© 1998 American Heart Association, Inc.
Original Contributions |
Immunization of LDL Receptor–Deficient Mice With Homologous Malondialdehyde-Modified and Native LDL Reduces Progression of Atherosclerosis by Mechanisms Other Than Induction of High Titers of Antibodies to Oxidative Neoepitopes
From the Department of Medicine, University of California San Diego, La Jolla, Calif.
Correspondence to Wulf Palinski, MD, University of California San Diego, Department of Medicine, 0682, 9500 Gilman Dr, BSB 1080, La Jolla, CA 92093-0682. E-mail wpalinski{at}ucsd.edu
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Abstract |
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Abstract—We and others previously showed that immunization of rabbits with different forms of oxidized low density lipoprotein (LDL) significantly reduced atherogenesis. We now investigated the effect of continued immunization on atherosclerosis in LDL receptor–deficient (LDLR-/-) mice to determine whether a similar reduction of atherosclerosis occurred in murine models and whether this was due to humoral immune responses, ie, formation of high titers of antibodies to oxidation-specific epitopes. Three groups of LDLR-/- mice were repeatedly immunized with homologous malondialdehyde-modified LDL (MDA-LDL), native LDL, or phosphate-buffered saline (PBS) for 7 weeks. Extensive hypercholesterolemia and accelerated atherogenesis were then induced by feeding a cholesterol-rich diet for 17 weeks, during which immunizations were continued. Binding of immunoglobulin (Ig) M and IgG antibodies, as well as IgG1 and IgG2a isotypes, to several epitopes of oxidized LDL were followed throughout the study. After 24 weeks of intervention, atherosclerosis in the aortic origin was significantly reduced by 46.3% and 36.9% in mice immunized with MDA-LDL and native LDL, respectively, compared with PBS (133 558 and 157 141 versus 248 867 µm2 per section, respectively). However, the humoral immune response to oxidative neoepitopes in the MDA-LDL group was very different from that of the LDL or PBS group. IgG antibody binding to MDA-LDL and other epitopes of oxidized LDL, such as oxidized phospholipid (cardiolipin), oxidized cholesterol, or oxidized cholesteryl linoleate, but not native LDL, increased markedly in mice immunized with MDA-LDL, but not in mice immunized with native LDL or PBS. In the MDA-LDL group, both T helper cell (Th)2–dependent IgG1 antibody and Th1-dependent IgG2a antibody binding to oxidative neoepitopes increased significantly over time. The fact that mice immunized with both MDA-LDL and native LDL had a significant reduction in atherosclerosis, whereas only the MDA-LDL group developed very high titers of antibodies to oxidation-specific epitopes, suggests that the antiatherogenic effect of immunization is not primarily dependent on very high titers of antibodies to oxidation-specific epitopes but is more likely to result from the activation of cellular immune responses.
Key Words: arteriosclerosis • oxidized lipoproteins • immune system • autoantibodies • T cells
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Introduction |
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Oxidation of lipoproteins and oxidative processes in general play an important role in the initiation and progression of atherosclerotic lesions. Oxidation of LDL occurs in a wide range of atherosclerotic lesions in humans and animal models,1 2 3 4 5 and by-products generated during this process, such as lipid peroxidation products and modified apoproteins, possess numerous properties that may contribute to atherogenesis (reviewed in References 6 through 86 7 8 ). These include cytotoxic damage to vascular cells, inhibition of vasodilation in response to nitric oxide, chemotactic recruitment of monocytes into the artery wall, phenotypic transformation of monocytes into macrophages, as well as the rapid uptake of oxidized LDL (OxLDL) by macrophages via scavenger receptors. More recently, it has also been recognized that oxidative processes may affect the regulation of gene expression by vascular cells and promote expression of a number of adhesion molecules and cytokines that may in turn contribute to lesion formation.8
The oxidation of LDL results in many structural modifications and the formation of a large number of neoepitopes.1 9 For example, reactive aldehydes generated during lipid peroxidation form covalent adducts with lysine and histidine residues of apoB or other closely associated proteins.6 7 These modified lysines are highly immunogenic, particularly when present on LDL.10 11 The immunogenicity of modified LDL has greatly facilitated generation of antibodies against "oxidation-specific epitopes," such as malondialdehyde (MDA)-lysines and 4-hydroxynonenal (4-HNE)–lysine.12 By analogy, OxLDL present in atherosclerotic lesions triggers a humoral immune response in vivo, and autoantibodies binding to various epitopes of OxLDL have been described in humans, rabbits, and mice.1 13 14 15 16 Extensive data have since accumulated suggesting that the titers of such antibodies may be of diagnostic and/or prognostic value (reviewed in Reference 1717 ). In a Finnish population in whom carotid atherosclerosis was followed by ultrasound over a 2-year period, the titer of autoantibody binding to MDA-LDL was significantly greater in subjects with rapid progression of atherosclerosis than in control subjects with minimal progression.18 Furthermore, the antibody titer was an independent predictor of progression of the disease. Most but not all subsequent epidemiological studies in humans also suggested that the titer of autoantibodies to MDA-LDL or copper-oxidized LDL (Cu-LDL) may be an indicator of the severity or rate of progression of the disease. For example, increased autoantibody titers were reported in patients with carotid atherosclerosis, coronary artery disease, diabetes, peripheral vascular disease, hypertension, and preeclampsia.19 20 21 22 23 24 25 26 27 Finally, a prospective study carried out in a more genetically homogeneous population, ie, LDL receptor–deficient (LDLR-/-) mice, demonstrated that autoantibody titers were directly correlated with the extent of aortic atherosclerosis.28
These observations raised the question whether humoral and/or cellular responses to OxLDL might not only reflect but also actively modulate the atherogenic process. The fact that lesions contain large numbers of immune-competent cells (macrophages and T cells), markers of their activation (interleukin-2 receptors, major histocompatibility complex [MHC] class II molecules), immunoglobulins, and terminal C5b-9 complement complexes has long suggested involvement of the immune system in atherogenesis (for a review, see Reference 2929 ). However, it was generally assumed that immune reactions would enhance lesion formation, as is clearly the case in transplant atherosclerosis.30 31 We and others have recently provided evidence that under particular circumstances, the opposite may be true. A study from our laboratory demonstrated that continuous immunization of LDLR-/- Watanabe heritable hyperlipidemic (WHHL) rabbits with MDA-modified homologous LDL resulted in very high titers of antibodies with specificity similar to that of naturally occurring autoantibodies and that this intervention significantly reduced the progression of atherosclerosis.32 Similar results were subsequently reported for cholesterol-fed New Zealand White rabbits33 and balloon-catheterized rabbits34 immunized with Cu-LDL. However, the mechanisms responsible for this beneficial effect remain unknown.
Crosses of atherosclerosis-susceptible murine strains, such as apoE-deficient and LDLR-/- mice, with murine strains with well-characterized immune deficiencies would offer excellent models in which to investigate whether the beneficial effects of immunization stem from the humoral and/or cellular system. In the current article, we demonstrate that immunization of LDLR-/- mice results in a reduction of atherogenesis and test the hypothesis that the beneficial effect is due to the formation of high-titered antibodies to oxidative neoepitopes.
