B-Lymphocyte Deficiency Increases Atherosclerosis in LDL
Receptor–Null Mice
Amy S. Major, Sergio Fazio, MacRae F. Linton
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Objective—Atherosclerosis is an inflammatory disease characterized by innate and adaptive immune responses. We
investigated the role of B cells and antibodies in the development of atherosclerosis in low density lipoprotein (LDL)
receptor– deficient (LDLR⫺/⫺) mice.
Methods and Results—Using wild-type and B-cell– deficient mice as bone marrow donors, we were able to generate
LDLR⫺/⫺ mice that possessed ⬍1.0% of their normal B-cell population. B-cell– deficient LDLR⫺/⫺ mice on a Western
diet showed marked decreases in total serum antibody and anti– oxidized LDL antibody. B-cell deficiency was
associated with a 30% to 40% increase in the lesion area in the proximal and distal aortas. Real-time reverse
transcription–polymerase chain reaction and enzyme-linked immunospot analyses showed a decrease in proatherogenic
(interferon-␥) and antiatherogenic (interleukin-10 and transforming growth factor-␤) cytokine mRNA and a decrease in
interleukin-4 – and interferon-␥–producing cells. Additionally, we observed a decrease in splenocyte proliferation to
oxidized LDL in the B-cell– deficient LDLR⫺/⫺ mice, suggesting that B lymphocytes may play a role in the presentation
of lipid antigen.
Conclusions—Collectively, these data demonstrate that B cells and/or antibodies are protective against atherosclerosis and
that this protection may be conferred by B-cell–mediated immune regulation. (Arterioscler Thromb Vasc Biol. 2002;
22:●●●-●●●.)
Key Words: B-lymphocyte deficiency 䡲 B cells 䡲 atherosclerosis 䡲 LDL receptors
I
12) are important in atherogenesis and that T helper-2–
associated cytokines (IL-10) are protective against lesion
development.7–9 Collectively, these reports strongly suggest a
role for acquired immunity in the development of
atherosclerosis.
Although T cells and their associated cytokines can be
proatherogenic or antiatherogenic, much less is known regarding the role of B cells in atherosclerosis. B cells produce
antibodies that are thought to be involved in atherosclerosis.
These antibodies are specific for self-antigens, such as heat
shock protein 60 and ␤2-glycoprotein, “modified-self” antigens, such as oxidized LDL (oxLDL) and its phospholipid
components, and bacterial antigens, such as Streptococcus
and Chlamydia.10 –13 Some of these antibodies have been
characterized as “natural” antibodies and are thought to be
derived from a specific subset of B cells, B-1 B cells.10
Autoantibodies against many of these antigens have been
found in humans with coronary heart disease (CHD) and in
animal models of atherosclerosis, and titers of anti– oxidized
LDL (anti-oxLDL) antibodies are directly correlated with the
severity of disease.14 Studies in animals have demonstrated
that humoral immunity leading to the production of autoantibodies against modified lipoproteins provides protection
t is well established that atherosclerosis is an inflammatory
disease involving the innate and acquired immune systems.
Much of the focus in studying immune-associated mediators
of atherosclerosis has been on macrophages. Recently, however, increasing attention has been given to determining the
role of T and B lymphocytes in the development of atherosclerosis. Early atherosclerotic lesions in humans and mice
have been shown to contain significant numbers of T cells
and some B cells.1,2 Although these lymphocytes are present
in the artery wall at most stages of disease, their relative
contribution to the development of atherosclerosis is not yet
well understood.
Many experiments show a decrease in lesion development
in lymphocyte-deficient mice compared with control mice,3–5
suggesting that T cells may be proatherogenic. When the role
of subsets of T cells is examined, it appears that CD4⫹ T cells
may contribute to the atherosclerotic process.6 Studies in
apoE-deficient (apoE⫺/⫺) mice and LDL receptor– deficient
(LDLR⫺/⫺) mice null for both T and B cells have demonstrated decreased lesion formation, supporting proatherogenic
effects of these lymphocytes.3–5 There is also sufficient
evidence that cytokines associated with a proinflammatory T
helper-1 response (interferon [IFN]-␥ and interleukin [IL]-
Received September 3, 2002; revision accepted September 16, 2002.
From the Department of Medicine, Division of Cardiovascular Medicine (A.S.M., S.F., M.F.L.), the Department of Pathology (S.F.), and the
Department of Pharmacology (M.F.L.), Vanderbilt University, Nashville, Tenn.
Correspondence to Dr Amy S. Major, Vanderbilt University, Department of Medicine, Division of Cardiovascular Medicine, 2220 Pierce Ave, Room
383, PRB, Nashville, TN 37232-6300. E-mail [email protected]
© 2002 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
DOI: 10.1161/01.ATV.0000039169.47943.EE
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Arterioscler Thromb Vasc Biol.
December 2002
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against atherosclerosis, usually in the absence of significantly
large changes in lipoprotein levels or distribution. Immunization of atherosclerosis-prone rabbits and mice with oxidative forms of LDL has resulted in the production of large
amounts of anti-oxLDL autoantibody and in protection from
atherosclerosis.15,16 Recently, it has been shown that splenectomized apoE⫺/⫺ mice have increased atherosclerotic lesion
areas compared with those lesion areas in sham-operated
mice, even in the presence of extreme hyperlipidemia.17
Adoptive transfer of B cells from atherosclerotic apoE⫺/⫺
mice has been found to be protective against this increased
atherosclerosis. Collectively, these data offer indirect evidence that B cells and antibodies can be beneficial in the
prevention of CHD.
