Nobuhiko Kubo, Hilde Cheroutre, Linda K. Curtiss, Judith A. Berliner and William A.

Boisvert
Laura J. Pinderski, Michael P. Fischbein, Ganesamoorthy Subbanagounder, Michael C. Fishbein,
Deficient Mice by Altering Lymphocyte and Macrophage Phenotypes ! LDL Receptor
Overexpression of Interleukin-10 by Activated T Lymphocytes Inhibits Atherosclerosis in
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Overexpression of Interleukin-10 by Activated
T Lymphocytes Inhibits Atherosclerosis in LDL
ReceptorDeficient Mice by Altering Lymphocyte
and Macrophage Phenotypes
Laura J. Pinderski, Michael P. Fischbein, Ganesamoorthy Subbanagounder, Michael C. Fishbein,
Nobuhiko Kubo, Hilde Cheroutre, Linda K. Curtiss, Judith A. Berliner, William A. Boisvert
AbstractPrevious studies demonstrated that interleukin-10 (IL-10) overexpression decreases formation of early
fatty-streak lesions in mice independent of lipoprotein levels. The present studies, using bone marrow transplantation,
demonstrate that overexpression of IL-10 by T cells inhibits advanced atherosclerotic lesions in LDL receptornull mice
fed an atherogenic diet. In mice receiving bone marrow from the IL-10 transgenic mice compared with those receiving
wild-type marrow, there was a 47% decrease in lesion size and a marked decrease in lesion complexity with an 80%
reduction in the necrotic core. Accumulation of cholesterol and phospholipid oxidation products in the aorta was
decreased by 50% to 80%, unrelated to plasma lipid or IL-10 levels. Our studies also provide insight into the mechanism
of the IL-10mediated decrease in lesion size. Although a strong influence toward a Th1 phenotype has previously been
demonstrated in atherosclerotic models, T lymphocytes in the IL-10 transgenic (Tg) group revealed a marked shift to
a Th2 phenotype, with decreased IFN- production and an increase in IL-10. Evaluation of specific immunoglobulin
subclasses demonstrated a preponderance of IgG
1
isotype, characteristic of a Th2 influence on B cell immunoglobulin
class-switching in the IL-10 Tg group. A major finding of these studies was altered monocyte/macrophage function in
the IL-10 Tg group. Monocytes showed a decrease in activation resulting in decreased expression of IFN-.
Furthermore, macrophage foam cells within lesions of the IL-10 Tg group exhibited markedly decreased apoptosis.
These studies demonstrate that T lymphocyte IL-10 can influence the function of other immune cells to reduce the
development of advanced atherosclerotic lesions in mice. (Circ Res. 2002;90:1064-1071.)
Key Words: cytokine

