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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:222-229.)
© 1996 American Heart Association, Inc.

Articles

Isolation and Characterization of Human Antioxidized LDL Autoantibodies

Marina Mironova; G. Virella; Maria F. Lopes-Virella

From the Department of Microbiology and Immunology (G.V.) and Division of Endocrinology, Diabetes and Medical Genetics, Department of Medicine (M.M., M.F.L.-V.), Medical University of South Carolina, and Ralph H. Johnson VA Medical Center (M.F.L.-V.), Charleston, SC.

Correspondence to Dr Maria F. Lopes-Virella, Ralph H. Johnson VA Medical Center, 109 Bee St, Charleston, SC 29401.

	Abstract

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Abstract Autoantibodies to oxidized LDL have been reported in normal subjects and in patients with arteriosclerosis, but their possible pathogenic role is not yet well defined. One important problem is the existence of contradictory data reported by different groups concerning the associations between antioxidized LDL autoantibodies and the presence or progression of arteriosclerotic lesions. Such contradictions led us to decide to isolate and characterize antioxidized LDL antibodies by affinity chromatography with the use of oxidized LDL cross-linked to Sepharose. Antioxidized LDL antibodies were isolated from selected serum samples obtained from eight subjects. Seven of them (six patients and one control subject) had high levels of antioxidized LDL antibody during screening. The other subject, a healthy volunteer, had a low level of antibody. All purified antibodies contained IgG (of subclasses 1 and 3) as the predominant isotype and were primarily specific for oxidized LDL but showed some cross-reactivity with malondialdehyde-modified LDL and native LDL. Two of the purified antibodies cross-reacted with cardiolipin. We determined average dissociation constants for the antioxidized LDL antibodies purified from five individuals, which varied between 2.4x10^-7 and 7.5x10^-7 mol/L, whereas the average dissociation constant of rabbit hyperimmune anti-LDL antibody was determined to be 2.7x10^-8 mol/L. In conclusion, we have purified human autoantibodies reactive with oxidized LDL that appear to be predominantly of moderate-to-low affinity and of variable cross-reactivity. The predominance of IgG1 and IgG3 antibodies is significant from the standpoint of potential pathogenicity, since these two subclasses activate the classic complement pathway system and have the highest binding affinities for Fc receptors on phagocytic cells.

Key Words: autoimmunity • antioxidized LDL autoantibody isolation • arteriosclerosis • affinity constants • isotypes

	Introduction

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In recent years, the hypothesis that lipoproteins can be modified in vivo by free radical–mediated oxidation and that these modified lipoproteins may initiate or contribute to the development of atherosclerosis has received considerable attention. It has been shown that endothelial cells, smooth muscle cells, and macrophages are able to oxidize LDL in vitro,^{1 2 3 4} that oxidized lipoproteins are present in atheromatous lesions,^{5 6 7} and that LDL extracted from human and rabbit atherosclerotic lesions exhibits nearly all of the physicochemical and immunologic properties of LDL oxidized in vitro.⁸ Strong support for the pathogenic role of oxidized lipoproteins in atherogenesis has also been obtained in studies showing that probucol and other antioxidants can inhibit plaque formation in cholesterol-fed animals^{9 10} and in clinical studies showing that LDL isolated from patients with confirmed arteriosclerosis has increased susceptibility to oxidation.^{11 12 13 14}

Several theories have been proposed to explain how oxLDL may contribute to atherosclerosis, on the basis of experimental data showing that this type of modified LDL stimulates macrophage transformation into foam cells, induces endothelial cell functional changes, such as the expression of cell adhesion molecules that leads to enhanced monocyte-endothelial interactions, and is cytotoxic in vitro to cultures of vascular endothelial and smooth muscle cells.^{15 16 17 18}

Furthermore, modified LDL can trigger an autoimmune response leading to the formation of autoantibodies and LDL-IC.^{5 19 20 21 22 23} Anti-oxLDL antibodies (oxLDL Ab) and LDL-IC have been demonstrated to be present in patients with vascular disease and in healthy subjects.^{20 21 22 23} The pathogenic role of LDL-IC has been suggested by experiments demonstrating that incubation of cells with LDL-IC significantly disturbs lipoprotein and cholesterol metabolism.^{24 25 26 27 28} We have carried out studies using human monocyte-derived macrophages in our laboratory^{24 26 27 28} and showed that incubation with LDL-IC is more efficient that any other known stimulus for the induction of foam-cell formation in vitro, leading also to the release of cytokines²⁹ and to a paradoxical increase in the expression of the LDL receptor.^{24 27 28}

The hypothesis that LDL-IC play a pathogenic role in atherosclerosis is supported by the evidence collected by several groups suggesting that oxLDL Ab may be formed in vivo.^{20 21 30 31} However, most of this supporting evidence is based on the results of screening immunoassays that do not seem to yield consistent results. Indeed, while some groups have reported that oxLDL Ab are more frequently detected in patients with confirmed atherosclerosis,^{21 30 31} we and others have been unable to demonstrate differences in antibody levels when comparing patients who have coronary disease and healthy volunteers.^{20 32}

One factor that may contribute to the discrepancies between different studies is the relative lack of specificity of the assays used for the detection of anti-modified LDL antibodies. With slight variations, most published studies are based on EIAs that compare the reactivity of a given serum sample with immobilized modified LDL and immobilized native LDL, with the results being expressed either as a difference or a ratio that reflects the increased binding to modified LDL. However, this approach may lead to erroneous values because it ignores the fact that oxidation of LDL adds negatively charged groups to the LDL molecule,³³ increasing the potential for nonspecific interactions with IgG. The possible interference of nonspecific interactions is aggravated by the fact that the antibodies to oxLDL appear to be of low affinity.³⁴

Thus, isolation of anti-oxLDL antibodies was of the utmost importance, not only to confirm their presence in sera found to be positive in screening assays but also to allow their adequate characterization.