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Methods |
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Mice and Diets
Forty-four male LDLR-/- mice (C57BL/6Jx129Sv background),35 age 21 to 28 weeks, were selected from our breeding colony established from animals originally provided by The Jackson Laboratories, Bar Harbor, Me. Mice were divided into 3 groups matched for age, body weight, and initial plasma cholesterol levels. The first group (n=15) was immunized with homologous MDA-modified LDL, as described below (MDA-LDL group). The second group (n=15) was immunized with homologous "native" LDL (LDL group). The third group (n=14) was immunized with PBS (control group). Mice were initially fed nonatherogenic rodent chow (Harlan Teklad W860) for 7 weeks. Under these conditions, LDLR-/- mice are known to develop only moderate hypercholesterolemia and limited atherosclerosis.28 During this period, mice were subjected to the primary and 3 biweekly booster immunizations. For the remaining 17 weeks of the intervention period, mice were fed an atherogenic diet free of cholate. This diet contained 21.2% milkfat and initially (weeks 8 through 10) 1% cholesterol and later (weeks 11 through 24) 1.25% cholesterol (TD96121, Harlan Teklad). Mice were kept on a 12-hour day/night cycle and had unrestricted access to water and food. Three weeks before the start of the experiment and
1 week after each booster immunization, 100 to 150 µL blood was
obtained in heparinized tubes from the retro-orbital plexus of
anesthetized (diethyl ether inhalation) mice. A plasma
aliquot of these samples was used for preliminary measurements of
antibody titers and for determination of plasma cholesterol
and triglyceride levels. The remainder of the plasma was
stored at -70°C for the final determination of all antibody titers
under identical assay conditions. Plasma cholesterol and
triglyceride levels were determined using an automated
enzymatic assay (Boehringer Mannheim Diagnostics).
The experimental protocol was approved by the Animal Subjects Committee
of UCSD.
Preparation of Antigens
Murine LDL used as the immunogen (either in its native form or
after modification with MDA) was freshly prepared for each
immunization. A typical preparation (3.5±0.9 mg apoprotein) was
obtained from 24 healthy LDLR-/- mice that had
been fed a diet containing 1% cholesterol (Harlan Teklad
TD95286) for 4 to 5 weeks. Blood was obtained from the vena cava of
anesthetized (diethyl ether inhalation) mice. LDL
(1.023<d<1.058 g/mL) was isolated by sequential
ultracentrifugation in the presence of antioxidants and
antiproteolytic agents.12 In brief, after the density was
adjusted to 1.022 mg/mL with NaBr, plasma was centrifuged at
65 000 rpm for 4 hours at 10°C using a Beckman L7-65
ultracentrifuge with a 65.2 rotor. The supernatant was pipetted
off, the density of the infranatant was adjusted to 1.063 with NaBr,
and the LDL fraction was isolated by centrifugation at
44 000 rpm for 22 hours using a 50.3 rotor. LDL was then extensively
dialyzed against PBS containing 2 mmol/L EDTA and sterile
filtered.
An aliquot of each LDL preparation was modified with MDA, as previously described.12 The degree of modification of the lysine residues of apoB was determined using the trinitrobenzenesulfonic acid assay36 and ranged from 72% to 82% (mean, 79%). The extent of modification was also verified by comparing the electrophoretic mobility of MDA-LDL to that of native LDL on agarose gels.37 Native and MDA-LDLs were stored at 4°C and used within 2 weeks.
To assess antibody titers in murine plasma throughout the intervention period, human LDL was isolated from a pool of healthy donors12 and modified with MDA, Cu2+, or 4-HNE. Human MDA-LDL was generated in the same way as murine MDA-LDL. Cu-LDL was generated by incubating 100 µg LDL per mL PBS, pH 7.35, with 5 µmol/L CuSO4 at 37°C for 2 to 16 hours,12 dialyzing it against PBS containing EDTA, and concentrating it with an Amicon Centriflo membrane cone-type CF25. For the screening of antibodies, a mixture of LDLs oxidized with Cu2+ for 2 hours or 16 hours was used to ensure that both early- and late-oxidation epitopes would be present. 4-HNE–LDL was generated under reducing conditions, as previously described.12
Immunization
The primary immunization consisted of 50 µg of homologous,
native LDL or MDA-LDL in 125 µL PBS suspended in an equal volume of
Freund's complete adjuvant. Mice were inhalation-anesthetized,
and the immunogen was injected subcutaneously into both inguinal
areas. Booster immunizations consisted of 25 µg antigen in Freund's
incomplete adjuvant injected intraperitoneally 2,
4, 6, and 8 weeks after the primary immunization and subsequently at
4-week intervals for 3 more months. Control animals were immunized with
an equal volume of sterile PBS in Freund's complete or incomplete
adjuvant to match immunizations with MDA-LDL and native LDL. Parallel
experiments not included in this report showed that the amount of
antigen and the frequency of booster immunizations were greater than
necessary to induce high-titered antibodies in mice, but boosting was
continued as described to maintain an experimental protocol similar to
our previous study in rabbits.32
Determination of Antibody Binding to Native and Oxidized
Lipoproteins
Titers of autoantibodies in murine sera binding to LDL, MDA-LDL,
Cu-LDL, and 4-HNE–LDL were determined using a chemiluminescence enzyme
immunoassay (CLEIA).16 38 In this assay, 5 µg/mL of the
antigen in 50 mmol/L Tris-buffered saline (TBS), pH 7.5,
containing 0.27 mmol/L EDTA, 0.02% NaN3,
and 20 µmol/L BHT (dilution buffer) was added to each well of a
96-well, white, round-bottomed MicroFluor microtitration plate (Dynex
Technologies) and incubated overnight at 4°C. Plates were washed 4
times with washing buffer (TBS containing 0.27 mmol/L EDTA,
20 µmol/L BHT, 0.02% NaN3, and 0.001%
aprotinin) in an automated plate washer. Murine sera were diluted 1:10
to 1:1000 (as indicated in individual experiments) in dilution buffer
containing 2% or 3% BSA, and 50 µL was added to each well and
incubated for 1 hour at room temperature. After 4 washes, plates were
incubated with 50 µL/well of an alkaline phosphatase–labeled goat
anti-mouse IgG (
-chain specific) or alkaline phosphatase-labeled
goat anti-mouse IgM (µ-chain specific) (Sigma) for 1 hour at room
temperature. These antibodies were diluted in 1% BSA/TBS according to
the supplier's specifications. IgG1 isotype antibody binding to the
plated antigen was detected using an alkaline phosphatase–labeled rat
monoclonal antibody to mouse IgG1 (clone LO-MG1-2, Zymed). IgG2a
antibodies were detected with an alkaline phosphatase–labeled
monoclonal rat antibody to mouse IgG2a (clone R19-15, PharMingen).
After the plates were washed, 25 µL of a 50% solution of Lumi-Phos
530 (Lumigen) was added to each well, and plates were incubated for 1
to 2 hours at room temperature in the dark. Luminescence was determined
using a Lucy 1 luminometer and WINLCOM software (Anthos
Labtec Instruments). Antibody binding was measured as relative light
units (RLUs) in 100 ms. Triplicate determinations were performed for
each plasma sample. Measurement of antibody binding to a given antigen
was done in a single assay. A high and a low standard serum was
included on each plate of a given assay to detect potential variations
between microtitration plates. The intra-assay coefficient of variation
for these assays was 6% to 10%.
