scan, magnetic resonance imaging, or angiography. Deep-vein thrombosis and pulmonary thrombosis were defined as venous thrombosis, confirmed by Doppler ultrasound, venography, or ventilation-perfusion scanning. Pregnancy morbidity was defined according to the preliminary criteria for APS (47). Any patients who had acute thrombosis within 2 months were excluded. Fifty age-matched British Caucasian healthy controls [40.7 ± 14.0 years (mean ± SD); range: 18–66 years] with no history of autoimmune, infectious, or thrombotic diseases were recruited. Informed consent was given for all subjects and the study was approved by both ethics committees of Okayama University Hospital and of St. Thomas' Hospital.


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  		TABLE 1. Patients' characteristics

 
Monoclonal Abs
Anti-human ß2-GPI Abs, Cof-22 (IgG1,{kappa}) and Cof-23 (IgG1,{kappa}), were established from BALB/c mice immunized with human ß2-GPI (31). They bind to monomeric ß2-GPI in solution. Anti-ß2-GPI auto-Ab, WB-CAL-1 (IgG2a,{kappa}), was derived from a WB F1 mouse (48). Anti-ß2-GPI auto-Ab, EY2C9 (IgM), was established from peripheral blood lymphocytes from an APS patient (49). Both WB-CAL-1 and EY-2C9 Abs bind only to ß2-GPI complexed with negatively charged PLs or with oxLDL, but not to monomeric ß2-GPI in solution. A mouse monoclonal anti-human apoB-100 Ab, 1D2 (IgG), was established from BALB/c mouse immunized with human apoB100. The 1D2 Ab reacts with both oxidized and native LDL.

Preparation of human ß2-GPI
ß2-GPI was purified from normal human plasma as described (50), with slight modification. Pooled plasma from healthy subjects was sequentially chromatographed on a heparin-Sepharose column, a DEAE-cellulose column, and an anti-ß2-GPI affinity column. To remove any contamination by IgGs, the ß2-GPI-rich fraction was further passed through a protein A Sepharose column. The final ß2-GPI fraction was delipidated by extensive washing with n-butanol.

Isolation and oxidation of LDL
LDL (d = 1.019–1.063 g/ml) was isolated by preparative ultracentrifugation from fresh normal human plasma, as described (51). The LDL was adjusted to 100 µg/ml of apoB equivalent and oxidized with 5 µM CuSO4 in 10 mM Hepes and 150 mM NaCl (pH 7.4) (Hepes buffer) for various periods at 37°C. To terminate the oxidation, EDTA (final concentration of 1 mM) was added and the LDL was dialyzed against Hepes buffer containing 1 mM EDTA. Protein concentration was determined using the BCA protein assay reagent (Pierce Chemical Co., Rockford, IL), and the degree of oxidation was estimated as thiobarbituric acid-reactive substances (TBARS) value (52) and by electrophoretic migration in agarose gels.

Agarose gel electrophoresis
Native or modified LDLs were spotted on an agarose gel film and subjected to electrophoresis in 0.05 M barbital buffer (pH 8.6) using the Pol-E-Film System kit (Herena Laboratories, Urawa, Japan).

Synthesis of oxysterol derivatives of 9-carboxynonanoate
oxLig-1 was synthesized, as previously reported (45). 22-Ketocholesteryl-9-carboxynonanoate (9-COOH-22KC) was synthesized in a similar way. Briefly, to a solution of 22-ketocholesterol (10 mg, 0.025 mmol) and azelaic acid (14.1 mg, 0.075 mmol) in acetone (1 ml) were added 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (19.2 mg, 0.10 mmol) and 4-(dimethylamino) pyridine (6.1 mg, 0.80 mmol). The mixture was stirred at room temperature for 2 days, concentrated, and extracted with chloroform. The extract was successively washed with 2 M hydrochloric acid and brine, dried over anhydrous magnesium sulfate, and evaporated. The residues were subjected to column chromatography on silica gel using toluene-ethyl acetate (3:1, v/v) to give 9-COOH-22KC (8.5 mg, 61% yield). 1H-NMR (300 MHz, CDCl3): {delta} = 5.35 (d, 1H, J 5.1 Hz, H-6), 4.59 (m, 1H, H-3).