	Methods

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Patients
We selected for antibody isolation eight serum samples, representing a wide range of antibody concentrations according to our screening assay, for the isolation studies (see Table 1). Two samples were obtained from normal healthy volunteers. One obtained from a 27-year-old woman contained a high concentration of anti–oxLDL Ab; whereas the other, obtained from a 51-year-old man, contained anti–oxLDL Ab in low concentrations. Both individuals were clinically asymptomatic and were not receiving any medications or taking vitamin supplements at the time of the study. The remaining samples contained high titers of antibody. One was obtained from a 57-year-old woman who had aortic atherosclerosis and hypercholesterolemia, treated with Lovastatin. Three were obtained from patients with diabetes mellitus: a 41-year-old woman with type I (insulin-dependent) diabetes, free of complications; and two men, 55 and 71 years old, with type II diabetes and established macrovascular disease (one of them with positive anti-cardiolipin antibodies and a history of treated syphilis). The other two patients (a 36-year-old woman and a 47-year-old man) who had high titers of antibody had histories of drug abuse and treated syphilis; both had positive results in the anti-cardiolipin antibody test.

View this table: [in this window] [in a new window]   Table 1. General Information About the Subjects From Whom the Serum Used for Isolation and Characterization of OxLDL Antibodies Was Obtained

Blood was collected from these patients as part of an ongoing study of anti–oxLDL Ab and other risk factors for arteriosclerosis. Informed consent was obtained from all participants in the study. The blood samples from each subject were collected under standard conditions and allowed to clot at 37°C for 1 hour, and serum was separated by centrifugation. All sera were screened for oxLDL Ab, seven were tested for anti-cardiolipin Ab, and six were tested for anti-phospholipid Ab. Antibody assays were carried out not later than 10 days after blood collection, and the serum samples were kept at 4°C until assayed. The samples used for isolation of anti–oxLDL Ab were stored at -70°C until chromatography was performed.

Measurement of OxLDL Ab
The concentrations of total oxLDL Ab in the serum and in fractions obtained after affinity chromatography were measured by the competitive EIA previously developed in our laboratory.²⁰ In brief, flat-bottomed Immulon type 1 plates were coated by adding 750 ng of oxLDL in 0.08 mol/L carbonate–0.17 mol/L bicarbonate buffer, pH 9.6, in each well. The plates were coated overnight at 4°C immediately before the assay was performed. After unbound oxLDL was washed off, the plates were blocked with 5% bovine serum albumin in PBS, pH 7.4, and washed with PBS-Tween 20. Serum samples and fractions isolated by affinity chromatography were tested both unabsorbed and absorbed with oxLDL (100 µg/mL). Serial dilutions of the absorbed and unabsorbed aliquots were prepared (1:10 to 1:40 for serum samples and 1:2 to 1:8 for fractions eluted from affinity chromatography columns) and added to the oxLDL-coated plates (100 µL/well). After incubation, the plates were washed and peroxidase-conjugated rabbit anti-human IgG (Organon Teknika/Cappel, Cat. No. 55221), reactive with both heavy and light chains, at a 1:5000 dilution, was added. After incubation and washing, a solution of 0.5 mmol/L 2.2'-azino-di-(3-ethylbenzthiazoline-6-sulfonate) in 45 mmol/L citric acid buffer, pH 4.0, was added to the plates and incubated in the dark for 10 minutes. The reaction was stopped with 0.1 mol/L citric acid, and the plates were read at 414 nm in a VMax enzyme-linked immunosorbent assay reader (Molecular Devices). The antibody concentrations are expressed as the difference between OD readings obtained from unabsorbed and absorbed samples. For serum oxLDL Ab, each value given is the average of three determinations at two different dilutions (1:10 and 1:20). For chromatography isolated fractions, the given values are averages of two determinations at two different dilutions (1:2 and 1:4).

IgA anti-oxLDL antibodies were measured using the same protocol but replacing the anti-human IgG conjugate with a peroxidase-conjugated anti-IgA, -chain specific (Organon Teknika/Cappel, Cat. No. 55251), used at a dilution of 1:4000.

Lipoprotein Isolation, Modification, and Characterization
Blood for lipoprotein isolation was collected in EDTA (1 mg/mL) after 12 hours of fasting. LDL (1.019<d<1.063) was isolated from plasma, after density adjustment with KBr^-, by preparative ultracentrifugation at 50 000 rpm/min for 22 hours on a Beckman L5-50 ultracentrifuge, using a type 50 rotor.³⁵ LDL preparations were washed by ultracentrifugation, dialyzed against a pH 7.4, 0.15 mol/L NaCl solution containing 1mmol/L EDTA, passed through an Acrodisc filter (0.22-µm pore size) to remove aggregates, and stored under nitrogen in the dark.

Oxidation of LDL was performed by incubation at 37°C for 18 hours of freshly isolated LDL diluted in PBS, pH 7.4, to a final concentration of 300 µg/mL in the presence of 10 µmol/L Cu²⁺.³⁶ The reaction was stopped by the addition of 200 µmol/L EDTA and 40 µmol/L butylhydroxytoluene (BHT), and the oxLDL was dialyzed against PBS containing 200 µmol/L EDTA and 40 µmol/L BHT.