In the present study, we directly examined the role of B
cells in atherosclerosis. We used B-cell– deficient (␮MT)
mice and C57BL/6 mice as bone marrow donors for transplantation into LDLR⫺/⫺ mice. ␮MT mice were generated by
disrupting the gene encoding the membrane ␮-chain of the
B-cell receptor.18 Using this transplantation approach, we
were able to generate LDLR⫺/⫺ mice that completely lacked
B cells and, subsequently, circulating antibodies. We show
that atherosclerosis is increased in B-cell– deficient LDLR⫺/⫺
mice and that the effect of B cells and/or autoantibody was
apparent in early and late atherosclerotic lesions. This increase in atherosclerosis was not due to an obvious increase
in inflammatory responses in the absence of B cells but might
be due to autoantibody production or immune regulation via
antigen presentation.
Methods
Animals
respectively. Macrophages were visualized by staining with PE–antiMac1. Staining was analyzed by using a FacsCalibur flow cytometer
and Cell Quest software (Becton Dickinson).
Serum Cholesterol and Triglycerides
Serum cholesterol and triglyceride levels were determined as previously described.20
ELISA for Total and OxLDL Antibody
Normal human serum was collected, and LDL was isolated by
density centrifugation and copper-oxidized by using 10 ␮mol/L
Cu2O4 as previously described.21 Ninety-six–well plates were coated
with 50 ␮L oxLDL (10 ␮g/mL) in 0.1 mol/L NaH4CO3 overnight at
4°C. The plates were washed twice with PBS containing 0.5%
Tween 20 (PBS-T) and blocked with 5% BSA/PBS for 2 hours at
room temperature. Plates were washed, and serum samples were
added, followed by incubation overnight at 4°C. Bound antibody was
detected with biotin-conjugated anti-mouse immunoglobulin secondary antibody (Southern Biotech), followed by incubation with
avidin-conjugated peroxidase (Sigma Chemical Co). Color was
developed by the addition of the substrate 2,2⬘-azino-bis(3ethylbenzthiazoline-6-sulfonic acid) (Sigma). Optical density was
determined at 405 nm.
Total serum antibody titers were determined by coating 96-well
plates with 50 ␮L of anti-mouse immunoglobulin (1 ␮g/mL). The
remaining ELISA procedure was conducted as described above for
oxLDL. Serum was diluted 1:300 and 1:105 for oxLDL and total
antibody ELISAs, respectively.
Quantification of Atherosclerosis in the
Proximal Aorta
At 4 and 12 weeks after initiation of the Western diet, mice were
euthanized and perfused with 20 mL PBS through the right ventricle.
Hearts and aortas were removed, and the hearts were embedded in
OCT and frozen in liquid nitrogen. Atherosclerosis was analyzed
according to the method of Paigen et al.22 Lesion area was measured
by using Imaging System KS300 2.0
LDLR⫺/⫺ (B6, 129S-Ldlrtm1Her) and ␮MT (B6, 129S-Igh-6tm1Cgn)
mice, both on the C57BL/6 background, were purchased from The
Jackson Laboratory (Bar Harbor, Me). Animals were kept in microisolator cages and, unless otherwise stated, were given normal
chow and acidified water (pH 2.5). Donor female C57BL/6 mice
were obtained from our mouse colony. All experiments involving
animals were conducted under the approval of Vanderbilt University’s Institutional Animal Care and Use Committee.
Quantification of Atherosclerosis of Distal Aorta
by En Face Preparations
Bone Marrow Transplantation
Histology
Bone marrow transplantation (BMT) was conducted as previously
described.19 Four weeks after BMT, the mice were started on a
Western diet containing 21% fat and 0.15% cholesterol.
Masson’s trichrome staining and macrophage staining using anti–
MOMA-2 were performed as previously described.20,24 B-cell staining was performed by fixing 5-mm cryosections with cold acetone
and incubating them with rat anti-CD19 (clone 1D3, Pharmingen) at
a 1:100 dilution in 5% horse serum/PBS. Slides were washed twice
in PBS-T and incubated with goat anti-rat biotin-conjugated antibody (1:100 dilution, Pharmingen). Positive cells were detected by
using FastRed (Sigma). Positive and negative controls for CD19
staining included staining of spleen sections from C57BL/6 and
␮MT mice (data not shown).
Cell Preparation and Flow Cytometry Analysis
Five weeks after BMT, peripheral blood mononuclear cells (PBMCs)
were collected from both groups of mice. PBMCs were isolated by
Ficoll gradient centrifugation at 800g for 30 minutes. Cells were
washed and stained for specific cell surface markers. Splenocytes
from recipients were isolated at various times after BMT and washed
in either PBS or RPMI-1640 containing 10% FBS, L-glutamine,
2␤-mercaptoethanol, and antibiotics (referred to hereafter as TCM).
Cells were counted and resuspended to the necessary concentration
in either flow cytometry medium (1% FBS/PBS and 0.02% sodium
azide) or TCM.
All antibodies used for flow cytometry were purchased from
Pharmingen. Cell surface markers were stained by incubation of
PBMCs with antibodies against Thy1.1, B220, and Mac-1. The
percentage of B cells in spleens was determined by staining with
biotin–anti-CD19 and avidin-conjugated PerCP. Percent CD4⫹ and
CD8⫹ T cells were determined by FITC–anti-CD4 and PE–anti-CD8,
Mice were perfused as described above. Aortas, from the aortic valve
to the iliac bifurcation, were collected, fixed in 4% paraformaldehyde, cleaned of any surrounding tissue, opened longitudinally,
pinned flat, and stained with Sudan IV, as previously described.23
Lesion area was measured by use of Imaging System KS300 2.0 and
is expressed as a percentage of total aorta area.