Similar to oxLig-1, the 1H-NMR spectrum of 9-COOH-22KC showed a signal assignable to H-3 at {delta} = 4.59 ppm as a multiplet, suggesting that the hydroxyl group at this position was esterified. Although the spectrum also revealed a signal of olefinic proton at H-6 in lower magnetic field, spin-spin coupling was observed between the neighboring methylene group. The molecular mass of 9-COOH-22KC was identical to that of oxLig-1. The 9-COOH-22KC was positive in the Lieberman-Burchard reaction, indicating a conjugated ketone at position 7 is not present.

Ligand blot analysis on a TLC plate
For TLC ligand blotting, lipids were spotted on a Polygram silica gel G plate (Machery-Nagel, Duren, Germany) and developed in chloroform-methanol (8:1, v/v). Ligand blot analysis was performed, as described previously (45, 46). Briefly, after drying and blocking with PBS containing 1% BSA, the plate was subsequently and simultaneously incubated with ß2-GPI and anti-ß2-GPI Ab (Cof-22 and EY2C9, respectively) for 1 h. Subsequently, the plate was incubated with horseradish peroxidase (HRP)-labeled anti-mouse IgG or anti-human IgM for 1 h. In between each step, the plates were extensively washed with PBS. The color was developed with H2O2 and 4-methoxy-1-naphthol. On a control TLC plate, separated ligands were stained with I2 vapor.

ELISA for ß2-GPI-oxLDL complexes
Anti-ß2-GPI Ab (WB-CAL-1) was adsorbed on a microtiter plate (Immulon 2HB, Dynex Technologies, INC., Chantily, VA) by incubating at 8 µg /ml (dissolved in Hepes buffer, 50 µl/well) at 4°C overnight. The plate was blocked with 1% skim milk for 1 h. Serum samples (100-fold diluted) or solutions containing ß2-GPI-oxLDL complexes or oxLDL were added to the wells (100 µl/well) and incubated for 2 h. For some experiments, exogenous ß2-GPI (25 µg/ml) was present in this step. The wells were subsequently incubated with biotinyl-anti-apoB-100 Ab (1D2) for 1 h and HRP-labeled avidin for 30 min. Color was developed with o-phenylenediamine and H2O2. The reaction was terminated by adding 2 N sulfuric acid, and the OD at 490 nm was measured. Between each step, extensive washing was performed using Hepes buffer containing 0.05% Tween 20. Raw OD of samples in individual assays was corrected by mean OD of the blank wells. When 1.0 U/ml was adjusted to 3 SD above the mean of serum samples from 50 normal subjects, 1.0 U/ml of the oxLDL12 h-ß2-GPI16 h complex was equilibrated to ~4.5 µg/ml of apoB equivalent. A sample was considered positive when its reactivity was higher than 1.0 U/ml.

ELISA for anti-ß2-GPI-lipid IgG Abs
CL (from bovine heart, Sigma Chemical Co.), oxLig-1, or 9-COOH-22KC (50 µg/ml in ethanol, 50 µl/well) was adsorbed by evaporation on a plain polystyrene plate (Immulon 1B), and the plate was then blocked with 1% BSA. Purified monoclonal auto-Abs or serum samples (100-fold diluted) were incubated in the wells with or without ß2-GPI (25 µg/ml) for 1 h, and HRP-labeled anti-mouse IgG or anti-human IgG or IgM was then added. Further steps were performed as described in "ELISA for ß2-GPI-oxLDL complexes." Raw OD of individual samples was corrected by mean OD of the blank wells. OD variation among plates was normalized by using a positive control. A sample was considered to be positive when its Ab titer was higher than 3 SD above the mean OD of plasma samples of 50 normal subjects.

ELISA for anti-ß2-GPI IgG Abs
ELISA for anti-ß2-GPI IgG Abs was performed as described (30). Briefly, ß2-GPI was adsorbed on polyoxygenated polystyrene plates (carboxylated, Sumilon C, Sumitomo Bakelite Co., Ltd., Tokyo, Japan) by incubating at 10 µg/ml (50 µl/well) at 4°C, overnight, and the plates were blocked with 3% gelatin. Serum samples were diluted 100-fold and incubated in the wells for 1 h. HRP-labeled anti-human IgG was then added to the plates. Further steps were performed as described in "ELISA for ß2-GPI-oxLDL complexes."

ELISA for IgG immune complexes
To determine ELISA for IgG immune complexes (IgG IC) formed with ß2-GPI or LDL, anti-ß2-GPI Ab (Cof-23) or anti-apoB100 Ab (1D2) was adsorbed on plain polystyrene plates (Immulon 1B) by incubating overnight at 5 µg/ml (50 µl/well) at 4°C. The plates were then blocked with 1% BSA. Serum samples (100-fold diluted) were incubated in the wells for 1 h and HRP-labeled anti-human IgG was added. Further steps were performed as described in "ELISA for ß2-GPI-oxLDL complexes."