MDA modification was carried out as described by Haberland et al,³⁷ by incubating freshly isolated LDL with 0.1 mol/L MDA for 3 hours at 37°C, followed by extensive dialysis against 0.15 mol/L NaCl with 0.001 mol/L EDTA, pH 7.4.

The degree of oxidation of oxLDL was measured by a modification of the TBARS assay³⁸ and by fluorescence spectroscopy.³⁹ The generation of TBARS increased from 1.13 (native LDL) to 14.8 nmol MDA equivalents/mg LDL protein (oxLDL). The fluorescence intensity of the oxLDL at 360 nm excitation/430 nm emission was 16.09-fold higher than that of native LDL.

Measurement of Anti-Cardiolipin and Anti-Phospholipid Antibodies
Serum anti-cardiolipin antibody was determined with an anti-cardiolipin semiquantitative test kit (Reaads Medical Products, Inc). The concentration of serum anti-phospholipid antibody was measured with the Asserachrom APA kit (Diagnostica Stago).

Isolation of Autoantibodies
Antibodies to oxLDL were isolated by an affinity chromatography protocol developed by us. To prepare immobilized oxLDL, we started by coupling 10 mg of freshly isolated LDL to 2 g of CNBr-Sepharose 4B (Pharmacia Biotech). Coupling was allowed to proceed for 18 hours at 4°C, with the tube containing the gel slurry placed on a rocking platform. Remaining residual active groups were blocked with 0.2 mol/L glycine, pH 8.0, for 2 hours at room temperature. After blocking, the slurry was transferred to a chromatography column. The excess of uncoupled ligand was removed by three cycles of washing with NaHCO₃ buffer (0.1 mol/L, pH 8.3) and acetate buffer (0.5 mol/L, pH 4) both containing 0.5 mol/L NaCl. The column was washed with PBS, equilibrated immediately thereafter with 10 µmol/L CuCl₂, and incubated under these conditions for 18 hours at 37°C. Oxidation was stopped by washing the column with PBS containing 200 µmol/L EDTA and 40 µmol/L BHT. After the oxidation step was completed, the column was washed in sequence with NaHCO₃ and acetate buffers, as described above, and then equilibrated with 0.01 mol/L NaHCO₃ buffer, pH 8.3. To isolate oxLDL Ab we used 1 mL of serum with known anti-oxLDL content diluted in 4 mL of the same bicarbonate buffer used to equilibrate the column. The diluted serum was allowed to diffuse into the column, and the serum-loaded column was incubated overnight at 4°C. Unbound proteins were washed off with the equilibrating buffer and two bound fractions were eluted in sequence, the first one with 0.1 mol/L, NaHCO₃ buffer containing 0.5 mol/L NaCl, pH 8.3, and the second with 0.5 mol/L acetate buffer also containing 0.5 mol/L NaCl, pH 4.0.

Immunoglobulin Isotype Distribution in Purified OxLDL Antibodies
The overall distribution of immunoglobulin isotypes in the fractions eluted from the column was determined by double immunodiffusion assay.⁴⁰ IgG, IgM, and IgA were quantified in the eluted fractions from five sera by radial immunodiffusion (low level RID kits, The Binding Site). The distribution of IgG subclasses in eluted antibody peaks was also determined by radial immunodiffusion using ultralow level RID plates obtained from The Binding Site.

Specificity of Purified OxLDL Antibodies
To determine the specificity of purified oxLDL antibodies a modification of our EIA for anti–oxLDL Ab was used. The reactivity of eluted autoantibodies with immobilized oxLDL was tested using Immulon plates prepared as described earlier, in which a series of aliquots containing purified antibody were tested. The concentrations of isolated antibody used in these studies ranged from 120 to 370 µgEq/L. Five aliquots with identical antibody concentration were studied for each purified antibody. One of the aliquots was unabsorbed and the remaining were absorbed with oxLDL, MDA-modified LDL, native LDL, and cardiolipin (all at final concentrations of 100 mg/L), as previously described.²⁰ The final volume of the aliquots (absorbed and unabsorbed) was 0.4 mL. The samples were incubated overnight at 4°C and then centrifuged at 5000 rpm for 10 minutes. Any visible precipitate and the bottom 100-µL layer of each tube were discarded. Two dilutions (1:2 to 1:4) were tested for each aliquot. The results were expressed as the percent in reduction of reactivity with oxLDL after absorption, determined by subtracting the OD from the two dilutions of the absorbed aliquots from the OD of identical dilutions of the unabsorbed aliquot. The degree of reduction of reactivity with oxLDL was considered a direct indication of the reactivity of the purified antibody with each of the lipids and lipoprotein preparations used for absorption.