Determination of Antibody Binding to Oxidized Phospholipids
and Cholesterol
The CLEIA used to determine antibody binding to cardiolipin
differed from the above with regard to the plating of the antigen. We
recently demonstrated that the standard assays used to measure
"anticardiolipin" antibodies actually measure antibodies to
oxidized cardiolipin (OxCL).38 In standard assays,
cardiolipin is added to microtiter wells in ethanol and is plated by
overnight evaporation of the solvent. Plated cardiolipin exposed to air
is rapidly oxidized within a few minutes.38 39 To
standardize the extent of oxidation, bovine heart cardiolipin (Avanti
Polar Lipids) was diluted to 20 µg/mL in 100% ethanol, and 25 µL
was added to each well. Plates were dried under air at room temperature
(30 minutes) and air exposure was then continued for another hour.
Under these conditions the unsaturated fatty acids of cardiolipin
undergo extensive oxidation, leading to formation of oxidative
neoepitopes recognized by anticardiolipin
antibodies,38 39 40 as well as by some induced monoclonal
antibodies to OxLDL.38 Absolute ethanol was added to blank
wells, and binding to these wells was subtracted to obtain specific
binding. Murine sera diluted with 2% BSA/TBS were added to the
antigen-coated wells after the plates were washed, and the antibody
binding to the OxCL was determined as described above.
Antibody binding to unoxidized and oxidized cholesterol, cholesteryl linoleate, and cholesteryl oleate was determined as follows: Antigens (Sigma) were solubilized in isopropanol at 10 µg/mL and plated at 25 µL/well. To determine binding to the oxidized forms of these antigens, cholesterol, cholesteryl linoleate, and cholesteryl oleate were oxidized by plating and allowing the antigens to be exposed to air overnight at room temperature after evaporation of the solvent. Plasma was then added the following morning. In contrast, binding to the unoxidized forms was achieved by adding the antigens to the wells, evaporating the solvent under argon, and then immediately adding plasma to the wells. Detection of bound antibodies was performed as described above, using labeled second antibodies to murine IgG or IgM.
Determination of Immune Complexes on Circulating Murine
LDL
To detect circulating immune complexes between antibodies
potentially induced by immunization or
physiologically present autoantibodies and
oxidative neoepitopes on LDL, a "sandwich" CLEIA was used. A rabbit
anti-mouse apoB antiserum (G-485-3; a kind gift from Dr Helen Hobbs,
University of Texas Southwestern Medical Center, Dallas, Tex)
was diluted to 10 µg/mL and plated overnight at 4°C (capture
antibody). Wells were then incubated with a 1:50 dilution of murine
plasma for 2 hours at room temperature. After thorough washing,
immunoglobulins bound to the captured LDL were detected with alkaline
phosphatase–labeled goat anti-mouse IgG or IgM antibodies, as
described above.
Evaluation of Atherosclerosis
The heart and aortic tree were perfusion-fixed with
formal sucrose, dissected, and prepared for morphometry, as previously
described.15 The extent of atherosclerosis
was determined in en face preparations of the entire aortic tree, as
well as in cross sections through the aortic origin, by
computer-assisted image analysis described in detail in
Reference 4141 . The measurement of cross sections through the aortic
origin was done to provide a measure of lesion thickening, because
surface measurements of Sudan IV–stained aortas cannot differentiate
between lesions of different stages. Lesion size in the aortic origin
was measured in ten 8-µm-thick hematoxylin/eosin-stained step
sections (every sixth serial section over a distance of 480 µm,
beginning with the appearance of at least 2 valve leaflets). All
measurements of aortic surface areas were performed by the same
investigator. Measurements of lesion areas in the cross sections of the
aortic origin were also performed by the same operator. Because of the
less-automated detection of intimal/medial delimitation, selected
sections from each group were double checked by a second
investigator.
Statistical Analysis
Results were analyzed by ANOVA and Student's unpaired
t test. Data shown are mean±SEM. Data on the extent of
atherosclerosis were also analyzed by
nonparametric tests.
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Results |
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Effect of Intervention on Body Weight and Plasma Lipid Levels
Mice were of fairly mature age (21 to 28 weeks) at the start of the experiment, and the matched baseline body weights in the 3 groups were at adult levels (30.8±0.1 g). None of the interventions had a significant effect on body weight, which showed only a mild increase after the animals were put on the high-fat, high-cholesterol diet. The final body weights were 36.0±1.7 g in the group immunized with MDA-LDL, 43.0±2.3 g in the group immunized with native LDL, and 34.9±1.5 g in the control group immunized with PBS.
Plasma cholesterol levels were not affected by the primary
immunization or the first 2 boosts and remained at the preimmunization
level in all 3 groups (Figure 1
).
After the start of the high-cholesterol diet at week 7,
plasma cholesterol levels increased in parallel to 1400
mg/dL. Levels in the control group then remained relatively constant,
whereas in the MDA-LDL and LDL groups a slight decrease was observed.
However, the difference between the 3 groups did not reach statistical
significance, except at the final time point. The time-averaged
cholesterol levels (area under the
cholesterol-versus-time curve divided by the entire length
of the study) were also similar (928±77, 937±47, and 1084±70 mg/dL
in the MDA-LDL, LDL, and PBS groups, respectively; NS).
Triglyceride levels rose from 106±5 mg/dL at the start of
the experiment to 608±140 mg/dL in the MDA-LDL group, 500±93 mg/dL in
the LDL group, and 713±114 mg/dL in the PBS group (NS).
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Effect of Intervention on Atherosclerosis
After 24 weeks of intervention, the extent of
atherosclerosis was determined by computer-assisted
image analysis of 10 step sections through the aortic origin of
each mouse. Statistical evaluation of data from all 3 groups suggested
a normal distribution, and ANOVA indicated that the groups were
different (P<0.01). Comparison of groups was therefore
performed using Student's unpaired t test. As shown in
Figure 2, immunization with
MDA-LDL led to a significant reduction of lesion size compared with the
control group (133 588±20 577 versus 248 867±38 207
µm2/section, P<0.01). Surprisingly,
immunization with LDL also reduced the lesions. The mean lesion size in
the LDL group was 157 141±61 680
µm2/section (P<0.05, compared with
the control group). Comparisons of differences between intervention and
control groups using nonparametric statistics also yielded
significant differences between groups. Although the reduction
in lesion size was less impressive in the LDL group than in the MDA-LDL
group (36.9% versus 46.3%), the difference between these 2 groups was
not statistically significant (P=0.49).
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In our experience, exposure of LDLR-/- mice41 42 to plasma cholesterol levels >1000 mg/dL for only 3 to 4 months, as in the current experiment, only induces mild atherosclerosis that involves 7% to 10% of the aortic tree. We nevertheless also determined the percent of aortic surface area covered by Sudan IV–positive lesions in the 3 treatment groups. Consistent with previous data,41 42 <10% of the aortic surface was involved, and no significant differences in the percent of atherosclerotic surface area were found between the 3 experimental groups (MDA-LDL, 8.14±1.1%; LDL, 9.08±1.2%; and control, 8.4±1.3%). Although we did not have a control group that was not subjected to any immunization, it should be noted that the degree of atherosclerosis observed in the PBS group in either location was very similar to what we had previously seen in LDLR-/- mice on similar diets.28 41 42
As reported above, the differences in plasma cholesterol
levels between the 3 groups were not statistically significant.
