Circulating Autoantibodies Recognizing Peroxidase-Oxidized Low Density Lipoprotein
Evidence for New Antigenic Epitopes Formed In Vivo Independently From Lipid Peroxidation
Oxidatively modified LDLs are antigenic and elicit the generation of autoantibodies often detected in plasma and within plaques of atherosclerotic patients. Although Cu2+-oxidized LDL and malondialdehyde (MDA)–modified LDL are usually used as antigens in immunoassays, other, still unrecognized epitopes may be formed in vivo during LDL oxidation and may induce antibody production. Antibodies recognizing LDL oxidatively modified by Cu2+, 2,2′-azobis-(2-amidino propane) hydrochloride (AAPH), and the combination of horseradish peroxidase and H2O2 (HRP) were detected in serum of a group of 90 unselected patients. HRP-oxidized LDL was the antigen that revealed the highest IgG titers, although the extent of LDL oxidation (evaluated as conjugated diene formation, loss of tryptophan fluorescence, production of fluorescent aldehydic adducts, and change in electrophoretic mobility) was comparable to that obtained with Cu2+ and AAPH. There was a highly statistically significant correlation between the IgG titers detected using Cu2+- and AAPH-oxidized LDLs as antigens, but no correlation was found between the IgG titers revealed by HRP and Cu2+ or AAPH. In addition, the antibody titers against MDA-modified LDL exhibited a significant correlation with those against Cu2+- or AAPH-oxidized LDL but did not correlate with those against HRP-oxidized LDL. Finally, immunocompetition experiments revealed that IgG recognizing HRP-oxidized LDL did not cross-react with Cu2+-oxidized LDL and vice versa. The possibility that lipid peroxidation–independent modifications could play a role in HRP-induced formation of antigenic epitopes in LDL was supported by two lines of evidence. First, in probucol-enriched LDL, despite the complete inhibition of lipid peroxidation, HRP, but not Cu2+ and AAPH, was still able to generate epitopes that were recognized by the same sera reacting with HRP-oxidized native (not probucol-enriched) LDL. In addition, the presence of autoantibodies against Cu2+- and AAPH-oxidized LDLs was negatively correlated with serum α-tocopherol concentration, whereas the titers against HRP-oxidized LDL did not exhibit any statistically relevant correlation with α-tocopherol levels. Together, these findings indicate that peroxidase(s)-dependent mechanisms can trigger peculiar lipid peroxidation–independent modifications of LDL in vivo.
- Received January 1, 1996.
- Revision received May 17, 1996.
Oxidative modifications generate molecular epitopes in LDL that exhibit peculiar biological activities.1 Oxidized LDLs activate endothelial cells to express surface adhesion molecules for circulating monocytes and lymphocytes,2 3 are chemotactic for the same cell types,4 and are more avidly taken up by resident macrophages in the subendothelial space to form foam cells.5 Oxidatively modified LDLs are also antigenic and elicit an immune response with the generation of circulating autoantibodies.6
Antibodies to Cu2+-oxidized or MDA-derivatized LDL7 8 have been detected in atherosclerotic plaques9 and in plasma of patients with overt atherosclerotic diseases10 11 or those with classic risk factors such as essential hypertension,12 non–insulin-dependent diabetes mellitus,13 primary hypercholesterolemia,14 and renal failure.15 Few longitudinal studies have provided evidence supporting a diagnostic role of the antioxidized LDL autoantibody titer as an independent predictor for the progression of carotid atherosclerosis16 or the occurrence of myocardial infarction.17
Little is known, however, about the molecular mechanisms responsible for LDL oxidation in vivo and about the nature of the initiating stimuli. A wide variety of in vitro models of LDL oxidation have been developed in the last few years (see Reference 18 for a review), but their adequacy to mimic in vivo oxidation is largely speculative. A considerable attention has been recently focused on the possibility that peroxidase-catalyzed reactions may play a role in oxidizing LDL in vivo.19 20 21 22 In the presence of hydrogen peroxide, several peroxidases, including myeloperoxidase, are able to promote the peroxidation of polyunsaturated fatty acids in LDL, although the molecular mechanism is still largely obscure and the requirement for apoB100 appears absolute. It is worth noting, however, that even indirect evidence supporting the occurrence of such a process in vivo is still lacking.