Real-Time RT-PCR
After the rats were fed a Western diet for 4 weeks, spleens were
removed from 4 mice per group, and total spleen RNA was isolated
by using TRIzol reagent (Invitrogen). mRNA levels for IL-4, IL-10,
IFN-␥, and transforming growth factor (TGF)-␤ were measured by
using the One-Step Reverse Transcription–Polymerase Chain Reaction (RT-PCR) Master Mix Reagents as per the manufacturer’s
instructions (Applied Biosystems) and the Sequence Detection System (ABI Prism 7700 Sequence Detection System and software,
Applied Biosystems). Primer and probes were designed according to
Major et al
B Cells Protect Against Atherosclerosis
3
Figure 1. Flow cytometric analysis of splenocytes in C57BL/63 LDLR⫺/⫺ (A) and ␮MT3 LDLR⫺/⫺ (B) mice. Twelve weeks after BMT,
spleen cells were isolated from 3 mice per group and analyzed for the pan B-cell marker CD19, as described in Methods. The x-axis
represents the logarithmic fluorescence intensity for cells stained positively for CD19. The y-axis represents the relative number of
CD19-positive and -negative cells of the 10 000 cells analyzed. One representative animal from each group is shown. Positively stained
cells are represented by a shift to the right in mean logarithmic fluorescence.
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Xia et al.25 Cycling conditions for IL-4, IFN-␥, and IL-10 were as
follows: step 1, 30 minutes at 48°C; step 2, 10 minutes at 95°C; step
3, 15 seconds at 95°C; and step 4, 1 minute at 58°C. Steps 3 and 4
were repeated for a total of 40 cycles. The cycling conditions for
TGF-␤ were the same as above except that step 4 was conducted at
60°C. The 18S mRNA was used as an internal control. The 18S
control was always amplified on the same plate as the specific
cytokine with the use of the same amount of total RNA. Relative
amounts of mRNA for each cytokine were quantified by the standard
curve method.
Results are expressed as the optical density value at 490 nm after
subtraction of the background (no oxLDL wells).
Statistical Analyses
Statistical analyses on serum lipids, atherosclerosis data, and splenocyte proliferation experiments were conducted with a Student t test.
Real-time RT-PCR data were tested by use of a Mann-Whitney rank
sum test comparing the ␮MT3 LDLR⫺/⫺ group with the C57BL/
63 LDLR⫺/⫺ group. Values of P⬍0.05 and P⬍0.06 were considered
significant for the Student t test and rank sum test, respectively.
ELISPOT Assay
Results
Enzyme-linked immunospot (ELISPOT) assay for the quantification
of numbers of cytokine-producing spleen cells was performed as
previously described.26 Briefly, 96-well nitrocellulose plates (Millipore) were coated overnight at 4°C with either anti–IL-4 or anti–
IFN-␥ (both from Pharmingen) diluted in PBS. Plates were blocked
with TCM for 2 hours at room temperature, and pooled splenocytes
from 4 mice per group were added to the wells in the presence or
absence of 10 ␮g/mL oxLDL. Plates were incubated 24 hours at
37°C and 5% CO2, and then the cells were lysed, and the plates were
washed with PBS-T. The plates were incubated for 2 hours at room
temperature with 100 ␮L of either biotin-conjugated anti–IL-4 or
IFN-␥ (Pharmingen). After another wash cycle, the plates were
incubated for 1 hour at room temperature with 100 ␮L avidinperoxidase followed by development with substrate solution containing 0.15 mg/mL 5-bromo-4-chloro-3-indolylphosphate. Spot development occurred for ⬇30 minutes and was stopped by adding dH2O.
Cytokine spot-forming cells were counted by use of a dissecting
scope.
Splenocyte Proliferation
After 4 weeks of the Western diet, spleen cells from 4 mice per group
were isolated as described above and pooled. Cells were plated at
3⫻105 cells per well in a 96-well plate in TCM and incubated in
either the presence or absence of oxLDL. Proliferation was measured
by using the CellTiter 96MDSU⫺ AQueous One Solution Cell Proliferation Assay as per the manufacturer’s specifications (Promega).
TABLE 1.
Splenocyte Number and Cell Surface Phenotype in Bone Marrow–Transplanted LDLRⴚ/ⴚ Mice
Cell No. (106)
%CD19⫹
No. CD19⫹(10⫺6)
C57BL/6
33.3 (3.0)
66.1 (2.8)
22.1 (2.6)
mMT
11.3 (0.8)*
Donor Strain
BMT results in the deficiency of B-cell and serum antibody
concentration. Mice were lethally irradiated and reconstituted
with either C57BL/6 (C57BL/63 LDLR⫺/⫺) or ␮MT
(␮MT3 LDLR⫺/⫺) bone marrow. Flow cytometric analyses
of PBMCs confirmed the reconstitution of irradiated
LDLR⫺/⫺ mice, demonstrating that at 5 weeks after BMT,
⬇5% of the circulating monocytes were B cells in C57BL/
63 LDLR⫺/⫺ mice, whereas ⬍1.0% of the circulating monocytes in ␮MT3 LDLR⫺/⫺ mice were B cells (data not
shown). Analysis of splenocytes 12 weeks after BMT showed
that the B-cell compartment was reconstituted in C57BL/
63 LDLR⫺/⫺ mice (Figure 1A) but was completely absent in
the ␮MT3 LDLR⫺/⫺ mice (Figure 1B). The percentages of
CD4⫹ and CD8⫹ cells and Mac-1⫹ cells were increased in the
␮MT recipients (Table 1). However, this did not result in an
increase in the absolute numbers of these cells because the
␮MT3 LDLR⫺/⫺ group had significantly lower numbers of
total spleen cells compared with the control group.