Nevertheless, the absolute plasma cholesterol
concentrations of the MDA-LDL and LDL groups were consistently
lower than those of the control group (Figure 1
), and their
time-averaged cholesterol levels were also lower by 150
mg/dL. To rule out the possibility that the reduction in
atherosclerosis in the MDA-LDL and LDL groups might be
accounted for by this difference in cholesterol exposure,
the correlation between the time-averaged plasma
cholesterol level (area under the curve divided by time;
Figure 1) and the average size of atherosclerotic lesions in the
aortic origin was determined by using the data of all mice from the 3
groups. As shown in Figure 3, a
weak but significant correlation existed between the 2
parameters even at the very high cholesterol
levels induced in this study (P<0.001). However, the slope
of the regression line was shallow, and a difference in plasma
cholesterol of 150 mg/dL could only have accounted for a
small part (<35 000 µm2/section) of the
reduction in atherosclerosis actually observed
(-115 279 and -91 726 µm2/section in
the MDA-LDL and LDL groups, respectively). Thus, immunization reduced
atherogenesis by a distinct mechanism, in addition to a potential
effect resulting from the slight lowering of plasma
cholesterol.
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Humoral Immune Response to Immunization
Plasma samples from each mouse were obtained before immunization
and throughout the study to follow the humoral immune response. Because
homologous LDL (or MDA-LDL) was used to immunize the mice, antibodies
against epitopes of native LDL should not be generated. Indeed, IgG
antibody binding to native murine or human LDL was extremely low at any
time point in all of the 3 experimental groups (data not shown).
The first model epitope of OxLDL tested was MDA-LDL. For these assays,
we postulated that human MDA-LDL could be used as the antigen, because
antibodies previously generated by immunizing mice and rabbits with
homologous MDA-LDL recognized MDA-LDL prepared from LDL of different
species.1 4 12 16 Preliminary binding assays indeed showed
similar binding to mouse MDA-LDL (the actual immunogen) as to human
MDA-LDL. As expected, immunization with MDA-LDL induced a
>200 000-fold rise of specific IgG binding to MDA-LDL (Figure
A) when plasma was tested at a
1:1000 dilution. In contrast, animals immunized with PBS showed only a
39% rise in absolute binding to MDA-LDL and animals immunized with
native LDL, a 50% increase. After the initial rise, the binding to
MDA-LDL remained fairly constant in the MDA-LDL group, despite
additional booster immunizations, the switch to
high-cholesterol diets, and the resulting
atherogenesis.
We then determined the antibody binding to other oxidative neoepitopes.
We began by determining binding to oxidized phospholipids by using OxCL
as the antigen. We previously demonstrated that oxidized phospholipid
epitopes are generated when LDL is oxidized.38 39 In the
current study, the MDA-LDL group also demonstrated a dramatic increase
in the binding of antibodies to OxCL, with a somewhat similar time
course as the binding to MDA-LDL (Figure 4B).
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Because autoantibodies to oxidized phospholipids were formed, we
investigated whether antibodies were also formed against oxidative
products of cholesterol and/or cholesteryl esters, the
predominant lipids of LDL. In these assays, we coated microtiter wells
with cholesterol or cholesteryl esters and compared their
recognition by antibodies to that obtained with the same antigen
oxidized by overnight exposure to air. As shown in Figure 4C, there was a dramatic rise in antibody binding to
oxidized cholesterol. In contrast, binding to unoxidized
cholesterol was very low and probably nonspecific (data not
shown).
Because the above assays used a high plasma dilution (1:1000) that may
not have permitted detection of differences between the LDL and PBS
groups with low antibody titers, we also compared the binding to
different oxidation products of IgG antibodies in the 3 groups at
the end of the intervention period by using lower plasma dilutions
(Figure 5). At low dilutions,
binding to MDA-LDL and Cu-LDL was again significantly greater in the
MDA-LDL group than in the other 2 groups (P<0.0001). As
shown in the right panels, which use a different scale, binding to both
antigens was also slightly but consistently higher in the LDL
than in the PBS group (P<0.05 at 1:80 dilution).
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Binding to oxidized cholesteryl linoleate was also markedly greater in
the MDA-LDL group than in the LDL and PBS groups (P<0.0001;
Figure 5, lower panels), whereas no difference in antibody
binding to unoxidized cholesteryl linoleate was detectable (data not
shown). Binding to either unoxidized or oxidized cholesteryl oleate was
very low and not different between groups (not shown).
In the previous assays, we had used a secondary antibody that detects
all IgG antibodies. To investigate the potential role of T helper (Th)1
and Th2 cells, which are involved in the formation of IgG2a and IgG1
antibodies, respectively,43 we determined the binding of
these 2 subclasses of antibodies to some of the same antigens. Figure 6 compares the binding of the 2
IgG isoforms to human MDA-LDL at the start (ie, before immunization)
and at the end of the study. Both IgG1 and IgG2a antibodies increased
substantially in the MDA-LDL group only, mirroring the increase in
total anti–MDA-LDL IgG (Figures 4 and 5). Similar
results were also obtained when OxCL was used as the antigen (Figure 7). This assay not only compared
antibodies at the first and last time points of the study but also
followed the IgG1 and IgG2a antibodies over time.
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We also examined the formation of IgM antibody binding to various
oxidative neoepitopes in the 3 experimental groups, because IgM
antibodies presumably reflect primarily a T-cell–independent process.
In contrast to what we saw for IgG antibodies, binding of IgM
antibodies to native LDL showed a small but significant increase of
equal magnitude in all 3 groups compared with the preimmune time point
(data not shown). Binding of IgM antibodies to MDA-LDL increased to a
much greater extent, but surprisingly there was no statistically
significant difference between the groups immunized with MDA-LDL, LDL,
or PBS (Figure 8A). Similarly,
there was a marked rise in antibody binding to oxidized
cholesterol, but again the rise was similar in all 3 groups
(Figure 8B). IgM antibody binding to Cu-LDL (Figure 9A) and to 4-HNE–LDL (Figure 9B) was higher in the MDA-LDL group than in the other 2 groups,
albeit the absolute binding was much lower than that seen in Figure 8. Surprisingly, binding of IgM antibodies to OxCL (Figure 9C) was minimal and did not increase much during the study. This
contrasts sharply with the strong IgG response to the same antigen
(Figure 4B).
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Finally, we sought to test the hypothesis that increased titers of antibodies to oxidation-specific epitopes lead to increased formation of immune complexes with LDL that had undergone minimal modification, because this might affect the removal of such LDL from plasma. IgG and IgM immune complexes on circulating LDL were determined in the terminal plasma samples of all mice. However, no significant differences were found between the 3 groups in either IgG or IgM immune complexes with LDL (for IgG, 23 407±2902, 23 428±4478, and 22 254±1770 RLUs/100 ms; for IgM, 33 829±4752, 28 926±6224, and 28 327±5339 RLUs/100 ms in the MDA-LDL, LDL, and PBS groups, respectively).