Here we report that HRP-oxidized LDLs are recognized by circulating autoantibodies detected in serum of humans. These antibodies specifically recognize HRP-oxidized LDLs even when the peroxidation of polyunsaturated fatty acids in LDL was inhibited by probucol. The titers of these autoantibodies do not correlate with those reacting with Cu2+- or AAPH-oxidized LDL or with MDA-derivatized LDL. These findings suggest that epitopes generated during peroxidase-catalyzed LDL oxidation are formed in vivo and differ from those generated during lipid peroxidation–dependent LDL oxidation triggered by Cu2+ or by AAPH.
Ninety unselected healthy subjects (62 men and 28 women, aged 48±12 years) living in the same geographic area and with comparable lifestyle were enrolled in this study. None of the subjects had a clinical history or detectable evidence for atherosclerotic diseases, hypertension, or diabetes mellitus, and none were taking drugs directly or indirectly affecting lipid metabolism or the antioxidant status. Venous blood was taken after overnight fasting, the serum was obtained by conventional centrifugation and stored at −80°C until used.
Venous blood was taken from normal healthy volunteers, after overnight fasting, in polypropylene tubes containing K-EDTA (final concentration, 1 mg EDTA/mL blood), and plasma was collected after centrifugation.10
EDTA-supplemented plasma was incubated at 37°C in a thermostated water bath with a continuous gentle stirring, with or without 1 mmol/L probucol dissolved in absolute ethanol (final ethanol concentration, <1%) for 3 hours. At the end of the incubation, nonenriched and enriched plasmas were processed for LDL isolation as described above.
Native LDLs, obtained after plasma ultracentrifugation, were kept in saline phosphate (10 mmol/L) buffer, pH 7.2, containing EDTA (1 mg/mL) and immediately used to coat ELISA plates.
EDTA was removed from the LDL fraction by rapid gel filtration,10 and LDLs were resuspended in oxygen-saturated PBS (10 mmol/L Pi, pH 7.2) at a concentration of 0.25 mg LDL mass/mL buffer (=50 μg LDL protein/mL=0.1 μmol/L). Three different experimental conditions were used to induce LDL oxidation: Cu2+, used as CuSO4, 2.5 μmol/L final concentration at 30°C; AAPH, 1 mmol/L (freshly-prepared) at 37°C; H2O2, 0.2 mmol/L and HRP, 5 U/mL at 37°C. After 18 hours the modified LDLs were directly used to cover ELISA plates.
Freshly isolated LDLs (2 mg/mL) were incubated for 3 hours at 37°C with 0.5 mol/L MDA or, when indicated, with increasing MDA concentrations ranging from 0.01 to 0.5 mol/L. Free MDA was obtained by acid hydrolysis of MDA–bisdimethyl-acetal. Unbound MDA was then removed by rapid gel filtration. Under these conditions large portions of the ε-amino group in lysine residues were derivatized.
Evaluation of Lipid Peroxidation, Tryptophan Loss, Changes in Electrophoretic Mobility, and MDA- and 4-Adducts in Modified Proteins
The evaluation of lipid peroxidation in LDL was performed after the formation of conjugated dienes at 234 nm, as described by Esterbauer et al.23 Tryptophan loss was measured fluorometrically at 282 nm excitation and 330 nm emission as described in Reference 24. Increases in electrophoretic mobility of modified lipoproteins were measured on Cellogen strips following the manufacturer's directions for electrophoretic conditions, gel staining, and destaining. Results were expressed as modified lipoprotein mobility relative to native lipoprotein mobility. The presence of MDA- and HNE-adducts in modified proteins was detected using scanning fluorescence spectroscopy. The quantitation of the fluorescence intensity at 360 nm excitation/430 nm emission was taken as an indirect measure of the HNE–protein adduct25 and at 400 nm excitation/470 nm emission as an indirect measure of the MDA–protein adduct.25
Serum levels of α-tocopherol were determined as described.23 Briefly, serum was precipitated with the use of ethanol and subsequently extracted with hexane. The hexane phase was then evaporated and the residue was dissolved in methanol and separated by HPLC.
Measurement of Antioxidized LDL and Anti-MDA–Modified LDL Autoantibodies
The quantitation of the different autoantibodies was performed using an ELISA method. Antigens for this assay included native LDL (protected against oxidation by EDTA), oxidized LDL (obtained after extensive oxidation with Cu2+, AAPH, or HRP), and LDL derivatized with MDA as described above. Each well was coated with 10 μg antigen in PBS for 4 hours. The remaining binding sites were then blocked with the use of 3% fetal bovine serum in PBS (coating buffer) for 2 hours at 37°C.