Determination of the Dissociation Constants (K_d) of Purified Antibodies
An estimate of the affinity of the purified antibody preparations was obtained through the measurement of K_ds by EIA, according to the method described by Friguet et al.⁴¹ For this purpose we also used flat-bottomed Immulon type 1 plates coated with 750 ng of oxLDL per well. Purified oxLDL Ab was used at a final concentration 200 µgEq/L (13.4^-10 mol/L), calculated by extrapolating the difference in OD between unabsorbed and absorbed aliquots of the purified human antibody from a calibration curve established with purified IgG from a rabbit anti-LDL antiserum.²⁰ The levels of anti-oxLDL calculated in this way are not quite exact but allowed us to calculate the dilutions necessary to bring the antibody concentration to the ideal range, which should be as low as possible but not lower than 10^-10 mol/L.⁴¹ A series of antibody aliquots was adsorbed using different concentrations of oxLDL (2.4^-7 to 1.85^-9 mol/L) and was tested together with the unabsorbed sample. Absorbed and unabsorbed samples were incubated in the oxLDL-coated plates overnight at 4°C. At the end of that period the samples were processed as described for the anti-oxLDL assay (see above). The concentrations of antigen and antibody along with the absorbance values measured in unabsorbed and absorbed samples were used to construct a plot of v/a versus v where v corresponds to bound antibody and v/a to bound antibody/free antigen. The slope of the plot was used to calculate the K_d for each tested sample. The plots showed the existence of high- and low-affinity antibody populations for four of the six purified antibodies, and we calculated both average affinities and the affinities of the two distinct components. The validity of the method was tested by calculating the fraction of antibody retained by overnight incubation in antigen-coated wells⁴¹ with the use of one of our purified anti-LDL antibodies (subject A). A value of 0.15 was obtained for the retained fraction at the concentrations used for K_d calculation, showing that the fraction of antibody trapped in the EIA represents a relatively small proportion of the total antibody and, therefore, should not affect significantly the antigen/antibody equilibrium in the samples adsorbed with oxLDL. All calculations were performed using the plotting and data analysis tools of the Microsoft Excel 4.0 program.

	Results

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Isolation of OxLDL Ab
All but two sera showed two elution peaks of antioxidized LDL antibody, after uncoupled protein was washed out from the oxLDL column. Fig 1 represents a typical elution profile. The two eluted peaks and the washout peak were tested by EIA to determine the concentration of oxLDL Ab. In all cases the first eluted peak, obtained with 0.1 mol/L, pH 8.3, NaHCO₃ buffer+0.5 mol/L NaCl, contained significantly greater concentrations of antibody than the second peak, eluted with 0.5 mol/L, pH 4.0, acetate buffer+0.5 mol/L NaCl (Table 2). For further characterization of purified antibodies we used the first elution peak because the second did not contain sufficient amounts of oxLDL Ab for additional studies.

View larger version (17K): [in this window] [in a new window]   Figure 1. Elution profile obtained when serum C was fractionated by affinity chromatography in a Sepharose-oxLDL column. The concentration of antibody in whole serum was estimated as 2452 µgEq/L; the antibody content of tubes 11 and 20 was estimated to be, respectively, 2070 µgEg/L and 504 µgEq/L.

View this table: [in this window] [in a new window]   Table 2. Concentrations of OxLDL Antibodies in the Different Peaks Eluted From the Immobilized OxLDL Column

Characterization of Isolated Anti-OxLDL Antibodies
We recovered anti-oxLDL antibodies from every serum tested, including that of subject B, a normal control subject with low antibody concentrations in the unfractionated serum (Tables 1 and 3). The concentration of antibody in the first and second elution peaks varied from patient to patient, and the absorption of anti-oxLDL antibody to the column was not complete, judging from the finding of antibody in the washout peaks (Table 2). This could reflect the fact that the capacity of absorption of the column was saturated or that even in the mild washing conditions used in our protocol anti-oxLDL antibodies of low affinity were eluted from the column.

View this table: [in this window] [in a new window]   Table 3. Immunoglobulin Isotype Distribution in Purified OxLDL Antibodies

Immunoglobulin isotype distribution was determined in the isolated antibodies (Table 3). According to double immunodiffusion assays, all purified antibodies showed predominance of IgG, although in five cases IgM was also detected. Quantification by RID confirmed that IgG was the predominant isotype, exceeding by a factor of 2 to 13 times the concentration of IgM. In subject E, a type II diabetic patient with macrovascular disease, IgA was also detected in the purified antibody fraction. The concentration of IgA anti-oxLDL antibody determined by EIA was also maximal in the antibody purified from this subject. Quantification of IgG subclasses revealed predominance of IgG1 and IgG3, which represented 89% to 93% of the total amount of IgG in all purified antibodies.

The specificity of the purified anti-oxLDL antibodies was determined by testing the reduction in reactivity with oxLDL caused by absorption with oxLDL, MDA-modified LDL, native LDL, and cardiolipin (Table 4). In all cases, the greatest reduction in reactivity (59% to 75%) was observed after absorption with oxLDL. Most isolated antibodies showed their highest degree of cross-reactivity with MDA-modified LDL, with reductions in reactivity with oxLDL ranging from 20% to 42% after absorption with MDA-modified LDL. The cross-reactivity with native LDL varied widely between different isolated antibodies (9% to 30%). Two purified antibodies appeared to cross-react with cardiolipin. One was purified from a patient with a history of treated syphilis and the other from a patient with anti-phospholipid antibodies in the serum (Table 1) but no other evidence suggesting a diagnosis of systemic lupus erythematosus or anti-phospholipid antibody syndrome.

View this table: [in this window] [in a new window]   Table 4. Specificity and Dissociation Constants of Purified OxLDL Antibodies

The K_d of anti-oxLDL antibodies purified from six different sera and of the isolated IgG fraction from a rabbit hyperimmune serum were measured by EIA. In four cases, the plots of v versus v/a were similar to the one represented in Fig 2, which shows two components of different affinities. In two cases, the plots of v versus v/a showed one single component (Table 4). In the four cases in which two components of different affinities were evident, we calculated both an average K_d and separate K_d values for the high and low affinity components (as shown in Table 4). The average K_d values for the human antibodies ranged from 2.4x10^-7 to 7.5x10^-7 mol/L. The high affinity components had K_d values ranging from 1.6x10^-7 to 2.9x10^-7 mol/L, whereas the low affinity components had K_d values ranging from 6.3x10^-7 to 8.3x10^-7 mol/L. For comparison we measured the average and component K_d values for rabbit anti-LDL antibody. The average K_d was 2.7x10^-8 mol/L, with a high affinity component with a K_d of 9.3x 10^-9 mol/L and a low affinity component with a K_d of 6.9x10^-8 mol/L. From these observations it is possible to conclude that human antibodies are of lower affinity than rabbit hyperimmune antibodies, the K_d values for human antibodies being about 10-fold lower than the K_d of rabbit antibodies.