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Discussion |
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Atherosclerosis fulfills many of the criteria of a chronic inflammatory process, and substantial histopathological evidence points toward an involvement of humoral and cellular components of the immune system in the disease process. However, the mechanisms involved are poorly understood. While there is little doubt that immune reactions to allografts increase atherogenesis in transplanted arteries,30 31 44 their role in conventional atherosclerosis is controversial, and both beneficial and detrimental consequences of immune modulation have been reported.
The assumption that the immune system may reduce atherogenesis is
supported by the increase in atherosclerosis observed
in a number of different immunocompromised animal models. For example,
elimination of T lymphocytes with monoclonal antibodies resulted in
larger proliferative lesions in balloon-catheterized rat
aortas.45 Cyclosporine treatment (which
suppresses T cells) of hypercholesterolemic mice and
rabbits also accelerated
atherosclerosis.46 47 MHC class
I–deficient C57BL/6 mice, which lack cytolytic T cells and have
impaired natural killer cell activity, also developed a 3-fold increase
in lesions in the aortic valve region when fed a high-fat
diet.48 In contrast, mice lacking either interferon
(IFN)-44 or the IFN- receptor,49 the
interleukin-8 receptor,50 or the p55 component of the
tumor necrosis factor receptor51 had decreased
atherogenesis. ApoE-/- mice crossed with Rag-1
knockout mice showed a 42% decrease in atherosclerosis
when fed a regular diet (cholesterol levels of 800
mg/dL) compared with apoE-/- mice. However, the
same immunodeficient apoE-/- mice showed no
significant decrease in atherosclerosis when fed a
high-fat diet that resulted in plasma cholesterol levels of
1800 mg/dL,52 53 suggesting that exceedingly high
cholesterol levels overwhelm any modulatory effect of
immune deficiency. By analogy, conflicting results have been obtained
with interventions inducing or enhancing the humoral immune response to
different antigens. For example, immunization with heat shock proteins
caused a transitory inflammatory form of lesion in
normocholesterolemic rabbits.54 55 In
contrast, immunization of hypercholesterolemic WHHL and
New Zealand White rabbits with different forms of OxLDL significantly
reduced progression of the disease.32 33 34
Mice are a particularly valuable animal model to study the mechanisms by which modulation of the immune system affects atherogenesis, because the murine immune system is well characterized and several immunodeficient strains exist that can be crossed with strains that develop extensive atherosclerosis. In the current article, we demonstrate that continued immunization of LDLR-/- mice with MDA-LDL, a model of OxLDL, yields a highly significant reduction of atherosclerosis in the aortic origin by mechanisms that appear to be independent of the modulation of plasma cholesterol levels. In view of the observations in immunodeficient apoE-/- mice discussed above, it is noteworthy that the extent of atherosclerosis in our experiment was reduced by immunization despite extremely high plasma cholesterol levels.
The current study also provides important mechanistic insights. When we first reported that immunization of WHHL rabbits inhibits the progression of atherogenesis, we proposed 3 potential mechanisms.32 The first of these was the possibility that high titers of antibodies to epitopes of OxLDL enhanced the removal of minimally oxidized (mm) LDL from the circulation. Although it has long been assumed that LDL is oxidized in the arterial wall6 rather than in plasma, where powerful antioxidant protection exists, there is increasing evidence that mmLDL is present in the circulation. For example, plasma LDL of human subjects with manifest cardiovascular disease and ß-VLDL from atherosclerotic rabbits contain significantly more oxidation-specific epitopes then does LDL from controls (References 56 and 5756 57 and J.L.W., unpublished data, 1998). It could therefore be hypothesized that high titers of antibodies to oxidation-specific epitopes enhance formation of immune complexes with mmLDL. This would lead to its rapid removal from the circulation, whereas in the absence of this process, mmLDL might penetrate the vascular wall, where it would be much more susceptible to further oxidation than native LDL. In general, the finding that immunization with MDA-LDL induced a high titer of IgG antibody binding to MDA-lysine epitopes, whereas immunization with native LDL and PBS did not, would not support this hypothesis, although it does not exclude the possibility that this event occurred to some extent in both the MDA-LDL and LDL groups, as discussed below.
In addition to increased anti–MDA-LDL titers, immunization with MDA-LDL also resulted in a strong increase of IgG antibody binding to a variety of other oxidative neoepitopes that one may expect to be present on OxLDL, such as oxidized phospholipids, oxidized cholesterol, and oxidized cholesteryl linoleate. Because MDA modification of LDL should not generate the latter epitopes in principle, this suggests that after immunization the MDA-LDL was rapidly taken up via scavenger receptors of macrophages and that many of these oxidative epitopes were actually generated within macrophages. Because the macrophage is a classic antigen-presenting cell, this would lead to efficient induction of high-titered antibodies. The superiority of antigens (even minimally modified autologous proteins) being taken up by scavenger receptors and inducing T-cell stimulation has previously been established.58 59 Antibodies generated by the above mechanism should bind well to various epitopes of OxLDL.
The humoral immune response to "native" LDL appeared to be far less
pronounced than that to MDA-LDL. Nevertheless, it is likely that
immunization with LDL also leads to a localized inflammatory condition
that resulted in the generation of some mmLDL that would be taken
up by macrophages. Indeed, we observed a small but significant
increase in IgG antibody binding to MDA-LDL and Cu-LDL in the LDL group
compared with the PBS group when the sera were tested at low dilutions
(Figure 5). A similar small increase in titers was previously
reported by Ameli et al.33
The fact that immunization with LDL led to a significant reduction in lesions that was only marginally smaller than that achieved by immunization with MDA-LDL (-36.9% versus -46.3%) suggests that the major beneficial effect of immunization did not result from enhanced removal of mmLDL from the circulation. However, it is possible that relatively small amounts of immunoglobulins formed in the LDL group are sufficient to achieve almost complete removal of mmLDL from the circulation and that a further dramatic increase in titers, as seen in the MDA-LDL group, does not substantially increase benefit. In this case, consumption of antibodies would have markedly reduced the plasma levels of antibodies in the LDL group, whereas the relative impact on the very high titers in the MDA-LDL group would be negligible. Our failure to detect higher antibody titers in mice immunized with LDL could also have resulted from the fact that the antigens used in our CLEIAs did not contain appropriate epitopes formed in vivo, in particular, epitopes that would be destroyed by more extensive oxidation.
The slightly lower plasma cholesterol levels observed in both the LDL and MDA-LDL groups compared with the PBS group would be consistent with the assumption that a small fraction of the LDL was removed from the circulation as a consequence of increased antibody binding to such epitopes. The failure to find differences in circulating immune complexes with plasma LDL does not support this idea, although rapid removal of such immune complexes could have prevented significant differences.
A second postulated mechanism by which high-titered antibodies against oxidation-specific epitopes could have reduced lesion formation would be by enhancing the uptake of OxLDL by macrophages and thereby preventing it from exerting proinflammatory and toxic effects in the vascular wall. We have previously demonstrated that circulating autoantibodies to OxLDL penetrate atherosclerotic lesions and form immune complexes with OxLDL,60 which could enhance the uptake of OxLDL by macrophages via Fc receptors or phagocytosis.14 61 However, the marked discrepancy between antibody titers in the MDA-LDL and LDL groups does not support a major role for this mechanism, either.