In the present study, a 1:11 dilution of serum from each subject was prepared and 200 μL was added in duplicate to wells coated with native and modified proteins. The 1:11 dilution used here was selected because it gave the highest sample-to-blank ratio. Highest dilutions (1:50, 1:100, and 1:500) gave comparable results. After incubation at 37°C for 2 hours, wells were decanted and washed four times before an appropriate peroxidase-conjugated antibody specific for IgG (diluted 1:2000) was added. After 1 hour of incubation at 37°C and extensive washing, the peroxidase activity was developed using phenylenediamine dihydrochloride and H2O2 as revealing reagents. The absorbance was measured at 492 nm in an automatic microplate reader.
To calculate antibody titers we used the difference between the spectrophotometric readings of anti-modified and anti-native antigen wells. With this approach, the spectrophotometric readings of anti-native antigen wells represent the corresponding blank of anti-modified antigen wells and minimize the possible detection of false-positive values due to cross-reactivity with both epitopes. The intra-assay variability was always lower than 3.4%, and the inter-assay variability was always lower than 7.1%.
Results are expressed as mean±SD of the overall data. The statistical significance of the difference between various parameters was calculated using Student's t test. The correlations were calculated using linear models exclusively for parameters whose distributions did not significantly differ (P<.02) from those predicted by normal distribution models. All the statistical computations were performed using the CSS:Statistica program for personal computers.
Oxidative Modifications Promoted by Cu2+, AAPH and HRP, in LDL
The oxidation of LDL promoted by Cu2+, AAPH, and HRP was associated with significant modifications of the physicochemical properties of the lipoprotein particle. They included the peroxidation of polyunsaturated fatty acids, the loss of tryptophan, the formation of covalent adducts between end products of lipid peroxidation and lysine residues in apoB100, and the increase of the negative charge26 (Table⇓). The oxidatively modified LDLs were then used as antigens to reveal the presence of circulating autoantibodies in the serum of patients.
Antibodies in Human Sera Recognize Cu2+-, AAPH-, and HRP-Oxidized LDL
As reported in Fig 1⇓, the immunoreactivity of oxidatively modified LDL was well detectable with sera from a certain number of patients. However, the overall signal was markedly higher when HRP-oxidized LDL were used as antigen (mean optical density [OD] at 492 nm, 0.680±0.545 ), as compared with both Cu2+- and AAPH-oxidized LDLs (mean ODs at 492 nm 0.451±0.239 and 0.443±0.199, P<.009 and P<.006, respectively). This discrepancy was not due to any unspecific binding of the secondary antibody to HRP-oxidized LDL containing plates. In fact there was no reactivity of the peroxidase-conjugated anti-IgG antibody toward wells exclusively coated with HRP-oxidized LDL. In addition, the presence, in the same population investigated, of patients with high reactivity together with patients with low reactivity is a further evidence against nonspecific events as major determinants of the differences observed. Furthermore, the sera specifically recognizing HRP-modified LDLs did not recognize HRP alone, thus excluding the possibility that HRP covalently linked to apoB100 could be the immune recognition site.
Cu2+, AAPH, and HRP all induce the peroxidation of LDL lipids and the derivatization of apoB100 with aldehydic products of lipid peroxidation. It is thus conceivable to hypothesize that the modified molecular epitopes generated by the three different stimuli would be, at least qualitatively, similar. A statistically significant correlation (r=.814, P<.0001) was in fact detected between the antibody titers against Cu2+-oxidized and AAPH-oxidized LDLs (Fig 2⇓). However, no correlation was detected between the antibody titers against HRP-oxidized LDL and Cu2+-oxidized LDL (r=−.02) or AAPH-oxidized LDL (r=.06).
A statistically significant correlation between the antibody titers against LDL derivatized with MDA-LDL and Cu2+-oxidized LDLs (r=.465, P<.001) or AAPH-oxidized LDL (r=.451, P<.002), respectively, was consistently observed. In addition, no correlation was found with the use of MDA-derivatized LDL and HRP-oxidized LDL as antigens (r=.07) (Fig 3⇓).