Concentrations of total serum antibody were similar for all
recipient mice before BMT. However, starting at 4 weeks on
the diet (8 weeks after BMT), the ␮MT3 LDLR⫺/⫺ mice
compared with C57BL/6 recipients had a significantly lower
2.1 (0.5)*
0.2 (0.1)*
%CD4⫹
No. CD4⫹(10⫺6)
%CD8⫹
No. CD8⫹(10⫺6)
%Mac1⫹
No. Mac1⫹(10⫺6)
19.7 (2.5)
6.6 (1.0)
9.0 (1.2)
3.0 (0.4)
9.5 (0.9)
3.2 (0.6)
48.8 (3.5)*
5.5 (0.3)
30.7 (3.3)*
3.5 (0.6)
28.3 (3.2)*
3.2 (0.5)
Data are represented as cell No. or percent (⫾SEM) of three animals in each group.
Splenocytes were isolated and analyzed at 12 weeks after BMT.
*Statistical significance (P⬍0.01) compared to the C57BL/6 group as determined by Student’s t test.
4
Arterioscler Thromb Vasc Biol.
December 2002
Figure 2. Total antibody (A) and anti-oxLDL antibody (B) were measured by ELISA before BMT and at 4 and 12 weeks after initiation of
Western diet in C57BL/63 LDLR⫺/⫺ (open bars) and ␮MT3 LDLR⫺/⫺ (closed bars) mice. Bars represent the mean⫾SE of 8 to 10 mice
per group.
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total serum antibody concentration (Figure 2A). In a similar
manner, antibodies against oxLDL were also dramatically
reduced in the ␮MT3 LDLR⫺/⫺ group compared with the
C57BL/63 LDLR⫺/⫺ control group (Figure 2B). These data
show that transplantation of B-cell– deficient marrow results
in LDLR⫺/⫺ mice that lack B cells and antibody without
change in the absolute numbers of other immune cells, such
as T cells and macrophages.
B-Cell Deficiency Does Not Result in Significant
Change in Serum Cholesterol or Triglycerides
There were no significant differences in fasting serum cholesterol or triglyceride levels, at any time during the study, in
LDLR⫺/⫺ mice that were transplanted with either ␮MT or
C57BL/6 bone marrow (Table 2). The ␮MT mice are on the
C57BL/6 background; thus, genetic differences in donor
strains is not a concern in these studies. Before the initiation
of the Western diet, mice in both groups had cholesterol and
triglyceride levels that ranged between 200 and 400 mg/dL
and between 100 and 200 mg/dL, respectively. After the
initiation of the Western diet, the levels of serum cholesterol
and triglycerides dramatically increased in both groups, as
expected. In addition, there were no significant differences in
body weight between the 2 groups at any time point
measured.
B-Cell Deficiency Increases Atherosclerotic Lesions
in Proximal and Distal Aortas in LDLRⴚ/ⴚ Mice
To determine the effect of B-cell deficiency on early and late
atherosclerotic lesions, transplanted LDLR⫺/⫺ mice were
euthanized at 4 and 12 weeks after the initiation of the
Western diet. Oil red O analyses of the proximal aorta
revealed a 2-fold increase in early fatty streak lesions after 4
weeks on the Western diet (8 weeks after BMT) in the
␮MT3 LDLR⫺/⫺ female mice (32 000⫾4000 ␮m2 per secTABLE 2.
Levels of Cytokine mRNA and Cell Proliferation
Are Reduced in Spleens of B-Cell–Deficient
LDLRⴚ/ⴚ Mice
To test the hypothesis that the absence of B cells in peripheral
secondary lymphoid organs affects T helper-1/2 responses
during atherogenesis, mRNA levels for IL-10, IL-4, TGF-␤,
and IFN-␥ were measured in total spleen RNA after 4 weeks
Body Weight and Serum Cholesterol and Triglyceride in BMT LDLRⴚ/ⴚ Mice
0 Weeks
Donor Strain
tion) compared with the C57BL/63 LDLR⫺/⫺ female control
mice (17 000⫾6000 ␮m2 per section; Figure 3A, left). This
difference was still apparent after 12 weeks on the diet (16
weeks after BMT), when lesions are more complicated
(Figure 3A, right). Male ␮MT3 LDLR⫺/⫺ mice (n⫽8) also
showed a significant increase in atherosclerotic lesion area
(P⫽0.046) compared with male C57BL/63 LDLR⫺/⫺ control
mice (n⫽7) after 4 weeks on the diet (10 221⫾2528 versus
2552⫾603 ␮m2 per section, respectively). This increase in
atherosclerosis after 4 weeks on the diet in B-cell– deficient
LDLR⫺/⫺ male mice also occurred without any differences in
serum cholesterol (data not shown).
En face analyses of atherosclerosis in the distal aorta
showed a 2-fold increase in lesion area after 12 weeks on the
diet in the ␮MT3 LDLR⫺/⫺ mice (0.46⫾0.11%) compared
with the control mice (0.22⫾0.04%, Figure 3B). However,
this difference did not quite reach statistical significance
(P⫽0.052).