Statistical analysis
Statistical analysis was performed by StatView software (Abacus Concepts, Berkeley, CA). Fisher's exact test was used to compare the occurrence of auto-Abs and clinical histories. Ninety-five percent confidence interval (95% CI) was calculated by Woolf's method.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Role of 7-ketone function as a ligand for ß2-GPI binding
We compared the binding of ß2-GPI to two positional ketone variants of {omega}-carboxyl oxysterol esters (i.e., oxLig-1 and 9-COOH-22KC) in ligand blot and ELISA using anti-ß2-GPI Abs as a probe. ß2-GPI preferentially bound to the 7-keto-variant (oxLig-1) but not to 9-COOH-22KC in the ligand blot as detected by Cof-22 or EY2C9 Ab (Fig. 1) . In the ELISA using a ligand-coated plate, ß2-GPI binding to solid-phase oxLig-1 rather than 9-COOH-22KC was detected with anti-ß2-GPI Abs (Cof-22, WB-CAL-1, or EY2C9) (Table 2). These data demonstrate that the ketone function at position 7 of the cholesterol backbone is a critical determinant for high-affinity interaction between ß2-GPI and its ligands, e.g., oxLig-1, derived from Cu2+-mediated oxLDL.



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  		Fig. 1. Ligand blot analysis on two {omega}-carboxyl variants of oxysterol ester 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) and 22-ketocholesteryl-9-carboxynonanoate. The developed ligands on a TLC plate were stained with I2 vapor (A) and ligand blot was performed with anti-ß2-glycoprotein I (ß2-GPI) antibodies (Abs), i.e., Cof-22 (B) and EY2C9 (C).

 

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  		TABLE 2. ß2-GPI binding to solid phase {omega}-carboxyl variants of oxysterol ester, detecting in ELISA with anti-ß2-GPI Abs

 
ß2-GPI interaction with LDL undergoing Cu2+-mediated oxidation
LDL (100 µg/ml of apoB equivalent) was oxidized by incubating with 5 µM CuSO4 for 12 h at 37°C (oxLDL12 h), and the oxidation was terminated by addition of EDTA. In ELISA for detecting ß2-GPI-oxLDL complexes, the OD was increased only when oxLDL12 h was incubated with exogenous ß2-GPI in the assay wells. The formation of complexes was dependent upon the concentration of both ß2-GPI and oxLDL (Fig. 2A, B) . Significant complex formation occurred only with oxLDL12 h and not with native LDL. Complex formation at pH 7.4 was almost completely inhibited in the presence of heparin or MgCl2 (Fig. 2C). The inhibition was also observed with CaCl2 in the same manner (data not shown). These data indicate that ß2-GPI can initially form dissociable noncovalent complexes with oxLDL12 h. In contrast, relatively stable complexes between oxLDL and ß2-GPI were consistently observed when oxLDL12 h was incubated at pH 7.4 with ß2-GPI for 16 h at 37°C (oxLDL12 h-ß2-GPI16 h). The subsequent addition of heparin or MgCl2 at pH 7.4 failed to disrupt oxLDL12 h-ß2-GPI16 h complex formation (Fig. 2D). LDL, ß2-GPI, and their complexes were applied to agarose gels for electrophoresis. As shown in Fig. 3 , the increased negative charge in LDL that was gained by the incubation with CuSO4 for 12 h at 37°C (oxLDL12 h) was neutralized by the interaction with ß2-GPI (oxLDL12 h-ß2-GPI16 h).



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  		Fig. 2. Profiles of complex formation between Cu2+-mediated oxidized LDL (oxLDL) and ß2-GPI. A: oxLDL12 h [0 (open triangles), 0.16 (open squares), or 2.5 µg/ml of apolipoprotein B (apoB) equivalent (open circles)] was incubated with various concentrations of ß2-GPI in the assay wells, and ELISA for ß2-GPI-oxLDL complexes was performed. B: Indicated concentrations of oxLDL12 h (LDL treated with 5 µM CuSO4 for 12 h at 37°C, circles) or native LDL (squares) were incubated in the absence (open symbols) or presence (25 µg/ml, closed symbols) of ß2-GPI, and the ELISA was performed. C: Indicated concentrations of oxLDL12 h and ß2-GPI (25 µg/ml) were incubated in the assay wells and the ELISA was performed in the absence (open circles), or presence of heparin (100 U/ml; closed squares) or MgCl2 (10 mM; closed diamonds). D: oxLDL12 h-ß2-GPI16 h complexes were prepared by incubating oxLDL12 h (100 µg/ml) with ß2-GPI (100 µg/ml) at 37°C for 16 h. The ELISA was performed with the complexes (2.5 µg/ml of apoB equivalent) in the absence (open circles) or presence of heparin (100 U/ml; closed squares) or MgCl2 (10 mM; closed diamonds). Results are expressed as the mean ± SD of triplicate samples.