A third potential mechanism by which immunization could have affected
atherosclerosis is through cell-mediated immune
mechanisms. Atherosclerotic lesions of mice resemble those of other
species in that they contain both CD4+ and CD8+ cells but few if any B
cells.62 63 Lesions also contain large numbers of
antigen-presenting cells, such as macrophages, bearing MHC
class II molecules on their surfaces, which colocalize with IFN- and
interleukins.29 49 Oxidation-specific epitopes are
abundant in lesions, both extracellularly and within
macrophages.15 28 It is therefore likely that
modified peptides of apoB derived from OxLDL are candidates for antigen
presentation by classic human leukocyte antigen A (HLA)
class II mechanisms. It is also likely that oxidized lipids or
lipid-protein adducts are presented as antigens, via mechanisms
such as the recently described CD1 molecule.64 All
cell-mediated immune mechanisms presumably result from initial T-cell
recognition of antigen,65 66 and indeed, 10% of CD4+ T
cells cloned from human lesions specifically proliferated in response
to OxLDL in an HLA class II–restricted manner.67
Once antigen-activated, T cells secrete a variety of cytokines that could have a profound effect on the progression of the atherogenic process. At present, we do not know whether differences in T-cell populations, such as cytolytic lymphocytes or natural killer cells, or differences in their secretory products exist between the MDA-LDL and LDL groups on 1 side and the PBS group on the other. Th1 and Th2 cells are known to have different patterns of cytokine secretion and have been postulated to play different roles in promoting inflammatory conditions, as well as in cross-regulation of each other's activity.43 68 69 Therefore, one could assume that a shift from 1 T-cell subset to another may have influenced the balance of proatherogenic and antiatherogenic factors secreted in lesions. Such shifts during the course of atherosclerosis have recently been described in apoE-/- mice.70 In response to immunization with MDA-LDL, there appeared to be a generalized increase in both Th1 and Th2 subtypes (judged by rises in both IgG1 and IgG2a antibodies). However, no comparable increase in IgG1 or IgG2a was seen in the LDL group (which also received Freund's adjuvant). Furthermore, it is unknown whether the differences in the level of plasma antibodies to the epitopes of OxLDL accurately reflect differences in Th1 and Th2 cell numbers or activity in the lesions. Only direct studies of the various lymphocyte populations in lesions, in lymph nodes draining atherosclerotic arteries, and of memory cells at other sites will determine their relative importance. Adoptive transfer experiments could also establish the role of cell-mediated immunity in the protective effect.
We have previously reported that IgM antibodies to oxidation-specific epitopes are prominent in atherosclerotic mice15 16 28 and human subjects with clinically manifest coronary heart disease. The generation of these antibodies is thought to be largely T-cell independent. In the current study there was a steady rise in IgM antibodies to a variety of oxidation-specific epitopes, including oxidized lipids. Because the rise was similar in all 3 experimental groups, it is not likely that IgM directly contributed to the protective effect observed, although they could participate in the natural history of lesion development.
The fact that a significant reduction in lesion size was observed in cross sections of the aortic origin, but not in the percentage of aortic surface stained with Sudan IV, suggests that immunization inhibited the growth of lesions but did not significantly impact lesion initiation. In LDLR-/- and apoE-/- mice, lesions first appear at a few very consistent predilection sites in the arch and near the orifices of the mesenteric arteries. In our experience, lesions at these sites undergo considerable intimal thickening before they begin to cover a larger percentage of the aortic tree. Later, horizontal spreading of lesions occurs mostly in the abdominal aorta and arch, whereas the thoracic aorta shows only isolated lesions.15 28 41 42 It could therefore be assumed that the limited lesions we observed had not reached a stage when they would rapidly spread. Consequently, in this setting, determination of the surface area would not be a very sensitive parameter to detect a beneficial effect of immunization. The conclusion that intervention affected progression rather than initiation of lesion formation is also supported by our previous immunization study in WHHL rabbits.32 In that study, immunization of young animals failed to yield a significant effect on early stages of atherogenesis in the aorta, whereas immunization of older animals with more extensive initial atherosclerosis reduced lesion formation by 35%.
|
Acknowledgments |
|---|
These studies were supported by grants HL14197 and HL5989 (La Jolla Specialized Center of Research in Molecular Medicine and Atherosclerosis; to J.L.W. and W.P.) from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md; grant HL57505 (to J.L.W.); and a grant from the German Academic Exchange Service (DAAD) (to S.F.). The current article is part of Stefan Freigang's Medical Doctorate Thesis, Humboldt University, Berlin, Germany. We thank Florencia Casanada, Joe Juliano, and Jennifer Pattison for excellent technical assistance.
Received May 26, 1998; accepted July 29, 1998.
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C. J. Binder, K. Hartvigsen, and J. L. Witztum Promise of Immune Modulation to Inhibit Atherogenesis J. Am. Coll. Cardiol., August 7, 2007; 50(6): 547 - 550. [Full Text] [PDF]
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 K.-Y. Chyu and P. K. Shah Choking off Plaque Neovascularity: A Promising Atheroprotective Strategy or A Double-Edged Sword? Arterioscler Thromb Vasc Biol, May 1, 2007; 27(5): 993 - 995. [Full Text] [PDF]
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 G. N. Fredrikson, B. Hedblad, G. Berglund, R. Alm, J.-A. Nilsson, A. Schiopu, P. K. Shah, and J. Nilsson Association Between IgM Against an Aldehyde-Modified Peptide in Apolipoprotein B-100 and Progression of Carotid Disease Stroke, May 1, 2007; 38(5): 1495 - 1500. [Abstract] [Full Text] [PDF]
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M. Torzewski, V. Ochsenhirt, A. L. Kleschyov, M. Oelze, A. Daiber, H. Li, H. Rossmann, S. Tsimikas, K. Reifenberg, F. Cheng, et al. Deficiency of Glutathione Peroxidase-1 Accelerates the Progression of Atherosclerosis in Apolipoprotein E-Deficient Mice Arterioscler Thromb Vasc Biol, April 1, 2007; 27(4): 850 - 857. [Abstract] [Full Text] [PDF]
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 S. Tsimikas, E. S. Brilakis, R. J. Lennon, E. R. Miller, J. L. Witztum, J. P. McConnell, K. S. Kornman, and P. B. Berger Relationship of IgG and IgM autoantibodies to oxidized low density lipoprotein with coronary artery disease and cardiovascular events J. Lipid Res., February 1, 2007; 48(2): 425 - 433. [Abstract] [Full Text] [PDF]