LDL receptor

B lymphocytes

antibodies

apoptosis
A
therosclerosis has been demonstrated to be a chronic
inflammatory process involving various immune cells,
particularly macrophages and T lymphocytes. We and others
have shown that interleukin (IL)-10 overexpression can
inhibit fatty-streak formation, independent of lipoprotein
levels, in C57BL/6J mice fed a cholate containing athero-
genic diet.
1,2
However, these studies were restricted to exam-
ining the development of early lesions in C57BL/6J mice on
a cholate-containing diet, which is known to alter lipid
metabolism and induce inflammatory cytokines in the liver.
3
Therefore, cholate may artificially predispose the athero-
sclerotic process in these animals to be highly inflamma-
tory, partially exaggerating the antiinflammatory role of
IL-10. Because of the therapeutic potential of IL-10 in
human atherosclerosis, the present studies were directed at
determining whether IL-10 can retard development of
advanced atherosclerotic lesions in an animal model that
did not require a cholate-containing diet and, more impor-
tantly, examining the mechanism of IL-10 inhibition of
atherogenesis.
Evidence that IL-10 may play a role in advanced lesions
was demonstrated by several in vivo studies. IL-10 has been
demonstrated within human atherosclerotic plaques,
4,5
pri-
marily in macrophages. Increased local levels of IL-10 has
been associated with decreased apoptotic activity as mea-
sured by TUNEL staining, as well as decreased local iNOS
expression within these diseased human arteries.
4
Decreased
levels of IL-10, contrasted with increased levels of IL-6, have
been demonstrated in patients with unstable coronary syn-
dromes.
6
In vitro studies have demonstrated that oxidized
lipids can alter IL-10 expression within human monocytes,
5
and IL-10 can alter human aortic endothelial cell function to
inhibit oxidized phospholipid-induced monocyte-endothelial
interactions in vitro.
2
Both the in vivo and the in vitro studies
Original received May 16, 2001; revision received April 3, 2002; accepted April 9, 2002.
From the Departments of Medicine (L.J.P, G.S., J.A.B.), Experimental Pathology (L.J.P., M.C.F., J.A.B.), Surgery (M.P.F.), and Microbiology and
Immunology (M.P.F.), University Of California Los Angeles; Department of Immunology (N.K., L.K.C., W.A.B.), The Scripps Research Institute, La
Jolla; and the Division of Developmental Immunology (H.C.), The La Jolla Institute For Allergy and Immunology, San Diego, Calif.
Correspondence to Laura J. Pinderski, University of Alabama, Birmingham, Division of Cardiology, THT 332, 1900 University Blvd, Birmingham,
AL 35294-0006. E-mail lpinderski@cardio.com.uab.edu
2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000018941.10726.FA
1064
Molecular Medicine
by guest on March 6, 2014 http://circres.ahajournals.org/ Downloaded from
suggested, but did not directly test, potential mechanisms by
which IL-10 might inhibit atherosclerosis.
Our present studies used transplantation of bone marrow
from either wild-type or IL-10 transgenic mice, in which the
murine IL-10 gene is under the control of a human IL-2
promoter,
7
into LDL receptordeficient (LDL-R
/
) mice.
This model allows for localized overexpression of IL-10,
because transgenic expression is restricted to T lymphocytes
that have encountered antigen or otherwise had their T-cell
receptor engaged. These studies tested the hypothesis that
IL-10 overexpression by T cells could reduce advanced
atherosclerotic lesion formation by shifting the T-cell pheno-
type from a T-helper 1 (Th1) phenotype, previously seen in
atherosclerosis,
8,9
to a Th2 phenotype. Previous studies in
inflammatory bowel disease suggested that such a switch
might be possible. Through cell-cell interaction and cytokine
secretion, T-helper lymphocytes can impact B lymphocyte
and monocyte/macrophage function. The data presented here
demonstrate that IL-10 overexpression in activated T lym-
phocytes inhibits development of advanced lesions and de-
creases accumulation of cholesterol and phospholipids in