 


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  		Fig. 3. The negative charge in oxLDL is partially neutralized by interaction with ß2-GPI. Native LDL, oxLDL, ß2-GPI, and oxLDL12 h-ß2-GPI16 h complexes were analyzed by electrophoresis on an agarose gel and visualized by staining with amido black.

 
To further examine the processes of complex formation, a series of time-course studies was performed. Figure 4A reveals the time-dependent generation of TBARS in CuSO4-treated LDL. The TBARS were rapidly generated in LDL preparations exposed to the Cu2+ ion at 37°C, with a peak at 4 h. In contrast, oxidation of LDL that generated the ß2-GPI binding proceeded with a lag and reached its maximum after ~12 h (Fig. 4B). The complex formation was almost completely inhibited by the addition of heparin or MgCl2. These data are consistent with our previous observations demonstrating that ß2-GPI binds to Cu2+-oxLDL but not to MDA-modified LDL (44).



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  		Fig. 4. Time-course study of complex formation between Cu2+-mediated oxLDL and ß2-GPI. A: Thiobarbituric acid reactive substances in LDL (treated with 5 µM CuSO4 for indicated period) were measured. B: ß2-GPI-oxLDL complexes formed by incubating Cu2+-oxLDL (2.5 µg/ml of apoB equivalent) with ß2-GPI (0 µg/ml, open circles; 25 µg/ml, closed circles) in the assay wells, and ELISA was performed. The ELISA with 25 µg/ml of ß2-GPI was also performed in the presence of heparin (100 U/ml, closed squares) or MgCl2 (10 mM; closed diamonds). C: ß2-GPI-oxLDL complexes generated by incubation of preformed oxLDL12 h (100 µg/ml) with ß2-GPI (100 µg/ml) during indicated periods at 4°C (dotted lines) or at 37°C (solid lines) were detected in the ELISA. The ELISA was also performed in the absence (open circles) or presence of heparin (100 U/ml; closed squares) or MgCl2 (10 mM; closed diamonds). D: ß2-GPI-oxLDL complexes were formed by the simultaneous incubation of LDL (100 µg/ml) and ß2-GPI (100 µg/ml) during the Cu2+ (5 µM) oxidation at 37°C and ELISA of ß2-GPI-oxLDL complexes (2.5 µg/ml equivalent of apoB) were detected in the ELISA. The ELISA was also performed in the absence (open circles) or presence of heparin (100 U/ml; closed squares) or MgCl2 (10 mM; closed diamonds). Results are expressed as the mean ± SD of triplicate samples.

 
The preformed oxLDL12 h (final concentration at 100 µg/ml of apoB equivalent) was also incubated with ß2-GPI (100 µg/ml) for different periods at 4°C or 37°C (Fig. 4C). The formation of ß2-GPI-oxLDL complexes was temperature- and time-dependent. The complexes were not dissociated by the addition of heparin or MgCl2 after the incubation at pH 7.4. Figure 4D indicates that the stable interaction between ß2-GPI and oxLDL was generated during the Cu2+-oxidation process even in the presence of ß2-GPI.

Stability of in vitro ß2-GPI-oxLDL complexes at different pHs
The stable complexes appeared at neutral pH and are possibly Schiff-base adducts formed between {varepsilon}-amines of lysine residues of ß2-GPI and oxidatively generated aldehydes on the Cu2+-mediated oxLDL vesicles. To test this, we analyzed the stability of nonreduced and NaCNBH3-reduced complexes at basic pH conditions. As shown in Fig. 5 , no dissociation was observed in the reduced oxLDL12 h-ß2-GPI16 h complexes at any pH conditions tested in the absence of MgCl2. In the presence of MgCl2, 82% of nonreduced complexes dissociated at pH 10, whereas 69% of reduced complexes dissociated. The stable complexes may be formed by both electrostatic interaction and Schiff-base formation between an oxidized moiety on Cu2+-oxLDL and the PL binding patch on the ß2-GPI molecule that is composed of 14 positively charged amino acid residues and a hydrophobic loop. These findings also indicate that the adduct is either not a Schiff base, or if it is a Schiff base, it resides in an environment that is not accessible to NaCNBH3 (e.g., a hydrophobic pocket).