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 S. Brandlein, N. Rauschert, L. Rasche, A. Dreykluft, F. Hensel, E. Conzelmann, H.-K. Muller-Hermelink, and H. P. Vollmers The human IgM antibody SAM-6 induces tumor-specific apoptosis with oxidized low-density lipoprotein Mol. Cancer Ther., January 1, 2007; 6(1): 326 - 333. [Abstract] [Full Text] [PDF]
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 P. C. Dimayuga, X. Zhao, J. Yano, and K.-Y. Chyu Changes in immune responses to oxidized LDL epitopes during aging in hypercholesterolemic apoE(-/-) mice Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2006; 291(6): R1644 - R1650. [Abstract] [Full Text] [PDF]
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 M McMahon, J Grossman, W Chen, and B H Hahn Inflammation and the pathogenesis of atherosclerosis in systemic lupus erythematosus Lupus, November 1, 2006; 15(11_suppl): 59 - 69. [Abstract] [PDF]
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T. Yamashita, S. Freigang, C. Eberle, J. Pattison, S. Gupta, C. Napoli, and W. Palinski Maternal Immunization Programs Postnatal Immune Responses and Reduces Atherosclerosis in Offspring Circ. Res., September 29, 2006; 99(7): E51 - E64. [Abstract] [Full Text] [PDF]
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A. F. Saad, G. Virella, C. Chassereau, R. J. Boackle, and M. F. Lopes-Virella OxLDL immune complexes activate complement and induce cytokine production by MonoMac 6 cells and human macrophages J. Lipid Res., September 1, 2006; 47(9): 1975 - 1983. [Abstract] [Full Text] [PDF]
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A. Tuominen, Y. I. Miller, L. F. Hansen, Y. A. Kesaniemi, J. L. Witztum, and S. Horkko A Natural Antibody to Oxidized Cardiolipin Binds to Oxidized Low-Density Lipoprotein, Apoptotic Cells, and Atherosclerotic Lesions Arterioscler Thromb Vasc Biol, September 1, 2006; 26(9): 2096 - 2102. [Abstract] [Full Text] [PDF]
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B. J. Bennett, M. Scatena, E. A. Kirk, M. Rattazzi, R. M. Varon, M. Averill, S. M. Schwartz, C. M. Giachelli, and M. E. Rosenfeld Osteoprotegerin Inactivation Accelerates Advanced Atherosclerotic Lesion Progression and Calcification in Older ApoE-/- Mice Arterioscler Thromb Vasc Biol, September 1, 2006; 26(9): 2117 - 2124. [Abstract] [Full Text] [PDF]
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G. Stoll and M. Bendszus Inflammation and Atherosclerosis: Novel Insights Into Plaque Formation and Destabilization Stroke, July 1, 2006; 37(7): 1923 - 1932. [Abstract] [Full Text] [PDF]
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 A. Tedgui and Z. Mallat Cytokines in Atherosclerosis: Pathogenic and Regulatory Pathways Physiol Rev, April 1, 2006; 86(2): 515 - 581. [Abstract] [Full Text] [PDF]
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X. Zhou, A.-K. L. Robertson, C. Hjerpe, and G. K. Hansson Adoptive Transfer of CD4+ T Cells Reactive to Modified Low-Density Lipoprotein Aggravates Atherosclerosis Arterioscler Thromb Vasc Biol, April 1, 2006; 26(4): 864 - 870. [Abstract] [Full Text] [PDF]
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C. Meisinger, J. Baumert, N. Khuseyinova, H. Loewel, and W. Koenig Plasma Oxidized Low-Density Lipoprotein, a Strong Predictor for Acute Coronary Heart Disease Events in Apparently Healthy, Middle-Aged Men From the General Population Circulation, August 2, 2005; 112(5): 651 - 657. [Abstract] [Full Text] [PDF]
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C. J. Binder, P. X. Shaw, M.-K. Chang, A. Boullier, K. Hartvigsen, S. Horkko, Y. I. Miller, D. A. Woelkers, M. Corr, and J. L. Witztum Thematic review series: The Immune System and Atherogenesis. The role of natural antibodies in atherogenesis J. Lipid Res., July 1, 2005; 46(7): 1353 - 1363. [Abstract] [Full Text] [PDF]
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S. J. Nicholls and S. L. Hazen Myeloperoxidase and Cardiovascular Disease Arterioscler Thromb Vasc Biol, June 1, 2005; 25(6): 1102 - 1111. [Abstract] [Full Text] [PDF]
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I. Goncalves, M.-L. M. Gronholdt, I. Soderberg, M. P.S. Ares, B. G. Nordestgaard, J. F. Bentzon, G. N. Fredrikson, and J. Nilsson Humoral Immune Response Against Defined Oxidized Low-Density Lipoprotein Antigens Reflects Structure and Disease Activity of Carotid Plaques Arterioscler Thromb Vasc Biol, June 1, 2005; 25(6): 1250 - 1255. [Abstract] [Full Text] [PDF]
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X. Zhou, A.-K. L. Robertson, M. Rudling, P. Parini, and G. K. Hansson Lesion Development and Response to Immunization Reveal a Complex Role for CD4 in Atherosclerosis Circ. Res., March 4, 2005; 96(4): 427 - 434. [Abstract] [Full Text] [PDF]
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F. J. Tinahones, J. M. Gomez-Zumaquero, L. Garrido-Sanchez, E. Garcia-Fuentes, G. Rojo-Martinez, I. Esteva, M. S. R. de Adana, F. Cardona, and F. Soriguer Influence of age and sex on levels of anti-oxidized LDL antibodies and anti-LDL immune complexes in the general population J. Lipid Res., March 1, 2005; 46(3): 452 - 457. [Abstract] [Full Text] [PDF]
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G. S. Getz Thematic review series: The Immune System and Atherogenesis. Immune function in atherogenesis J. Lipid Res., January 1, 2005; 46(1): 1 - 10. [Abstract] [Full Text] [PDF]
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J. Nilsson, G. K. Hansson, and P. K. Shah Immunomodulation of Atherosclerosis: Implications for Vaccine Development Arterioscler Thromb Vasc Biol, January 1, 2005; 25(1): 18 - 28. [Abstract] [Full Text] [PDF]
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 B. Ludewig, P. Krebs, and E. Scandella Immunopathogenesis of atherosclerosis J. Leukoc. Biol., August 1, 2004; 76(2): 300 - 306. [Abstract] [Full Text] [PDF]
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C. A. Reardon, E. R. Miller, L. Blachowicz, J. Lukens, C. J. Binder, J. L. Witztum, and G. S. Getz Autoantibodies to OxLDL fail to alter the clearance of injected OxLDL in apolipoprotein E-deficient mice J. Lipid Res., July 1, 2004; 45(7): 1347 - 1354. [Abstract] [Full Text] [PDF]
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S. Tsimikas, H. K. Lau, K.-R. Han, B. Shortal, E. R. Miller, A. Segev, L. K. Curtiss, J. L. Witztum, and B. H. Strauss Percutaneous Coronary Intervention Results in Acute Increases in Oxidized Phospholipids and Lipoprotein(a): Short-Term and Long-Term Immunologic Responses to Oxidized Low-Density Lipoprotein Circulation, June 29, 2004; 109(25): 3164 - 3170. [Abstract] [Full Text] [PDF]
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P. A. VanderLaan, C. A. Reardon, and G. S. Getz Site Specificity of Atherosclerosis: Site-Selective Responses to Atherosclerotic Modulators Arterioscler Thromb Vasc Biol, January 1, 2004; 24(1): 12 - 22. [Abstract] [Full Text] [PDF]