Masson’s trichrome staining demonstrated that the lesions
in both groups of animals after 12 weeks on the diet had
significant but similar collagen deposition (blue) that was
deposited adjacent to, but not within, the macrophage–foam
cell area of the lesion (Figure 4A through 4D). In addition,
immunostaining for CD19 showed no presence of B lymphocytes in atherosclerotic lesions of the proximal aorta in either
the ␮MT3 LDLR⫺/⫺ or the C57BL/63 LDLR⫺/⫺ mice (Figure 4E and 4F).
4 Weeks
12 Weeks
Weight
Chol
Trig
Weight
Chol
Trig
Weight
Chol
Trig
C57BL/6
14.3 (0.3)
298.2 (5.5)
107.4 (4.0)
19.6 (0.3)
853.3 (46.7)
230.3 (15.0)
19.7 (0.6)
1147.5 (78.9)
319.9 (18.2)
mMT
13.1 (0.5)
309.3 (23.9)
103.2 (2.3)
19.2 (0.3)
818.9 (48.4)
263.7 (21.3)
20.8 (0.9)
1253.6 (39.2)
355.7 (8.1)
Body weight, serum cholesterol, and triglyceride were measured at 0, 4, and 12 weeks after the initiation of Western diet.
Body weight is expressed as g (⫾SEM); serum cholesterol (Chol) and triglyceride (Trig) are expressed as mg/dL (⫾SEM).
There were no significant differences between groups in body wt or serum cholesterol and triglyceride at any time point as determined by Student’s t-test.
Major et al
B Cells Protect Against Atherosclerosis
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Figure 3. Atherosclerotic lesion area was determined in the proximal aorta by oil red O staining (A) and in the distal aorta by en face
analysis (B) 12 weeks after initiation of the Western diet as described in Methods. Bars represent the mean⫾SE of the C57BL/
63 LDLR⫺/⫺ (open bars) and ␮MT3 LDLR⫺/⫺ (closed bars) groups. *P⬍0.05 and **P⫽0.052 by Student t test. Numbers of mice per
group are labeled on bars.
on the diet by quantitative real-time RT-PCR. There was a
30% to 50% decrease in all cytokine mRNA levels in the
spleens of ␮MT3 LDLR⫺/⫺ mice compared with control
mice (Figure 5A). ELISPOT analyses on splenocytes from
mice on the diet for 12 weeks showed that the frequency of
IL-4 –producing cells in the ␮MT3 LDLR⫺/⫺ group was 270
per 107 cells compared with 507 per 107 cells in the C57BL/
63 LDLR⫺/⫺ group. Likewise, IFN-␥–producing cells were
reduced in the ␮MT3 LDLR⫺/⫺ splenocytes compared with
control splenocytes (6 per 107 cells versus 22 per 107 cells,
respectively). In addition to a decrease in cytokine production
in the spleen, ex vivo, we also observed a significant decrease
in spleen cell proliferation after restimulation with oxLDL
(Figure 5B).
Figure 4. Histological analyses of proximal aortic lesions in C57BL/63 LDLR⫺/⫺ mice (A, C, and E) and ␮MT3 LDLR⫺/⫺ mice (B, D, and
F) after 12 weeks on Western diet. A and B, Masson’s trichrome staining showing collagen (blue) and elastin (black) deposition. C and
D, Staining for macrophages by use of an antibody against MOMA-2. E and F, B-cell staining by use of anti-CD19 antibody. Sections
are serial sections from 1 representative mouse in each group and are shown at ⫻100.
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Arterioscler Thromb Vasc Biol.
December 2002
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Figure 5. Real-time RT-PCR analysis of cytokine mRNA and cell proliferation in spleen cells. A, Total RNA from the spleens was isolated, and relative amounts mRNA levels for IL-10, IL-4, TGF-␤, and IFN-␥ were determined by quantitative real-time RT PCR. B,
Spleen cells were isolated and pooled from 3 mice per group and incubated in the presence or absence of oxLDL. Bars in panel A are
mean⫾SE values of 4 mice per group, with open bars representing C57BL/63 LDLR⫺/⫺ mice and closed bars representing
␮MT3 LDLR⫺/⫺ mice. Bars in panel B are the mean⫾SD values of 6 wells per group and treatment. **P⬍0.06 by Mann-Whitney rank
sum test; *P⬍0.001 by Student t test.
Discussion
The present study is the first to examine the effect of a
specific and total B-cell deficiency on atherogenesis. We
describe a protective role for B cells and/or antibody during
atherosclerotic lesion development in LDLR⫺/⫺ mice on a
Western diet. This protection was observed without any
obvious difference in serum cholesterol or triglyceride levels
between experimental and control animals. B-cell deficiency
was deleterious at times, representing early fatty streak
lesions (after 4 weeks on the diet) and, later, more advanced
lesions (after 12 weeks on the diet) in the proximal aorta.
Increased atherosclerosis was also evident in the distal aorta
after 12 weeks on the Western diet. The increased atherosclerosis in ␮MT3 LDLR⫺/⫺ mice did not appear to be due to
increased inflammatory responses in these mice because we
observed similar decreases in “antiatherogenic” (IL-10 and
TGF-␤) and “proatherogenic” (IFN-␥) cytokine mRNA levels as well as cytokine-producing cells in the spleen. We also
demonstrate a decrease in splenocyte proliferation in response to oxLDL in the absence of available B cells as
antigen-presenting cells.
Recently, Caligiuri et al17 showed that compared with
sham-operated control mice, removal of the spleen (which
serves as a large reservoir for T and B lymphocytes) from
apoE⫺/⫺ mice increased atherosclerosis. This difference was
observed in a severe hyperlipidemic environment, inasmuch
as the animals were placed on a Western diet for 12 weeks.