A further and more convincing support to the view that Cu2+-oxidized and HRP-oxidized LDLs were recognized by different antibodies was given by immunocompetition studies. As reported in Fig 4⇓, the binding of specific sera to Cu2+-oxidized LDL–coated wells was specifically inhibited by adding increasing amounts of Cu2+-oxidized LDL but not native or HRP-oxidized LDL. Conversely, the binding of specific sera to HRP-oxidized LDL–coated wells was specifically inhibited by adding increasing amounts of HRP-oxidized LDL but not native or Cu2+-oxidized LDL. These findings clearly indicate that the molecular epitopes recognized by sera reacting with HRP-oxidized LDLs differ from those generated in Cu2+-oxidized LDLs.
Antigenic Epitopes Generated by HRP in LDL and Recognized by Circulating Antibodies Are Formed by Lipid Peroxidation–Independent Mechanisms
It is generally believed that, among the various antigenic epitopes formed during Cu2+-induced LDL oxidation, those generated by derivatization of amino groups in aoB100 with breakdown products of lipid peroxidation are quantitatively more relevant.27 The possibility that lipid peroxidation–independent mechanisms may play a significant role in promoting immunologically relevant modifications in HRP-oxidized LDL was investigated using probucol-enriched LDL. Probucol enrichment completely inhibited the formation of conjugated dienes and MDA- or HNE-apoB100 fluorescent adducts upon incubation with either Cu2+, AAPH, or HRP (Table⇑).
As shown in Fig 5⇓, the reactivity of human sera against Cu2+- and AAPH- oxidized LDLs was almost completely abolished when the LDLs used for antigen preparation were previously enriched with probucol. On the other hand, a comparable inhibition was not observed with HRP-oxidized LDL, where probucol enrichment, although efficient in preventing lipid peroxidation, did not prevent the formation of the antigenic epitope(s) recognized by circulating antibodies.
Correlations Between Serum α-Tocopherol and Circulating Antioxidatively Modified LDL Autoantibodies
The role of α-tocopherol in antagonizing LDL oxidation is still a matter of debate, although evidence has been obtained from both small supplementation studies and large epidemiological investigations supporting a protective role of this antioxidant.28 29 30 The results reported in Fig 6⇓ indicate that patients with high levels of α-tocopherol in serum have the lowest titers of autoantibodies against Cu2+-oxidized LDL, AAPH-oxidized LDL, and MDA-derivatized LDL. This suggests that a stronger antioxidant defense (provided by α-tocopherol) would give a better protection against the peroxidation of LDL in vivo and against the generation of autoantibodies recognizing lipid peroxidation–dependent antigenic epitopes in LDL. However, this was not the case of the antigenic epitopes generated during HRP-induced LDL oxidation and recognized by specific autoantibodies. In fact, the highest titers of anti-HRP–oxidized LDL antibodies were not associated with the lowest levels of α-tocopherol in serum, again suggesting that the molecular epitopes recognized by these antibodies were not generated by processes antagonized by α-tocopherol (ie, lipid peroxidation).
Oxidative modifications in LDL can be initiated in vitro by a variety of stimuli (see Reference 18 for a review). However, no clear-cut evidence has been obtained for the suitability of these models to mimic the oxidative process(es) occurring in vivo. A common feature of most of the in vitro systems is the extensive peroxidation of polyunsaturated fatty acids in LDL and the derivatization of amino acid residues in apoB100 by reactive aldehydic products of lipid peroxidation, such as MDAand 4-OH nonenal.18 Thus, the demonstration of circulating autoantibodies against aldehyde-modified LDL, although highly diagnostic for the occurrence of in vivo LDL oxidation, does not give valuable information about the nature of the initiating stimuli. We now report that, despite similar levels of lipid peroxidation achieved in LDLs oxidized by Cu2+, AAPH, and HRP, the recognition of the differently oxidized LDL by antibodies present in human sera is not always comparable. In particular, the antigenic epitopes present in LDL oxidized with HRP, and recognized by human autoantibodies, are qualitatively and quantitatively different from those generated by Cu2+.
The assumption that autoantibodies against HRP-oxidized LDL in serum indicates that similar oxidative mechanisms exist in vivo can be criticized. However, several lines of evidence indicate that at least some of the antigenic epitopes generated by HRP are rather specific and substantially differ from those generated by other common initiators. They include (1) the specific immunocompetition, (2) the lack of correlation between the various antibody titers in the population study, and (3) the inefficiency of probucol in preventing the generation of recognizable antigenic epitopes.