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J. Karvonen, M. Paivansalo, Y. A. Kesaniemi, and S. Horkko Immunoglobulin M Type of Autoantibodies to Oxidized Low-Density Lipoprotein Has an Inverse Relation to Carotid Artery Atherosclerosis Circulation, October 28, 2003; 108(17): 2107 - 2112. [Abstract] [Full Text] [PDF]
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C. A. Reardon, L. Blachowicz, J. Lukens, M. Nissenbaum, and G. S. Getz Genetic Background Selectively Influences Innominate Artery Atherosclerosis: Immune System Deficiency as a Probe Arterioscler Thromb Vasc Biol, August 1, 2003; 23(8): 1449 - 1454. [Abstract] [Full Text] [PDF]
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G. N. Fredrikson, B. Hedblad, G. Berglund, R. Alm, M. Ares, B. Cercek, K.-Y. Chyu, P. K. Shah, and J. Nilsson Identification of Immune Responses Against Aldehyde-Modified Peptide Sequences in ApoB Associated With Cardiovascular Disease Arterioscler Thromb Vasc Biol, May 1, 2003; 23(5): 872 - 878. [Abstract] [Full Text] [PDF]
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G. N. Fredrikson, I. Soderberg, M. Lindholm, P. Dimayuga, K.-Y. Chyu, P. K. Shah, and J. Nilsson Inhibition of Atherosclerosis in ApoE-Null Mice by Immunization with ApoB-100 Peptide Sequences Arterioscler Thromb Vasc Biol, May 1, 2003; 23(5): 879 - 884. [Abstract] [Full Text] [PDF]
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A. S. Major, S. Fazio, and M. F. Linton B-Lymphocyte Deficiency Increases Atherosclerosis in LDL Receptor-Null Mice Arterioscler Thromb Vasc Biol, November 1, 2002; 22(11): 1892 - 1898. [Abstract] [Full Text] [PDF]
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R. Maron, G. Sukhova, A.-M. Faria, E. Hoffmann, F. Mach, P. Libby, and H. L. Weiner Mucosal Administration of Heat Shock Protein-65 Decreases Atherosclerosis and Inflammation in Aortic Arch of Low-Density Lipoprotein Receptor-Deficient Mice Circulation, September 24, 2002; 106(13): 1708 - 1715. [Abstract] [Full Text] [PDF]
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G. K. Hansson, P. Libby, U. Schonbeck, and Z.-Q. Yan Innate and Adaptive Immunity in the Pathogenesis of Atherosclerosis Circ. Res., August 23, 2002; 91(4): 281 - 291. [Abstract] [Full Text] [PDF]
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C. Buono, C. E. Come, J. L. Witztum, G. F. Maguire, P. W. Connelly, M. Carroll, and A. H. Lichtman Influence of C3 Deficiency on Atherosclerosis Circulation, June 25, 2002; 105(25): 3025 - 3031. [Abstract] [Full Text] [PDF]
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K. Tanaga, H. Bujo, M. Inoue, K. Mikami, K. Kotani, K. Takahashi, T. Kanno, and Y. Saito Increased Circulating Malondialdehyde-Modified LDL Levels in Patients With Coronary Artery Diseases and Their Association With Peak Sizes of LDL Particles Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 662 - 666. [Abstract] [Full Text] [PDF]
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G. K. Hansson Immune Mechanisms in Atherosclerosis Arterioscler Thromb Vasc Biol, December 1, 2001; 21(12): 1876 - 1890. [Abstract] [Full Text] [PDF]
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P. Thiagarajan Atherosclerosis, Autoimmunity, and Systemic Lupus Erythematosus Circulation, October 16, 2001; 104(16): 1876 - 1877. [Full Text] [PDF]
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G. S. Getz The First Human Monoclonal Antibody to Oxidized LDL Arterioscler Thromb Vasc Biol, August 1, 2001; 21(8): 1254 - 1255. [Full Text] [PDF]
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P. X. Shaw, S. Horkko, S. Tsimikas, M.-K. Chang, W. Palinski, G. J. Silverman, P. P. Chen, and J. L. Witztum Human-Derived Anti-Oxidized LDL Autoantibody Blocks Uptake of Oxidized LDL by Macrophages and Localizes to Atherosclerotic Lesions In Vivo Arterioscler Thromb Vasc Biol, August 1, 2001; 21(8): 1333 - 1339. [Abstract] [Full Text] [PDF]
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 D. M. Wuttge, P. Eriksson, A. Sirsjo, G. K. Hansson, and S. Stemme Expression of Interleukin-15 in Mouse and Human Atherosclerotic Lesions Am. J. Pathol., August 1, 2001; 159(2): 417 - 423. [Abstract] [Full Text]
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S. Stemme Plaque T-Cell Activity : Not So Specific? Arterioscler Thromb Vasc Biol, July 1, 2001; 21(7): 1099 - 1101. [Full Text] [PDF]
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C. A. Reardon, L. Blachowicz, T. White, V. Cabana, Y. Wang, J. Lukens, J. Bluestone, and G. S. Getz Effect of Immune Deficiency on Lipoproteins and Atherosclerosis in Male Apolipoprotein E-Deficient Mice Arterioscler Thromb Vasc Biol, June 1, 2001; 21(6): 1011 - 1016. [Abstract] [Full Text] [PDF]
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 D. Pratico, R. K. Tangirala, S. Horkko, J. L. Witztum, W. Palinski, and G. A. FitzGerald Circulating autoantibodies to oxidized cardiolipin correlate with isoprostane F2{alpha}-VI levels and the extent of atherosclerosis in ApoE-deficient mice: modulation by vitamin E Blood, January 15, 2001; 97(2): 459 - 464. [Abstract] [Full Text] [PDF]
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S. Tsimikas, W. Palinski, and J. L. Witztum Circulating Autoantibodies to Oxidized LDL Correlate With Arterial Accumulation and Depletion of Oxidized LDL in LDL Receptor-Deficient Mice Arterioscler Thromb Vasc Biol, January 1, 2001; 21(1): 95 - 100. [Abstract] [Full Text] [PDF]
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X. Zhou, G. Caligiuri, A. Hamsten, A. K. Lefvert, and G. K. Hansson LDL Immunization Induces T-Cell-Dependent Antibody Formation and Protection Against Atherosclerosis Arterioscler Thromb Vasc Biol, January 1, 2001; 21(1): 108 - 114. [Abstract] [Full Text] [PDF]
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P. M. Morganelli, S. M. Kennedy, and T. I. Mitchell Differential effects of interferon-{gamma} on metabolism of lipoprotein immune complexes mediated by specific human macrophage Fc{gamma} receptors J. Lipid Res., March 1, 2000; 41(3): 405 - 415. [Abstract] [Full Text]
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O Vaarala Antibodies to oxidised LDL Lupus, March 1, 2000; 9(3): 202 - 205. [Abstract] [PDF]
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J George, A Afek, B Gilburd, D Harats, and Y Shoenfeld Autoimmunity in atherosclerosis: lessons from experimental models Lupus, March 1, 2000; 9(3): 223 - 227. [Abstract] [PDF]
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K. Tanaga, H. Bujo, M. Inoue, K. Mikami, K. Kotani, K. Takahashi, T. Kanno, and Y. Saito Increased Circulating Malondialdehyde-Modified LDL Levels in Patients With Coronary Artery Diseases and Their Association With Peak Sizes of LDL Particles Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 662 - 666. [Abstract] [Full Text] [PDF]
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