View larger version (10K): [in this window] [in a new window]   Figure 2. Plot of the v vs v/a values calculated for the antibody isolated from patient's C serum. It is clear from the plot that there are two antibody populations with different Kd values.

As mentioned earlier, we always eluted two fractions from the immobilized oxLDL column (see Fig 1). The peak eluted with 0.5 mol/L, pH 4.0, acetate buffer+0.5 mol/L NaCl could contain a small subpopulation of antibodies of higher affinity. We have not been able to test this hypothesis due to the very low antibody content in these peaks. However, one can safely conclude that this fraction represents a very small proportion of the total antibody population.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Evidence suggesting the involvement of humoral and cellular immune reactions in different stages of development of the atherosclerotic process has been recently reviewed by several different groups,^{42 43 44} reflecting a recent surge of interest in the investigation of immunologic factors that may contribute to the pathogenesis of atherosclerosis.⁴³ Another area in which the attention of investigators has been focused is the study of the biological implications of LDL modification. Strong evidence has been published in recent years suggesting that modified LDL is generated in vivo. OxLDL has been demonstrated in the peripheral blood,⁴⁵ in the arterial wall,⁴⁶ and in atheromatous plaques.^{6 7 47} It is believed that the oxidation of LDL occurs in the arterial walls where there is an increased level of redox-active metal ions and LDL is sequestered from circulating antioxidants.⁴³ Most authors have concentrated their attention on the study of the atherogenic properties of oxLDL,^{6 16 30 37} but there is also good evidence that oxLDL is immunogenic, since antibodies to Cu-oxidized LDL and MDA-modified LDL have been detected by several groups.^{20 21 30 31 33 34} A potential pathogenic role for autoantibodies to LDL is supported by the evidence that has been collected by several groups showing that LDL-IC prepared in vitro or isolated from patient's sera can induce intracellular accumulation of cholesterol esters,^{22 23 24 25 26 27 28 29} by the finding of higher concentrations of oxLDL Ab in patients with confirmed atherosclerosis,^{21 30 31} and by studies suggesting that the titer of MDA-modified LDL Ab might be an independent predictor of carotid atherosclerosis progression.²¹ However, we²⁰ and others³² have failed to observe correlations between anti-oxLDL antibody levels and clinical or angiographic indices of arteriosclerosis. Given the obvious interest that surrounds the pathogenic role of anti-oxLDL antibodies, we decided to attempt to isolate and characterize such antibodies from a group of serum samples in which we had detected them by our screening immunoassay.^{20 34} In keeping with our previous observations, the samples with higher concentrations of antibody included sera from healthy volunteers and from patients with a variety of diagnoses, including patients with macrovascular atheromatous disease. For this purpose we devised an original affinity chromatography protocol that allowed us to isolate antibodies to oxLDL Ab from all the sera selected for the purpose.

The success of our isolation protocol was largely due to our initial assumption that the human antibodies to oxLDL should be of relatively low affinity, on the basis of the fact that absorption of antibody-containing sera with oxLDL fails to reduce the reactivity of the absorbed sample by more than 46%. Thus, we used a low ionic strength buffer for washing the column after incubation of the antibody-containing serum sample, and eluted most of the antibody just by eluting the column with a higher molarity buffer. A small proportion of antibody remained bound to the immobilized oxLDL and required elution with 0.5 mol/L acetate+0.5 mol/L NaCl, pH 4. This suggests that there is heterogeneity in the affinity of oxLDL Ab, which was also obvious in the peak eluted with 0.1 mol/L NaHCO₃ buffer containing 0.5 mol/L NaCl. The plots of v versus v/a obtained in the K_d studies with the use of purified anti-oxLDL antibody clearly suggest that the main antibody peak contains components of higher and lower affinities.

The average K_ds of the six isolated oxLDL Ab showed some variation, ranging from 2.4x10^-7 to 7.5x10^-7 mol/L, although as a group these values indicate that the antibodies to oxLDL are predominantly of moderate-to-low affinity. In contrast, the average K_d for a hyperimmune rabbit anti-LDL antibody was 2.7x10^-8 mol/L, a value indicative of moderately high affinity, as expected from an hyperimmune serum. The K_d for the high affinity component of the isolated human anti–oxLDL Ab also showed some variability, ranging from 1.6x10^-7 to 2.9x10^-7 mol/L. It is possible that differences in affinity may have an impact in the pathogenic potential of oxLDL-IC, since those immune complexes formed with high-affinity antibodies are likely to be more stable and more likely to cause complement fixation or to interact with Fc receptors.