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  		Fig. 5. Stability of oxLDL12 h-ß2-GPI16 h complexes and their NaCNBH3-reduced forms at different pH conditions. oxLDL12 h-ß2-GPI16 h complexes (100 µg/ml apoB equivalent) were treated with 200 mM NaCNBH3 in PBS at pH 7.4 for 16 h to be reduced. The nonreduced and reduced complexes were incubated at indicated pH for 16 h at 37°C in the absence or presence of 10 mM MgCl2. ß2-GPI-oxLDL complexes were measured in these preparations containing 300 ng/ml of LDL (apoB equivalent) in the ELISA. Results are expressed as the mean ± SD of triplicate samples.

 
Nondissociable ß2-GPI-oxLDL complexes exist in patient sera
We screened serum samples from patients with APS and/or SLE for high levels of ß2-GPI-oxLDL complexes. ß2-GPI-oxLDL complexes were previously characterized in 20 sera. This group showed high concentrations of serum complexes with a range of 2.1–13.7 U/ml, and a mean concentration of 4.48 U/ml (cutoff value: 1.0 U/ml). As shown in Fig. 6 , native LDL did not form complexes upon incubation with ß2-GPI at 37°C for 16 h. In contrast, oxLDL12 h-ß2-GPI16 h complexes were stable at pH 7.4, even in the presence of heparin or MgCl2. The typical binding pattern was also shown for preexisting oxLDL-ß2-GPI complexes detected in five serum samples at pH 7.4. In all 20 tested samples, the complexes that were preformed in vivo were stable at neutral pH, even in the presence of heparin and MgCl2 (The ODs in the cases with heparin and MgCl2 were 121 ± 25.1% and 128 ± 13.6% of control condition, respectively). The preformed complexes present in serum samples were also consistently observed after the 16 h-incubation with MgCl2 at pH 10 at 37°C (104 ± 10.9%), that can dissociate the complexes formed in vitro (Fig. 5). We interpret these findings to indicate that nondissociable and covalent adducts between ß2-GPI and in vivo oxLDL are formed. We propose that our in vitro adducts are intermediates in the formation of the nondissociable complexes.



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  		Fig. 6. ß2-GPI-oxLDL complexes present in patient sera. A: Native LDL (nLDL)-ß2-GPI16 h (the reaction mixture of native LDL and ß2-GPI by the 16 h incubation at 37°C, as a negative control) (300 ng/ml apoB equivalent), oxLDL12 h-ß2-GPI16 h (300 ng/ml), or 100-fold diluted ß2-GPI-oxLDL complex-positive sera were incubated in the absence or presence of heparin (100 U/ml) or MgCl2 (10 mM) at pH 7.4. B: The ß2-GPI-oxLDL complex-positive sera were preincubated at pH 7 or pH 10 in the presence of MgCl2 (10 mM) for 16 h at 37°C, and the ELISA was performed. Results are expressed as the mean of duplicate samples.

 
Clinical significance of ß2-GPI-oxLDL complex and its auto-Abs
In the ELISA, we obtained an apparent calibration curve for oxLDL12 h-ß2-GPI16 h complexes within a range of 10 ng/ml to 1.25 µg/ml of apoB equivalent. The ELISA was not affected by the high concentration of endogenous and monomeric ß2-GPI in serum samples, because WB-CAL-1 Ab used in the ELISA is highly specific for ß2-GPI complexed with oxLDL. In the present study, the ß2-GPI-oxLDL complexes were positive in 58.7% (27/46), 54.1% (20/37), and 56.8% (25/44) of patients with the primary APS, APS with SLE (secondary APS), and SLE without APS, respectively (Fig. 7) .



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  		Fig. 7. Serum levels of ß2-GPI-oxLDL complexes detected in ELISA. ß2-GPI-oxLDL complexes were detected in 100-fold diluted sera from normal subjects and patients with the primary antiphospholipid syndrome (PAPS), APS with systemic lupus erythematodes (SLE) (the secondary APS), and SLE without APS. Cutoff value (1 U/ml) was adjusted to 3 SD above the mean levels of 50 normal subjects. A number indicates mean level in each subject group.