hyperlipidemic LDL-R
/
mice. These data suggest that this
inhibition is mediated by the specific shift toward a Th2
phenotype with subsequent alteration in monocyte and lym-
phocyte function.
Materials and Methods
Animal Models
LDL-R
/
mice were initially obtained from Jackson Laboratory
(Bar Harbor, Maine). All mice were weaned at 4 weeks and initially
fed using a chow diet (No. 5015; Harlan-Teklad). Mice were housed
under a 12-hour light/dark cycle in specific pathogen-free conditions.
Marrow cells used for repopulation of the irradiated mice were
isolated from either 8-week-old C57BL/6J male mice (WT) or IL-10
transgenic male mice on a C57BL/6J background (IL-10 Tg).
7
Nineteen 6-week-old male LDL-R
/
mice were subjected to bone
marrow transplantation (BMT), as previously described.
10
The re-
cipient mice received bone marrow from either WT (n10) or IL-10
Tg (n9) mice. After BMT, mice were fed chow diet (No. 5015) for
4 weeks, and blood was obtained for baseline lipid determination.
Circulating white cells were counted to confirm reconstitution of the
peripheral leukocyte population and marrow engraftment. For 20
weeks, the mice were then fed an atherogenic diet (15.8% fat; 1.25%
cholesterol; no cholate; diet No. 94059 Harlan-Teklad) previously
used by our group and others.
10
All studies were carried out under
institutional review board approval.
Polymerase Chain Reaction
Confirmation of appropriate bone marrow engraftment was per-
formed using polymerase chain reaction (PCR). For details, see the
expanded Materials and Methods section found in the online data
supplement available at http://www.circresaha.org.
Lesion Morphology
At euthanasia, the hearts and aortas were prepared as previously
described.
11
The heart and proximal aorta were immediately frozen
in OCT medium and sectioned. Every third section was stained with
Oil Red O, with total lesion area quantified using these sections as
previously described.
2
For this analysis, 10 WT and 9 IL-10 Tg mice
were used. The remainder of the frozen sections were utilized for
immunostaining or staining with Massons trichrome (for details,
please see the expanded Materials and Methods in the online data
supplement).
Lipid Analysis
For details concerning liquid analysis, please see the expanded
Materials and Methods in the online data supplement.
IL-10 ELISA
Total circulating IL-10 was measured in the plasma of the mice at 0,
10, and 20 weeks on the atherogenic diet utilizing a Mouse IL-10
Quantikine Immunoassay kit (R&D Systems).
Intracellular Cytokine Staining and Flow
Cytometric Analysis
These studies were performed using methods similar to those
described previously.
12
For details, please see the expanded Materi-
als and Methods in the online data supplement.
Immunoglobulin Subclass Determination
Total IgG, IgG
2a
, IgG
1
, and malondialdehyde-specific
13
IgG
2a
and
IgG
1
in the plasma of the mice was quantitated with a sandwich
ELISA technique as described.
8
Immunostaining
Immunohistochemistry was performed on 5-m thick cryostat sec-
tions, as discussed in the expanded Materials and Methods in the
online data supplement.
Statistical Analysis
All statistical analysis was performed using 1-way ANOVA with
StatView (Abacus Concepts, Inc). Data presented are meanSE.
Results
Confirmation of BMT Engraftment
To confirm successful engraftment of donor bone marrow
cells, circulating white peripheral blood cells (WPBCs) from
all mice (WT, n10; IL-10 Tg, n9) were examined 4 weeks
after BMT. Total cell number and mononuclear cell popula-
tions were comparable for mice from both groups (data not
shown). PCR performed on DNA extracted from circulating
WPBC indicated the expected IL-10 transgene-derived
507-bp product in all the IL-10 Tg marrowrecipient mice
and complete absence of this product in the WT marrow
recipient mice (Figure 1).
IL-10 Overexpression Does Not Alter Circulating
Lipoprotein Profiles
Total cholesterol and triglyceride levels were similar at all
time points between the two groups (Figure 2). HDL choles-
terol was not significantly different at baseline between the