The isotypic characterization of the isolated anti-oxLDL antibodies showed that IgG was the predominant immunoglobulin isotype, representing 68% to 93% of the total amount of recovered immunoglobulins. A small amount of IgM was also detectable in most purified antibodies, whereas IgA was detected only in the antibody purified from one diabetic patient with macrovascular disease. This is in contrast to earlier reports by Beaumont and Beaumont⁴⁸ who suggested that IgA was the predominant immunoglobulin isotype of anti-LDL autoantibodies. We also determined the IgG subclass distribution in the purified antibodies and found that the most biologically active IgG1 and IgG3 subclasses predominated. This is a significant finding because IgG1 and IgG3 antibodies are efficient activators of the complement system, interact with the three known types of FcR,⁴⁹ and, consequently, are more likely to induce the formation of immune complexes with inflammatory properties.⁵⁰

There is limited information concerning the epitopes recognized by oxLDL Ab. Two epitopes characteristic of oxLDL have been identified thus far: lysine-MDA and lysine-4-hydroxy-nonenal.⁵¹ The demonstration of these epitopes in association to apolipoprotein B localized in arteriosclerotic lesions^{5 6 8} supports their generation under in vivo oxidizing conditions, and, as such, these epitopes appear to be prime candidates for recognition by anti–oxLDL Ab. On the other hand, Vaarala et al⁵² have published data suggesting that patients with systemic lupus erythematosus have antibodies that react both with phospholipids and MDA-modified LDL. The antibodies purified by our protocol cross-reacted with MDA-modified LDL but to a different extent (the reductions in reactivity after absorption with MDA-modified LDL ranged from 19% to 42%), probably reflecting the reactivity with epitopes common to both types of oxLDL. There was variable cross-reactivity with normal LDL, and two samples were found to cross-react with cardiolipin. It seems obvious that most oxLDL Ab are not directed to phospholipids, and it is possible that the cross-reactivity observed with cardiolipin may result, in fact, from the copurification of anti-phospholipid antibodies with antibodies directed to other epitopes of oxLDL. The existence of cardiolipin antibodies that apparently do not cross-react with oxLDL could indicate that those are antibodies of narrow specificity, as described by Vaarala et al.⁵² The different degree of cross-reactivity with MDA-modified LDL and native LDL is another factor that may be related to different pathogenic properties of oxLDL Ab. Obviously, questions such as these cannot be answered by our study—since it contained a very small number of samples and these questions were not our primary concern—but they certainly seem to emerge from the results obtained in this study.

It must be stressed that we are reporting the first successful effort to isolate and characterize human autoantibodies reactive with oxLDL. Orekhov et al¹⁹ have published data concerning the purification of anti-LDL antibodies, but their protocol involved affinity chromatography that used immobilized native LDL. The data of Orekhov et al also suggest that the antibodies were of moderate affinity, IgG being the predominant isotype; in contrast to the relative specificity of oxLDL Ab, anti-LDL antibodies appear to cross-react extensively with most types of modified LDL.

Thus, we have proved that humans can produce autoantibodies specifically directed to oxLDL. These antibodies are of relatively low affinity and can be isolated from the sera of patients with a variety of conditions, as well as from the sera of normal, healthy individuals. Comparison of the purified antibodies from different individuals revealed heterogeneity in average affinity, ratio of moderate- to low-affinity antibodies, cross-reactivity with native LDL and phospholipids, and/or isotype distribution. These differences may explain some of the discrepancies in the report by several groups that have been attempting to study the correlations between the existence of anti–oxLDL Ab and clinical evidence and/or evolution of arteriosclerosis. For example, it is possible that the different modalities of EIA used for screening of the anti–oxLDL Ab may be affected by antibody heterogeneity in different ways. Thus, more detailed characterizations of circulating antibodies in larger groups of patients or, perhaps, the detection and characterization of circulating LDL–anti-LDL IC may help clarify the pathogenic significance of the autoimmune response to modified LDL.

	Selected Abbreviations and Acronyms

 

Ab = antibody EIA = enzyme immunoassay LDL-IC = LDL-containing immune complexes MDA = malondialdehyde OD = optical density oxLDL = oxidized LDL PBS = phosphate-buffered saline TBARS = thiobarbituric acid–reactive substances

	Acknowledgments

 
This research was supported in part by the Research Service of the Ralph H. Johnson Department of Veterans Affairs Medical Center and by grant HL46815 from the National Institutes of Health. The authors wish to thank Alva Mullins for her editorial assistance and Elizabeth C. Fair and Candace Enockson for their technical help.

Received July 26, 1995; accepted December 1, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Steinbrecher UP, Parthasarathy S, Leake DS, Witztum JL, Steinberg D. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids. Proc Natl Acad Sci U S A. 1984;81:3883-3887. [Abstract/Free Full Text]

2. Morel DW, DiCorleto PE, Chisolm GM. Endothelial and smooth muscle cells alter low-density lipoprotein in vitro by free radical oxidation. Arteriosclerosis. 1984;4:357-364. [Abstract/Free Full Text]

3. Cathcart MK, Morel DW, Chisolm GM. Monocyte and neutrophils oxidize low density lipoprotein making it cytotoxic. J Leukoc Biol. 1985;38:341-350. [Abstract]

4. Parthasarathy S, Printz DJ, Boyd D, Joy L, Steinberg D. Macrophage oxidation of low-density lipoprotein generates a modified form recognized by the scavenger receptor. Arteriosclerosis. 1986;6:505-510. [Abstract/Free Full Text]

5. Palinski W, Rosenfeld ME, Ylä-Herttuala S, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL. Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci U S A. 1989;86:1372-1376. [Abstract/Free Full Text]

6. Haberland ME, Fong D, Cheng L. Malonyldialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits. Science. 1988;241:215-218. [Abstract/Free Full Text]

7. Yla-Herttuala S, Palinski W, Rosenfeld ME, Steinberg D, Witztum JL. Lipoproteins in normal and atherosclerotic aorta. Eur Heart J. 1990;11(suppl E):88-99.