The authors were able to show reduction of atherosclerosis
when splenic B cells from atherosclerotic apoE⫺/⫺ mice were
adoptively transferred to splenectomized as well as shamoperated animals. These data suggest that during atherosclerosis development, protective effector and/or memory B cells
are generated in the spleen. However, because only splenic B
cells were removed in splenectomized mice, the importance
of other B cell pools in sites such as lymph nodes, bone
marrow, and the peritoneal cavity (as well as nonimmune
functions of the spleen) in protection against atherosclerosis
was not addressed.
In the present study, we were able to assess the modulation
of atherosclerosis after complete, not partial, B-cell and
antibody deficiency. Furthermore, we demonstrated possible
antiatherogenic properties of B cells beyond antibody production. The overall reduction in cytokine production and cell
proliferation in spleens of ␮MT3 LDLR⫺/⫺ mice compared
with control mice (Figure 4A and 4B) indicates that B cells
may regulate immunity during the development of atherosclerosis, perhaps via antigen presentation of lipid antigen to T
cells.
The exact role of B cells or autoantibodies in atherosclerosis is not well defined. We focus mainly on the production
of oxLDL antibodies because this most likely would be one
of the major “autoantigens” present in LDLR⫺/⫺ mice on an
atherogenic diet. Several studies have demonstrated the
beneficial effects of anti-oxLDL antibody on the progression
of atherosclerosis.10,15,16,27 Shaw et al27 showed that a humanderived anti-oxLDL antibody could block the uptake of
oxLDL and apoptotic cells by macrophages, 2 mechanisms
that would be considered antiatherogenic. This could most
likely occur without affecting total cholesterol levels because
our data show increased atherosclerosis in the absence of
antibody, with no differences in total serum cholesterol
(Table 2). In addition to blocking oxLDL uptake, antibodyantigen complexes have been shown to regulate macrophage
and other immune cell responses via Fc␥ receptors.28 Much
evidence has accumulated to implicate a role for Fc␥ receptor
signaling in atherosclerosis. Several classes of Fc␥ receptors
have been detected in human atherosclerotic lesions,29 and
patients with advanced atherosclerosis have reduced levels of
CD32 (Fc␥ receptor IIA) expression on circulating monocytes, a molecule known to downregulate cell activation.30
Major et al
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These studies, in addition to the present study, support a
protective role of antibody-mediated effects on
atherosclerosis.
The effects of antibody may be proatherogenic or antiatherogenic, depending on the target antigen studied. Immunization of animals with heat shock protein 65 or ␤2-glycoprotein has been shown to lead to specific humoral immunity
against these antigens with increased lesion development.31–33
However, whether the increase in antibody production is a
cause or effect of atherosclerosis is not known. It would be
interesting to determine whether immunization of B-cell–
deficient mice with proatherogenic antigens would lead to
further increases in atherosclerosis. Indirect in vitro evidence
that antibody function could be proatherogenic demonstrated
that oxLDL immune complexes, via Fc␥ receptor binding,
activated a human monocyte/macrophage cell line.34 In addition, antibodies are also important activators of the complement system, which has been shown to be associated with
C-reactive protein in atherosclerotic lesions.35 Therefore, it
appears that the role of autoantibody in CHD is complicated
and may be system and antigen dependent.
B cells can regulate immune responses, independent of
antibody production, in other inflammatory situations, such
as ulcerative colitis and type-1 diabetes.36,37 Adoptive transfer
of activated B cells mediated protection in type-1 diabetes by
the induction of apoptosis of diabetogenic T cells via the
Fas/FasL signaling pathway.37 In addition, B cells are important for the development of memory CD4⫹ T cells in the
spleen and peripheral populations of a specific subset of
regulatory T cells (CD4⫹CD25⫹) that serve to decrease
inflammatory responses.38,39 These data are consistent with
our observations that T-cell–associated immune responses to
oxLDL are decreased in the spleen in the absence of B cells
(Figure 5A and 5B). The observation of lower numbers of
cytokine-producing cells and less proliferation in response to
oxLDL indicates that B cells act as a major lipid antigen–
presenting cell to other immune cells, such as T cells. Our
present data and the studies of others demonstrate that CD19⫹
B cells are not present in the aortic lesion (Figure 4E and 4F
and Zhou and Hansson2). Because CD19 is not expressed on
plasma cells, it is possible that fully differentiated plasma
cells are present in the lesions of LDLR⫺/⫺ mice after
transplantation. At any rate, our data suggest that B cells may
not have a direct local effect in the artery wall. One may
hypothesize that in the absence of B cells, a subset of
regulatory cells that serve to decrease inflammatory responses in the growing lesion or that inhibit lymphocyte
migration to atherosclerotic plaques is not activated in the
periphery. This hypothesis is supported by data showing that
splenectomized apoE⫺/⫺ mice had increased numbers of
CD4⫹ T cells in arterial lesions, which was decreased to
control levels after splenic B cell transfer.17 Additional
studies examining B cells as antigen-presenting cells for
oxLDL and as regulators of specific immune responses are
necessary to make further conclusions.
In summary, we have definitively shown that in LDLR⫺/⫺
mice, B cells are protective against the early and late stages of
atherosclerosis. This protection is likely related to antibody
production but may also involve the regulation of cellular
B Cells Protect Against Atherosclerosis
7
immunity against antigens such as oxLDL. Therefore, by
increasing our understanding of the mechanisms by which B
cells and antibodies confer protection against atherosclerosis,
it may be possible to develop future immune-modulating
therapies for individuals suffering from CHD.