two groups. There was a mildly lower HDL cholesterol in the
IL-10 Tg group at the time of euthanasia.
IL-10 Overexpression Decreases Atherosclerotic
Lesion Development
Analysis of the lesion area in the proximal aortas (using Oil
Red O staining) revealed a significant decrease in atheroscle-
rotic lesion development in the LDL-R
/
mice that had
received bone marrow from IL-10 transgenic donor mice,
compared with those recipient mice that had received bone
marrow from wild-type mice (WT 144 95581 m
2
versus
IL-10 Tg 77 12956 m
2
; P0.0001; Figure 3).
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IL-10 Overexpression Decreases Aortic
Accumulation of Lipids in LDL-R
/
Mice
Analysis of the distal aorta showed a significantly greater
amount of total cholesterol in the WT-recipient mice
(803.3213.0 g/mg wet weight tissue; n10) compared
with the IL-10 Tgrecipient mice (540.9210.6 g/mg wet
weight tissue; P0.01, n9). We and others have previously
shown that certain oxidized phospholipids, particularly
POVPC and PGPC, increase monocyte-endothelial interac-
tions in vitro and are present in significant amounts in
atherosclerotic lesions in animals.
14
We observed an increase
in these particular oxidized phospholipids within the WT-
recipient mice, compared with the IL-10 Tg BMrecipient
mice. POVPC and PGPC were significantly reduced in the
IL-10 transgenic group with respect to wet weight (POVPC
668.3%, P0.04; PGPC 83.79.5%, P0.009; n6) and
to a lesser extent with respect to total phosphorus in the lipid
extract, an indicator of total phospholipid (POVPC
5411.2%, P0.09; PGPC 7813%, P0.017; n6).
These results suggest that differences in bioactive phospho-
lipids probably arise from both a decrease in total phospho-
lipid accumulation in the IL-10 transgenic mice (as reflected
by decrease in bioactive lipids with respect to wet weight), as
well as a lesser but significant IL-10mediated decrease in
oxidation in the IL-10 group (as measured with respect to
phosphorus), where the PGPC decrease was significant and
the POVPC decrease nearly significant.
Circulating IL-10 levels Are Not Different Between
WT- and IL-10 TgRecipient Mice
Plasma IL-10 levels were assayed by ELISA in all mice at 0,
10, and 20 weeks on the atherogenic diet. There was no
significant difference between the two groups at any time
point (data not shown).
Figure 1. PCR conrms bone marrow engraftment in each
group. DNA was extracted from circulating white blood cells in
each mouse in both groups 4 weeks after BMT. PCR was per-
formed with primers specic for the IL-10 transgenic construct.
PCR products were run on a 1.5% agarose gel, and the
transgene-derived 507-bp product was visualized in all the IL-10
transgenic BMT recipients. Despite equal initial amounts of
DNA, the WT recipients did not display the specic product. A
representative gel is shown.
Figure 2. Lipoprotein proles do not differ between the WT and
IL-10 Tg group. Plasma was obtained from all mice at 0 and 20
weeks on the atherogenic diet. Total cholesterol and triglycer-
ides did not differ signicantly between the two groups at any
time point. HDL cholesterol was not signicantly different at
baseline, but was mildly decreased in the IL-10 Tg BMrecipient
group at the time of euthanasia. *Signicant change from base-
line to time of euthanasia; signicant difference between WT
and IL-10 Tg group. Data presented as meanSE; WT n10,
IL-10 Tg n9. Solid bar indicates WT group; hatched bar, IL-10
Tg recipient group.
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Intracellular Cytokine Staining/Flow Cytometry
Reveals a Th2 Lymphocytic Shift in the IL-10
Tg Group
Because the IL-10 transgene in this murine construct is under
the control of the IL-2 promoter, the expression of transgene-
derived IL-10 is restricted to activated T lymphocytes.
Expression of cell-surface CD69 and intracellular IL-2 were
examined as markers of activation and were similar between
the two groups of mice (74% versus 78% and 34% versus
33%, respectively). Despite comparable activation of CD4