 
Anti-ß2-GPI-oxLig-1 IgG Abs were found in 71.7% (33/46), 59.5% (22/37), and 11.4% (5/44) of patients with the primary APS, APS with SLE (secondary APS), and SLE without APS, respectively. The individual anti-ß2-GPI-oxLig-1 IgG Ab titers from this group of 127 patients are strongly correlated with both ß2-GPI-dependent IgG aCL and anti-ß2-GPI IgG Abs (correlation coefficient; r 2 = 0.69 and 0.81, respectively) (Fig. 8) . As shown in Fig. 9 , there also was a good correlation between IgG IC with ß2-GPI and anti-ß2-GPI IgG Abs (r 2 = 0.50) (Fig. 9A), IgG IC with ß2-GPI and anti-ß2-GPI-oxLig-1 IgG Abs (r 2 = 0.50) (Fig. 9B), and IgG IC with LDL and IgG IC with ß2-GPI (r 2 = 0.40) (Fig. 9C). However, a good correlation between levels of ß2-GPI-oxLDL complex and titers of any of these Abs was not observed (data not shown).



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  		Fig. 8. Correlation among ß2-GPI-related IgG Ab titers detected in three different ELISA systems. A: ß2-GPI-dependent IgG anticardiolipin Abs (anti-ß2-GPI-cardiolipin IgG Abs) versus anti-ß2-GPI-oxLig-1 IgG Abs. B: Anti-ß2-GPI IgG Abs (detected in ELISA using a ß2-GPI-coated polyoxygenated plate) versus anti-ß2-GPI-oxLig-1 IgG Abs.

 


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  		Fig. 9. Correlation among IgG Ab titers and levels of IgG immune complexes. A: IgG immune complex with ß2-GPI (IgG IC w/ ß2-GPI) versus anti-ß2-GPI IgG Abs. B: IgG IC with ß2-GPI versus anti-ß2-GPI-oxLig-1 IgG Abs. C: IgG immune complex with LDL (IgG IC with LDL) versus IgG IC with ß2-GPI.

 
In Table 3, the correlation between anti-ß2-GPI-oxLig-1 IgG Abs (not ß2-GPI-oxLig-1 complex antigen) and thrombosis was calculated and the relative risk of having thrombosis was approximated by odds ratio. The first line showed the correlation between Abs and all thrombosis in all 127 patients; therefore the referent was patients without any thrombosis. Abs were correlated with thrombosis among ß2-GPI-oxLDL the complex antigen-positive patients' group (the second line) and among the antigen-negative patients' group as well (the third line). The correlation between anti-ß2-GPI-oxLig-1 IgG Abs and arterial and venous thrombosis was presented in (B) and (C) in the same fashion, respectively. The relative risk in the ß2-GPI-oxLDL antigen-positive patients' group was higher than that in the antigen-negative patients' group. It is of interest because the presence of ß2-GPI-oxLDL antigen may be an additional risk of having arterial thrombosis in patients with anti-ß2-GPI-oxLDL Abs.


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  		TABLE 3. Association with anti-ß2-GPI-oxLig-1 IgG Abs and thrombosis in APS

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We previously reported that the major lipid ligands responsible for ß2-GPI binding to Cu2+-mediated oxLDL are {omega}-carboxylated 7-ketocholesterol esters such as oxLig-1, and that the {omega}-carboxyl moiety is also essential for ß2-GPI recognition (45, 46). The in vitro interaction between ß2-GPI and Cu2+-oxLDL is initially reversible by Mg2+ treatment but progresses to a much more stable interaction requiring Mg2+ and a high pH to be dissociated. In contrast, stable and nondissociable complexes between oxLDL and ß2-GPI are found in serum samples from patients with APS and/or SLE. We further detected the complexes as IgG-immune complexes containing LDL and ß2-GPI in sera from those patients, and statistical analysis indicates that the serum ß2-GPI-oxLDL complexes are associated with arterial thrombosis.

Foam cell formation is regarded as the hallmark of early atherogenesis, and LDL is the major source of lipids deposited in these cells. The binding of modified LDL to scavenger receptors on macrophages leads to unregulated cholesterol accumulation and the formation of foam cells with development of atherosclerotic lesions. Recently, we identified the structure of two major ligands, which provide ß2-GPI binding to Cu2+-oxLDL and anti-ß2-GPI Ab mediated-phagocytosis by macrophages, to be oxLig-1 and 7-ketocholesteryl-12-carboxy (keto) dodecanoate (oxLig-2) (45, 46). In the present study, we demonstrated that the conjugated ketone function at position 7 of the cholesterol backbone of the ligands is required for high-affinity binding for ß2-GPI and cannot be replaced by a ketone at the 22 position (Fig. 1 and Table 2).