Acknowledgments
This work was supported by National Institutes of Health (NIH)
grants HL-53989 and HL-57986 awarded to Drs Linton and Fazio,
respectively, and by the Lipid, Lipoprotein, and Atherosclerosis Core
of the Vanderbilt Mouse Metabolic Physiology Center (NIH grant
DK59637-01). Dr Major is supported by an NRSA Individual
Postdoctoral Fellowship (HL-10206-02). Drs Linton and Fazio are
Established Investigators of the American Heart Association. We
thank Drs Yan Ru Su and Alyssa H. Hasty for critical review of the
manuscript and Dr Su for advice regarding the real-time RT-PCR.
References
1. Roselaar SE, Kakkanathu PX, Daugherty A. Lymphocyte populations in
atherosclerotic lesions of apoE -/- and LDL receptor -/- mice: decreasing
density with disease progression. Arterioscler Thromb Vasc Biol. 1996;
16:1013–1018.
2. Zhou X, Hansson GK. Detection of B cells and proinflammatory cytokines in atherosclerotic plaques of hypercholesterolaemic apolipoprotein
E knockout mice. Scand J Immunol. 1999;50:25–30.
3. Daugherty A, Pure E, Delfel-Butteiger D, Chen S, Leferovich J, Roselaar
SE, Rader DJ. The effects of total lymphocyte deficiency on the extent of
atherosclerosis in apolipoprotein E-/- mice. J Clin Invest. 1997;100:
1575–1580.
4. Song L, Leung C, Schindler C. Lymphocytes are important in early
atherosclerosis. J Clin Invest. 2001;108:251–259.
5. Reardon CA, Blachowicz L, White T, Cabana V, Wang Y, Lukens J,
Bluestone J, Getz GS. Effect of immune deficiency on lipoproteins and
atherosclerosis in male apolipoprotein E-deficient mice. Arterioscler
Thromb Vasc Biol. 2001;21:1011–1016.
6. Zhou X, Nicoletti A, Elhage R, Hansson GK. Transfer of CD4(⫹) T cells
aggravates atherosclerosis in immunodeficient apolipoprotein E knockout
mice. Circulation. 2000;102:2919 –2922.
7. Lee TS, Yen HC, Pan CC, Chau LY. The role of interleukin 12 in the
development of atherosclerosis in apoE-deficient mice. Arterioscler
Thromb Vasc Biol. 1999;19:734 –742.
8. Pinderski Oslund LJ, Hedrick CC, Olvera T, Hagenbaugh A, Territo M,
Berliner JA, Fyfe AI. Interleukin-10 blocks atherosclerotic events in vitro
and in vivo. Arterioscler Thromb Vasc Biol. 1999;19:2847–2853.
9. Whitman SC, Ravisankar P, Elam H, Daugherty A. Exogenous
interferon-gamma enhances atherosclerosis in apolipoprotein E-/- mice.
Am J Pathol. 2000;157:1819 –1824.
10. Shaw PX, Horkko S, Chang MK, Curtiss LK, Palinski W, Silverman GJ,
Witztum JL. Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity. J Clin Invest.
2000;105:1731–1740.
11. Sherer Y, Tenenbaum A, Praprotnik S, Shemesh J, Blank M, Fisman EZ,
Motro M, Shoenfeld Y. Autoantibodies to cardiolipin and beta-2-glycoprotein-I in coronary artery disease patients with and without hypertension. Cardiology. 2002;97:2–5.
12. Kawamoto R, Doi T, Tokunaga H, Konishi I. An association between an
antibody against Chlamydia pneumoniae and common carotid atherosclerosis. Intern Med. 2001;40:208 –213.
13. Wick G. Atherosclerosis: an autoimmune disease due to an immune
reaction against heat-shock protein 60. Herz. 2000;25:87–90.
14. Tsimikas S, Palinski W, Witztum JL. Circulating autoantibodies to
oxidized LDL correlate with arterial accumulation and depletion of
oxidized LDL in LDL receptor– deficient mice. Arterioscler Thromb Vasc
Biol. 2001;21:95–100.
15. Freigang S, Horkko S, Miller E, Witztum JL, Palinski W. Immunization
of LDL receptor– deficient mice with homologous malondialdehydemodified and native LDL reduces progression of atherosclerosis by mechanisms other than induction of high titers of antibodies to oxidative
neoepitopes. Arterioscler Thromb Vasc Biol. 1998;18:1972–1982.
16. Palinski W, Miller E, Witztum JL. Immunization of low density
lipoprotein (LDL) receptor-deficient rabbits with homologous
8
17.
18.
19.
20.
21.
22.
23.
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
24.
25.
26.
27.
Arterioscler Thromb Vasc Biol.
December 2002
malondialdehyde-modified LDL reduces atherogenesis. Proc Natl Acad
Sci U S A. 1995;92:821– 825.
Caligiuri G, Nicoletti A, Poirier B, Hansson GK. Protective immunity
against atherosclerosis carried by B cells of hypercholesterolemic mice.
J Clin Invest. 2002;109:745–753.
Kitamura D, Roes J, Kühn R, Rajewsky K. A B cell-deficient mouse by
targeted disruption of the membrane exon of the immunoglobulin ␮ chain
gene. Nature. 1991;350:423– 426.
Linton MF, Atkinson JB, Fazio S. Prevention of atherosclerosis in apoE
deficient mice by bone marrow transplantation. Science. 1995;267:
1034 –1037.
Fazio S, Major AS, Swift LL, Gleaves LA, Accad M, Linton MF, Farese
RV Jr. Accelerated atherosclerosis in LDL receptor-null mice lacking
ACAT1 in macrophages. J Clin Invest. 2001;107:163–171.