T
lymphocytes in both groups, there was a marked shift from a
Th1 to a Th2 phenotype in CD4

T lymphocytes in the IL-10

Tg BM group, demonstrated by a 1.5-fold decrease in IFN-
production and a 9-fold increase in IL-10 production com-
pared with the CD4

lymphocytes from the WT BM

recipient mice (Figure 4A). Importantly, not only was the
proportion of T lymphocytes producing the cytokines altered,
but the level of cytokine expression by the lymphocytes was
also markedly affected. An even more prominent shift in
cytokine expression was seen in CD4

splenocytes between
the two groups (Figure 4B).
Examination of Immunoglobulin Subclasses
Reveals Opposite CD4

T-Helper Influence on B
Lymphocytes in the IL-10 Tg Group Compared
With the WT Group
During atherogenesis, antibodies against malondialdehyde-
LDL (antiMDA-LDL) are formed
15
and have been demon-
strated to undergo a class shift from IgG
2a
to IgG
1
during
lesion progression.
8
We measured antiMDA-LDL antibod-
ies to determine whether the T-lymphocyte phenotypic dif-
ference identified above impacted immunoglobulin subclass
production. As in previous studies,
8
the total levels of IgG did
not differ between the 2 groups of mice at 10 or 20 weeks on
the atherogenic diet (data not shown). The ratio of Th2 (IgG
1
)
to Th1 (IgG
2a
) antiMDA-LDL antibodies, however, was
significantly higher at both time points in the recipient mice
that had received IL-10 Tg BM compared with the WT
BMrecipient mice (Figure 5). This B-lymphocyte immuno-
globulin subclass shift is additional support for an enhanced
Th2 state in the IL-10 Tg group of mice.
Microscopic Analysis of Lesion Components
Although the atherosclerotic lesions of both groups of mice
contained lipid-laden cells, fibrous caps, and at least some
necrotic cores, the lesions of WT BMrecipient mice were
Figure 3. Atherosclerotic lesions are signicantly decreased in
the IL-10 Tg BMrecipient group. Aortas were harvested at the
time of euthanasia, sectioned, and stained with Oil Red O. Prox-
imal aortic atherosclerotic lesion area was quantitated and
found to be approximately 47% decreased in the IL-10 Tg
group (P0.001). Data presented as meanSE; WT n10, IL-10
Tg n9.
Figure 4. Intracellular cytokine staining reveals a Th2 phenotype
in the IL-10 Tg BMrecipient group. Peripheral blood lympho-
cytes (A) and splenocytes (B) were analyzed from mice in each
group at the time of euthanasia, and representative plots are
shown. T lymphocytes were identied by size, granularity, and
positive staining for CD4 (quadrants 2 and 4). After partial cell
membrane permeabilization, phycoerythrin-conjugated antibod-
ies against either IL-10 or IFN- were introduced, and cells were
then subjected to 2-color ow cytometry. Cells with positive
staining for cytokines are in quadrants 1 and 2; cells with dou-
ble positive staining cells are in quadrant 2. The WT group
made negligible amounts of IL-10, but actively produced IFN-,
whereas the IL-10 Tg group had a marked shift toward elabora-
tion of IL-10, with decreased IFN.
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considerably more complex and advanced (Figure 6A). In
contrast, lesions in the IL-10 Tg group were highly cellular,
resembling large fatty streaks, with small to absent necrotic
cores. MOMA-2 staining revealed these cellular areas to
contain mainly macrophages (data not shown). Morphometric
analysis of these sections confirmed significantly greater
cellularity of the atherosclerotic lesions in the IL-10 Tg
BMrecipient mice (Figure 6B). Conversely, necrotic cores
made up a much higher percentage of lesional area in the WT
BMrecipient mice (Figure 6B). Because the formation of the
necrotic core precedes smooth muscle replication and matrix
synthesis in mice, it is not surprising that the matrix area is
reduced in the IL-10 transgenic group (Figure 6B). This
difference in lesion composition suggested a possible alter-
ation in macrophage fate within the lesions of the two groups
of mice. Therefore, apoptotic activity was assessed using an
antibody to the activated form of caspase-3. For this analysis,
sections from fatty streaks of comparable lesion area were
compared between the two groups (8 sections per group).
There was a striking difference in caspase staining in the WT
group, primarily within MOMA-2positive cells (Figure 6C).
All sections of the WT group showed heavy staining, whereas
it was difficult to find caspase staining in the IL-10 group.
Comparison of fatty-streak lesions in the two groups revealed
that individual macrophages appeared larger in the lesions of
the IL-10 Tg group. This qualitative size difference may
reflect an ability of these macrophages to load greater
amounts of lipid without undergoing apoptosis and could
potentially account in part for the differences in oxidative
lipid products seen within the aortas of the two groups.
Immunostaining with anti-CD4, -CD8, and -CD3 antibodies
revealed no lymphocytes within the lesions of either group of
mice after 20 weeks on the atherogenic diet (data not shown).
This finding is consistent with previous documentation that
although T lymphocytes are present in significant numbers
early in lesion development in LDL-R
/
mice, the presence
of intralesional T lymphocytes decreases as lesions progress
over time.
16
Thus, there appeared to be a protective effect of
the T cellderived IL-10 in this BMT mouse model of
atherosclerosis, in terms of both macrophage apoptosis and
ultimate lesion development, despite the absence of T lym-
phocytes within the advanced lesions.
Intracellular Cytokine Staining/Flow Cytometry
Indicates Marked Activation of Circulating