A patch consisting of 14 positively charged amino acid residues, and a flexible loop in domain V of ß2-GPI were reported to be critical for interaction with amphiphilic compounds such as CL, phosphatidylserine, phosphatidic acid, and phosphatidylglycerol (38–40). We previously reported that an interaction with oxLDL was also provided by the same binding site of ß2-GPI (53). The conjugated ketone of the ligands may orient to hydrophilic space together with {omega}-carboxyl function, which results in providing specific binding to ß2-GPI. In general, a conjugated ketone is less likely to actively form Schiff-base adducts than an {omega}-aldehyde. The ß2-GPI ligands may be involved in a noncovalent and electrostatic interaction between oxLDL and ß2-GPI at neutral pH because the interaction is inhibited either by MgCl2, CaCl2, or heparin.

It is now well established that anti-ß2-GPI Abs found in sera from APS patients bind to a complex of ß2-GPI and negatively charged PLs, such as CL, phosphatidylserine, and phosphatidic acid, in ELISA using a PL-coated microtiter plate (50). Hörkkö et al. (54) recently demonstrated that aCLs present in APS patients react with the Schiff-base adducts formed between oxidized CL and ß2-GPI. However, such negatively charged PLs are very minor components of LDL. The Cu2+-mediated oxidative products in LDL include cholesterol or oxysterols esterified with 9- or 13-hydroperoxy (or hydroxy)-octadecadienoate, 9-oxononanoate, or with 9-carboxynonanoate, some of which have also been shown to be present in atherosclerotic plaques (55–57). As we previously reported (45, 46), {omega}-carboxyl-oxysteryl esters such as oxLig-1 and oxLig-2, but not oxidized PLs, were detected in Cu2+-oxLDL as major ligands for ß2-GPI binding. The nature of these in vitro and in vivo adducts has not yet been chemically defined, but conjugates between ß2-GPI and some oxidatively modified forms of cholesteryl esters, as well as oxidized PLs, are the most likely candidates. In the present study, treatment of oxLDL12 h-ß2-GPI16 h complex with excess NaCNBH3 (i.e., 200 mM) was ineffective for reduction of the imine in Schiff-base adducts. This result raises the possibility that the stable and nondissociable complexes between oxLDL and ß2-GPI may be generated by other mechanisms, such as the Michael reaction or direct oxidation of lysine residues by alkoxyl radicals of polyunsaturated fatty acids.

In the present study, we demonstrate that oxLDL circulates in patients with APS and/or SLE (54.1–58.7%) in stable and nondissociable complexes with ß2-GPI (Fig. 7). Many reports demonstrate that oxLDL is preferentially taken up by macrophages via scavenger receptors and lead to foam cell formation and development of atherosclerotic lesions. However, there is incomplete information about oxLDL circulating in the blood stream of patients with atheroscrelosis. Even though we did not measure the free form of oxLDL in patient sera, it is likely that oxLDL generated in vivo is complexed with endogenous ß2-GPI (the plasma concentration of ß2-GPI is ~200 µg/ml). As shown in Fig. 4D, in the presence of ß2-GPI, LDL that underwent in vitro oxidation formed stable adducts with increasing incubation time at neutral pH. Furthermore, the stable interaction between ß2-GPI and oxLDL was observed under several different in vitro conditions, including in buffer alone or in buffer containing 1% BSA or 50% human serum (data not shown). Thus, ß2-GPI ligands related to oxLig-1 and oxLig-2 provide specific interaction between ß2-GPI and oxLDL to form stable complexes in the presence of excess levels of other plasma/serum proteins.