Little is known about the possible antigenic determinants formed in LDL oxidized by HRP, but a few possibilities can be considered. It was rather surprising that HRP-oxidized LDLs did not compete with Cu2+-oxidized LDLs in the immune recognition of Cu2+-oxidized LDLs by IgG present in human sera, despite the generation of comparable MDA- or HNE-adducts in apoB100. Thus, the occurrence of major conformational changes in apoB100 caused by HRP must be hypothesized, and these changes could have made the aldehyde adducts less accessible to the binding of specific antibodies. Moreover, it has been reported that, in the presence of free tyrosine, the combination of myeloperoxidase and H2O2 can generate free and bound dityrosines31 that could be, at least theoretically, immunogenic. Since no free tyrosine was present in the incubation medium during the preparation of HRP-oxidized LDL, the formation of bound dityrosine must be negligible. It cannot be excluded, however, that the generation of intramolecular dityrosine could participate in promoting both large molecular rearrangements and generation of antigenic epitopes. This possibility was further substantiated by preliminary experiments demonstrating that HRP-oxidized LDLs exhibited a significant increase of dityrosine fluorescence (Reference 32 and M.S. and G.B., unpublished results, 1996).
Kalyanaraman et al have recently described the formation of an apoB100 free radical during HRP- induced LDL oxidation whose nature and reactivity was not characterized.21 It can be hypothesized, however, that the molecular rearrangement caused by this free radical in apoB100 could generate immunogenic sites different from the classic aldehyde-derivatized residues.
A relevant consequence of the findings reported in this study is that LDLs may undergo, in vivo, lipid peroxidation–independent modifications making them immunogenic. The correlation between serum α-tocopherol concentration and the titers of anti-Cu2+– and anti-AAPH–oxidized LDLs or anti-MDA–derivatized LDL autoantibody is in agreement with the role of this lipophilic antioxidant in preventing lipid peroxidation–dependent changes in vivo. On the other hand, the lack of correlation between α-tocopherol and anti-HRP–oxidized LDL antibody titers suggests that α-tocopherol may not be so efficient in preventing lipid peroxidation–independent changes. Few studies have reported that oxidatively modified LDLs, even in the absence of lipid peroxidation, possess some typical atherogenic properties, such as an increased uptake by macrophages and an impaired intracellular catabolism.33 34 35 . Altogether these findings could help to explain, at least in part, the failure of many interventional studies in which α-tocopherol was used to prevent the progression of atherosclerosis in humans and suggest the need for new and more efficient antioxidants able also to prevent lipid peroxidation–independent oxidative modifications in LDL.
High titers of circulating autoantibodies to MDA-derivatized LDL and to other epitopes of oxidatively modified LDL are frequently found in patients with severe carotid atherosclerosis, diabetes, and peripheral vascular disease10 11 12 13 14 15 and in LDL receptor–deficient mice with accelerated atherosclerosis.36 Thus, it appears that the detection of autoantibodies against oxidatively modified LDL may be used as a useful biochemical assay in the clinical setting of the patients prone to atherosclerosis or those with overt atherosclerotic disease. Although this approach is already operative and is currently being validated for antibodies to LDL oxidized with Cu2+ or modified with MDA or HNE,18 additional work is required to characterize the possible clinical significance of detecting antibodies to peroxidase-modified LDL.
In conclusion, the results obtained in this study demonstrate that (1) autoantibodies directed against peroxidase-oxidized LDL are present in the serum of humans, (2) the molecular epitopes recognized by autoantibodies in HRP-oxidized LDLs differ from those generated in Cu2+- or AAPH-oxidized LDL and MDA-derivatized LDL, and (3) the generation of these epitopes is largely lipid peroxidation independent.
Selected Abbreviations and Acronyms
|AAPH||=||2,2′-azobis-(2-amidino propane) hydrochloride|
|HPLC||=||high-performance liquid chromatography|
|HRP||=||combination of horseradish peroxidase and H2O2|
This work was supported by grants from Ministero dell'Università e della Ricerca Scientifica e Tecnologica. The excellent technical assistance of Maria Grazia Moretti is greatly acknowledged.
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