8. Yla-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086-1095.

9. Carew TE, Schwenke DC, Steinberg D. Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: evidence that antioxidants in vivo can selectively inhibit LDL degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. Proc Natl Acad Sci U S A. 1987;84:7725-7729. [Abstract/Free Full Text]

10. Kita T, Nagano Y, Yokode ML, Ishii K, Kume N, Ooshima A, Yoshida H, Kawai C. Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia. Proc Natl Acad Sci U S A. 1987;84:5928-5931. [Abstract/Free Full Text]

11. Regnstrom J, Nilsson J, Tornvall P, Landou C, Hamsten A. Susceptibility to LDL oxidation and coronary atherosclerosis in man. Lancet. 1991;339:1183-1186.

12. Chiu H-C, Jeng J-R, Shieh S-ML. Increased oxidizability of plasma LDL from patients with coronary heart disease. Biochim Biophys Acta. 1994;225:200-208.

13. Cominacini L, Garbi U, Pastorino AM, Davoli A, Campagnola ML, De Santis A, Pasini C, Faccini GB, Trevisan MT, Bertozzo L, Pasini F, Lo Cascio V. Predisposition to LDL oxidation in patients with and without angiographically established coronary artery disease. Atherosclerosis. 1993;99:63-70. [Medline] [Order article via Infotrieve]

14. Andrews B, Burnand K, Paganga G, Browse N, Rice-Evans C, Sommerville K, Leake D, Taub N. Oxidizability of LDL in patients with carotid or femoral artery atherosclerosis. Atherosclerosis. 1995;112:77-84.[Medline] [Order article via Infotrieve]

15. Steinberg D, Witztum JL. Lipoproteins and atherogenesis. JAMA. 1990;264:3047-3052.[Abstract/Free Full Text]

16. Berliner JA, Territo MC, Sevanian A, Ramin S, Kim JA, Bamshad B, Esterson ML, Fogelman A. Minimally modified low density lipoprotein stimulates monocyte-endothelial interactions. J Clin Invest. 1990;85:1260-1266.

17. Jeng JR, Chang CH, Shieh SM, Chiu HC. Oxidized low-density lipoprotein enchances monocyte-endothelial cell binding against shear stress-induced detachment. Biochim Biophys Acta. 1993;1178:221-227. [Medline] [Order article via Infotrieve]

18. Morel DW, Hessler JR, Chisolm GM. Low density lipoprotein cytotoxicity induced by free-radical peroxidation of lipid. J Lipid Res. 1983;24:1070-1076. [Abstract]

19. Orekhov AN, Tertov VV, Kabakov AE, Adamova IY, Pokrovsky SN, Smirnov VN. Autoantibodies against modified low density lipoprotein: nonlipid factor of blood plasma that stimulates foam cell formation. Arterioscler Thromb. 1991;11:316-326. [Abstract/Free Full Text]

20. Virella G, Virella I, Leman RB, Pryor MB, Lopes-Virella MF. Anti-oxidized low-density lipoprotein antibodies in patients with coronary heart disease and normal healthy volunteers. Int J Clin Lab Res. 1993;23:95-101. [Medline] [Order article via Infotrieve]

21. Salonen JT, Ylä-Herttuala S, Yamamoto R, Butler S, Korpela H, Salonen R, Nyyssönen K, Palinski W, Witztum JL. Autoantibody against oxidized LDL and progression of carotid atherosclerosis. Lancet. 1992;339:883-887. [Medline] [Order article via Infotrieve]

22. Klimov AN, Denisenko AD, Vinogradov AG, Nagornev VA, Pivovarova YI, Sitnikova OD, Pleskov AG. Accumulation of cholesteryl esters in macrophages incubated with human lipoprotein-antibody autoimmune complex. Atherosclerosis. 1988;74:41-46. [Medline] [Order article via Infotrieve]

23. Tertov VV, Orekhov AN, Kacharava AG, Sobenin IA, Perova NV, Smirnov VN. Low density lipoprotein-containing circulating immune complexes and coronary atherosclerosis. Exp Mol Pathol. 1990;52:300-308. [Medline] [Order article via Infotrieve]

24. Lopes-Virella MF, Griffith RL, Shunk KA, Virella G. Enhanced uptake and impaired intracellular metabolism of low density lipoprotein complexed with anti-low density lipoprotein antibodies. Arterioscler Thromb. 1991;11:1356-1367. [Abstract/Free Full Text]

25. Klimov AN, Denisenko AD, Popov AV, Nagornev VA, Pleskov VM, Vinogradov AG, Denisenko TV, Magracheva EY, Kheifes GM, Kuznetzov AS. Lipoprotein-antibody immune complexes: their catabolism and role in foam cell formation. Atherosclerosis. 1985;58:1-15. [Medline] [Order article via Infotrieve]

26. Gissinger C, Virella GT, Lopes-Virella MF. Erythrocyte-bound low density lipoprotein (LDL) immune complexes lead to cholesteryl ester accumulation in human monocyte derived macrophages. Clin Immunol Immunopathol. 1991;59:37-51. [Medline] [Order article via Infotrieve]

27. Gisinger C, Lopes-Virella MF. Lipoprotein-immune complexes and diabetic vascular complications. Diabetes. 1992;41:92-96.