The association of aPL with serious clinical complications such as arterial and/or venous thrombosis, recurrent fetal loss, and thrombocytopenia has been established in patients with APS. aCLs were initially considered to be directed to acidic PLs such as CL, but now it is widely accepted that ß2-GPI is the true antigen for aCL. In 1998, we showed that anti-ß2-GPI IgG Abs could be a serologic marker for arterial thrombosis in SLE patients, while anti-MDA-LDL IgG Abs were not associated with arterial thrombosis (43). In the present study, we demonstrate a good correlation among titers of anti-ß2-GPI-CL IgG Abs, anti-ß2-GPI IgG Abs, and anti-ß2-GPI-oxLig-1 IgG Abs (Fig. 8). The appearance of anti-ß2-GPI-oxLig-1 IgG Abs was better correlated with a history of arterial thrombosis rather than with venous thrombosis (Table 3). These findings suggest that ß2-GPI-oxLig-1 (i.e., ß2-GPI-oxLDL) complexes may be the true target antigen for the previously characterized aCL. The anti-ß2-GPI-oxLig-1 IgG Abs appears to be an excellent candidate for inducing autoimmune-mediated atherothrombosis/atherosclerosis.

However, when all tested APS/SLE patients were divided into two subgroups, i.e., the ß2-GPI-oxLDL complex positive and negative, a stronger association between anti-ß2-GPI-oxLig-1 Ab and episodes of those clinical manifestations was observed in the positive subgroup than in the negative one. In auto-Ab-positive APS patients, IgG immune complexes with ß2-GPI and LDL were also detected. Although the mechanisms of in vivo oxidation of LDL remain unclear, the resultant ß2-GPI-oxLDL complexes may have a pathogenic role as an autoantigen to induce the development of thrombosis, especially arterial thrombosis, in APS.

The ELISA methodology using solid-phase native or oxLDL to measure Abs against oxLDLs and/or to measure IC with LDL is problematic. In previous reports (58–60), competitive ELISA for anti-oxLDL Abs and prepurification of samples with polyethyleneglycol for their detection have been proposed to minimize nonspecific binding of Abs to LDL solid phases. The system described in this report has relatively low nonspecific binding because the stable oxLDL-ß2-GPI complexes formed in vitro do not have the high negative charge of Cu2+-oxLDL. Furthermore, we applied two types of Ab-capture ELISAs using anti-ß2-GPI Ab and anti-apoB100 for detecting IgG IC with ß2-GPI and IgG IC with LDL, respectively. These two ELISAs are not affected by high titers of rheumatoid factors and endogenous levels of ß2-GPI. Although extremely high levels of lipids (>350 mg/dl of total cholesterol, i.e., in cases of familial hypercholesterolemia) can exert a dose-dependent effect on IC levels, this was not a problem for the current study, since none of the patients were hypercholesterolemic (>300 mg/dl). As shown in Fig. 9, there were statistically significant correlations between anti ß2-GPI IgG and IgG IC with ß2-GPI, between anti-ß2-GPI-oxLig-1 IgG and IgG IC with ß2-GPI, and between IgG IC with ß2-GPI and IgG IC with LDL (oxLDL). All of these correlations indicate that the presence of IgG (anti-ß2-GPI) IC with the ß2-GPI-LDL (oxLDL) complexes in the APS sera. In addition, the contaminated IgG (anti-oxLDL) IC with LDL could not be excluded.

George et al. reported that LDL-receptor-deficient mice fed a chow diet and immunized with ß2-GPI had accelerated atherosclerosis (61). ß2-GPI was abundant within subendothelial regions and intimal-medial borders of human atherosclerotic plaques, and colocalized with monocytes and CD4-positive lymphocytes (62). Thus, there is increasing circumstantial evidence of an autoimmune mechanism involving ß2-GPI and oxLDL in the atherogenesis of APS.

This is the first report that stable and nondissociable ß2-GPI-oxLDL complexes are found in patient sera and that the complexes may be a quantifiable risk factor for arterial thrombosis in APS. However, the ß2-GPI-oxLDL complexes were found not only in APS but also in the Ab-negative and nonthrombolic SLE and chronic nephritis (data not shown). The observation indicates that the serum complex level alone does not predict clinical manifestation in APS. It is understood that abnormalities in lipid and lipoprotein metabolism are commonly associated with diverse renal diseases and that hyperlipidemia and increased plasma lipoproteins such as LDL contribute to the high incidence of atherosclerotic cardiovascular events and mortality noted in patients with renal disease. These findings also raise important new issues about the clinical significance of circulating ß2-GPI-oxLDL complexes in blood stream of patients with coronary artery diseases.


   ACKNOWLEDGMENTS
 
This work was supported in part by a grant for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan; and by a grant from the Ministry of Health, Labor and Welfare of Japan. The authors thank Dr. Luis Lopez for thoughtful discussion and Dr. Qingping Liu for technical assistance.

Manuscript received August 16, 2002 and in revised form December 27, 2002.


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