Lopes-Virella MF, Koskinen S, Mironova M, Horne D, Klein R,
Chassereau C, Enockson C, Virella G. The preparation of copperoxidized LDL for the measurement of oxidized LDL antibodies by EIA.
Atherosclerosis. 2000;152:107–115.
Paigen B, Morrow A, Holmes PA, Mitchell D, Williams RA. Quantitative
assessment of atherosclerotic lesions in mice. Atherosclerosis. 1987;68:
231–240.
Tangirala RK, Rubin EM, Palinski W. Quantitation of atherosclerosis in
murine models: correlation between lesions in the aortic origin and in the
entire aorta, and differences in the extent of lesions between sexes in LDL
receptor-deficient and apolipoprotein E-deficient mice. J Lipid Res. 1995;
36:2320 –2328.
Shi C, Lee WS, Russell ME, Zhang D, Fletcher DL, Newell JB, Haber E.
Hypercholesterolemia exacerbates transplant arteriosclerosis via
increased neointimal smooth muscle cell accumulation: studies in apolipoprotein E knockout mice. Circulation. 1997;96:2722–2728.
Xia D, Sanders A, Shah M, Bickerstaff A, Orosz C. Real-time polymerase
chain reaction analysis reveals an evolution of cytokine mRNA production in allograft acceptor mice. Transplantation. 2001;72:907–914.
Major AS, Cuff CF. Enhanced mucosal and systemic immune responses
to intestinal reovirus infection in beta2-microglobulin-deficient mice.
J Virol. 1997;71:5782–5789.
Shaw PX, Horkko S, Tsimikas S, Chang MK, Palinski W, Silverman GJ,
Chen PP, Witztum JL. Human-derived anti-oxidized LDL autoantibody
blocks uptake of oxidized LDL by macrophages and localizes to atherosclerotic lesions in vivo. Arterioscler Thromb Vasc Biol. 2001;21:
1333–1339.
28. Ravetch JV, Bolland S. IgG Fc receptors. Annu Rev Immunol. 2001;19:
275–290.
29. Ratcliffe NR, Kennedy SM, Morganelli PM. Immunocytochemical
detection of Fcgamma receptors in human atherosclerotic lesions.
Immunol Lett. 2001;77:169 –174.
30. Pfeiffer JR, Howes PS, Waters MA, Hynes ML, Schnurr PP, Demidenko
E, Bech FR, Morganelli PM. Levels of expression of Fcgamma receptor
IIA (CD32) are decreased on peripheral blood monocytes in patients with
severe atherosclerosis. Atherosclerosis. 2001;155:211–218.
31. George J, Afek A, Gilburd B, Blank M, Levy Y, Aron-Maor A, Levkovitz
H, Shaish A, Goldberg I, Kopolovic J, Harats D, Shoenfeld Y. Induction
of early atherosclerosis in LDL-receptor-deficient mice immunized with
beta2-glycoprotein I. Circulation. 1998;98:1108 –1115.
32. Afek A, George J, Gilburd B, Rauova L, Goldberg I, Kopolovic J, Harats
D, Shoenfeld Y. Immunization of low-density lipoprotein receptor
deficient (LDL-RD) mice with heat shock protein 65 (HSP-65) promotes
early atherosclerosis. J Autoimmun. 2000;14:115–121.
33. Afek A, George J, Shoenfeld Y, Gilburd B, Levy Y, Shaish A, Keren P,
Janackovic Z, Goldberg I, Kopolovic J, Harats D. Enhancement of atherosclerosis in beta-2-glycoprotein I-immunized apolipoprotein
E-deficient mice. Pathobiology. 1999;67:19 –25.
34. Huang Y, Jaffa A, Koskinen S, Takei A, Lopes-Virella MF. Oxidized
LDL-containing immune complexes induce Fc gamma receptor
I-mediated mitogen-activated protein kinase activation in THP-1 macrophages. Arterioscler Thromb Vasc Biol. 1999;19:1600 –1607.
35. Torzewski J, Torzewski M, Bowyer DE, Frohlich M, Koenig W,
Waltenberger J, Fitzsimmons C, Hombach V. C-reactive protein frequently colocalizes with the terminal complement complex in the intima
of early atherosclerotic lesions of human coronary arteries. Arterioscler
Thromb Vasc Biol. 1998;18:1386 –1392.
36. Mizoguchi A, Mizoguchi E, Takedatsu H, Blumberg RS, Bhan AK.
Chronic intestinal inflammatory condition generates IL-10-producing
regulatory B cell subset characterized by CD1d upregulation. Immunity.
2002;16:219 –230.
37. Tian J, Zekzer D, Hanssen L, Lu Y, Olcott A, Kaufman DL. Lipopolysaccharide-activated B cells down-regulate Th1 immunity and prevent autoimmune diabetes in nonobese diabetic mice. J Immunol. 2001;167:
1081–1089.
38. Suto A, Nakajima H, Ikeda K, Kubo S, Nakayama T, Taniguchi M, Saito
Y, Iwamoto I. CD4(⫹)CD25(⫹) T-cell development is regulated by at
least 2 distinct mechanisms. Blood. 2002;99:555–560.
39. Ngo VN, Cornall RJ, Cyster JG. Splenic T zone development is B cell
dependent. J Exp Med. 2001;194:1649 –1660.
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
B-Lymphocyte Deficiency Increases Atherosclerosis in LDL Receptor-Null Mice
Amy S. Major, Sergio Fazio and MacRae F. Linton
Arterioscler Thromb Vasc Biol. published online September 26, 2002;
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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