28. Griffith RL, Virella GT, Stevenson HC, Lopes-Virella MF. Low density lipoprotein metabolism by human macrophages activated with low density lipoprotein immune complexes. J Exp Med. 1988;168:1041-1059. [Abstract/Free Full Text]

29. Virella G, Muñoz JF, Galbraith GMP, Gissinger C, Chassereau C, Lopes-Virella MF. Activation of human monocyte-derived macrophages by immune complexes containing low density lipoprotein. Clin Immunol Immunopathol. 1995;75:179-189. [Medline] [Order article via Infotrieve]

30. Maggi E, Chiesa R, Melissano G, Castellano R, Astore D, Grossi A, Finardi G, Bellomo G. LDL oxidation in patients with severe carotid atherosclerosis: a study of in vitro and in vivo oxidation markers. Arterioscler Thromb. 1994;14:1892-1899. [Abstract/Free Full Text]

31. Bergmark C, Wu R, De Faire U, Lefvert AK, Swedenborg J. Patients with early-onset peripheral vascular disease have increased level of autoantibodies against oxidized LDL. Arterioscler Thromb Vasc Biol. 1995;15:441-445. [Abstract/Free Full Text]

32. Schumacher ML, Eber B, Tatzber F, Kaufmann P, Esterbauer H, Klein W. LDL oxidation and coronary atherosclerosis. Lancet. 1992;340:123. Letter.

33. Salmon S, Maziere C, Theron L, Beucler I, Ayrault-Jarrier ML, Goldsten S, Polonovski J. Immunological detection of low-density lipoproteins modified by malondialdehyde in vitro or in vivo. Biochim Biophys Acta. 1987;920:215-220. [Medline] [Order article via Infotrieve]

34. Virella G, Mironova M, Lopes-Virella MF. Comparing assays of antibodies to modified low-density lipoproteins. Clin Chem. 1995;41:324-325. [Free Full Text]

35. Lopes-Virella MF, Sherer GK, Lees AM, Wohltmann H, Mayfield R, Sagel J, LeRoy EC. Surface binding, internalization, and degradation by cultured human fibroblasts of LDL isolated from type 1 (insulin-dependent) diabetic patients: changes with metabolic control. Diabetologia. 1982;22:430-436. [Medline] [Order article via Infotrieve]

36. Steinbrecher UP. Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products. J Biol Chem. 1987;262:3603-3608. [Abstract/Free Full Text]

37. Haberland ME, Fogelman AM, Edwards PA. Specificity of receptor-mediated recognition of malondialdehyde-modified low density lipoproteins. Proc Nat Acad Sci U S A. 1982;79:1712-1716. [Abstract/Free Full Text]

38. Kim RS, LaBella FS. Comparison of analytical methods for monitoring auto-oxidation profiles of authentic lipids. J Lipid Res. 1987;28:1110-1117. [Abstract]

39. Cominacini L, Garbin U, Davoli A, Micciolo R, Bosello O, Gaviraghi G, Scuro LA, Pastorino AM. A simple test for predisposition to LDL oxidation based on the fluorescence development during copper-catalyzed oxidative modification. J Lipid Res. 1991;32:349-358. [Abstract]

40. Virella G. Antigen-antibody reactions. In: Virella G, ed. Introduction to Medical Immunology. 3rd ed. New York, NY: Marcel Dekker; 1993:126-127.

41. Friguet B, Chaffotte AF, Djavadi-Ochaniace L, Goldberg ME. Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-immunosorbent assay. J Immunol Methods. 1985;77:305-319. [Medline] [Order article via Infotrieve]

42. Libby P, Hansson GK. Involvement of the immune system in human atherogenesis: current knowledge and unanswered questions. Lab Invest. 1991;64:5-15. [Medline] [Order article via Infotrieve]

43. Lopes-Virella MF, Virella G. Short analytical review: atherosclerosis and autoimmunity. Clin Immunol Immunopathol. 1994;73:155-167. [Medline] [Order article via Infotrieve]

44. Wick G, Schett G, Amberger A, Kleindienst R, Xu Q. Is atherosclerosis an immunologically mediated disease? Immunology Today. 1995;16:27-33. [Medline] [Order article via Infotrieve]

45. Avogaro P, Bittolo Bon G, Cazzolato G. Presence of a modified low density lipoprotein in humans. Arteriosclerosis. 1988;8:79-87. [Abstract/Free Full Text]

46. Hoff FH, Gaubatz JW. Isolation, purification, and characterization of a lipoprotein containing apo B from the human aorta. Atherosclerosis. 1982;42:273-297. [Medline] [Order article via Infotrieve]

47. Hoff H, O'Neil J. Lesion-derived low density lipoprotein and oxidized low density lipoprotein share a lability for aggregation, leading to enchanced macrophage degradation. Arterioscler Thromb. 1991;11:1209-1222. [Abstract/Free Full Text]

48. Beaumont JL, Beaumont V. Immunological aspects of atherosclerosis. Atherosclerosis Rev. 1978;3:133-146.

49. Virella G, Wang AC. Biosynthesis, metabolism and biological properties of immunoglobulins. In: Virella G, ed. Introduction to Medical Immunology. 3rd ed. New York, NY: Marcel Dekker; 1993:91-102.

50. Virella G. Immune complex diseases. In: Virella G, ed. Introduction to Medical Immunology. 3rd ed. New York, NY: Marcel Dekker; 1993:379-396.

51. Palinski W, Yla-Herttuala S, Rosenfeld ME, Butler SW, Socher SA, Parthasarathy S, Curtiss LK, Witztum JL. Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein. Arteriosclerosis. 1990;10:325-335. [Abstract/Free Full Text]

52. Vaarala O, Alfthan G, Jauhiainen ML, Leirisalo-Repo ML, Aho K, Palosuo T. Crossreaction between antibodies to oxidized low-density lipoprotein and to cardiolipin in systemic lupus erythematosus. Lancet. 1993;341:923-925.[Medline] [Order article via Infotrieve]

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