Monocytes Within the WT-Reconstituted Mice
Compared With Minimal Activation in the IL-10
Tg BMReconstituted Group of Mice
Intracellular cytokine staining with flow cytometric analysis
was used to characterize the monocyte population. Initially,
the monocyte population was identified by size, granularity,
NK-negative, and CD11b-positive selection. Gated mono-
cytes were subsequently assessed for activation states via the
measurement of IFN-, previously shown to be expressed in
activated monocytes.
17,18
At the time of euthanasia, there was
a 6-fold decrease in the amount of IFN- produced by the
monocytes in the group of mice that had received IL-10 Tg
bone marrow compared with monocytes from the WT group
of mice. This dramatic difference was evident both in terms
of the number monocytes producing IFN-, as well as the
amount of IFN- expressed. Finally, this pattern was noted in
both the circulating monocytes (Figure 7), as well as those
residing within the spleen (data not shown).
Discussion
These studies demonstrate that IL-10 overexpression in lym-
phocytes leads to a significant decrease in the development of
advanced atherosclerotic lesions. Previous in vivo work
examining IL-10 in atherogenesis was restricted to studying
the development of early fatty-streak lesions in C57BL/6J
mice on a cholate-containing diet.
1,2
Our present studies
utilized the LDL-R
/
mouse, which develops complex le-
sions on a cholate-free atherogenic diet. Even after 20 weeks
on this diet, the majority of lesions in the mice transplanted
with bone marrow from IL-10 transgenic donors were ar-
rested at the fatty-streak stage. In marked contrast, animals
transplanted with wild-type bone marrow formed advanced
atherosclerotic lesions with large necrotic cores, as has been
observed in our previous studies.
10
We have shown recently
that LDL-R
/
mice subjected to BMT and fed the athero-
genic diet for 16 weeks had significantly larger aortic sinus
lesion areas compared with mice that were not subjected to
BMT.
19
Whether this increase was due to the effect of BMT
on initiation or progression is not known because only 1 time
point was examined. However, because both groups of mice
in our present study were subjected to BMT in an identical
manner, the comparison of their lesion formation should be
valid.
Whereas the previously documented effect of IL-10 in
decreasing atherosclerotic initiation
1,2
may play a role in this
observed decrease in advanced lesions, our present findings
suggest that IL-10 also affects processes associated with
Figure 5. Immunoglobulin subclass analysis reveals a Th2 inu-
ence on B lymphocytes in the IL-10 Tg BMrecipient group.
Sandwich ELISAs were performed on plasma from mice in each
group to quantify immunoglobulin subclasses. The total IgG did
not differ between the WT and IL-10 Tg groups (data not
shown). The ratio of antiMDA-LDL IgG
1
to IgG
2a
, however, sug-
gests a Th2 inuence on the B lymphocytes in the IL-10 Tg
BMrecipient group (n7) at both time points measured, com-
pared with the WT (n8) group. Differences between WT and
IL-10 Tg groups are statistically signicant.
1068 Circulation Research May 31, 2002
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lesion progression. A high degree of apoptosis was detected
among the intralesional cells of the WT mice, primarily
within the macrophages surrounding the lipid-laden cores. In
contrast, little to no apoptosis was seen within lesions of
similar cellularity in the IL-10 Tg BMrecipient mice. This
difference in lesions of comparable size suggests a decrease
in progression as well as initiation. Monocyte migration into
arterial walls with subsequent transformation into macro-
phages and then foam cells characterizes the early fatty-streak
stage in atherosclerosis. Apoptosis of foam cells, which is
influenced by cytokine expression and macrophage activation
state,
20
contributes to formation of the necrotic core and
occurs during progression of atherosclerotic lesions. Reduc-
tion in macrophage apoptosis most likely accounts for the
decreased formation of necrotic cores in the transgenic group.
Interestingly, IL-10 has been shown in other systems to
inhibit apoptosis.
21
Moreover, IL-10 increases stimulation as
well as production of reactive oxygen species and IL-6 while
inhibiting the incidence of apoptosis in human monocytes.
22
Previous studies of human atherosclerotic plaques revealed
an inverse association between presence of IL-10 and
TUNEL staining in atherosclerotic lesions,
4
suggesting our
murine results are applicable to the human disease process.
Lastly, because necrotic cores participate in destabilization of
plaques,
23
our murine results may have direct therapeutic
implications.
Because plasma levels of IL-10 were not altered in the
IL-10 Tg group, the observed effects on lesion progression
cannot be attributed to systemic alterations in this cytokine,
but are instead mediated by alterations in lymphocyte func-
tion. We demonstrate that IL-10 overexpression can promote
development of the Th2 phenotype despite ongoing hyperlip-
idemia, previously shown to induce a Th1 phenotype.
8,24
We
show this phenotypic switch results in decreased production
of IFN- by lymphocytes and splenocytes in the IL-10 Tg
group. Underscoring the relevance of this IFN- decrease are
Figure 6. Microscopic analysis of the
lesions in the WT and IL-10 Tg groups.
A, Trichrome staining of lesions in the
proximal aorta revealed a higher cellular-
ity in the lesions of the IL-10 Tg group,
contrasted with more extensive lipid-
lled necrotic cores within the WT
lesions. Nearly all intralesional cells were
MOMA-2 positive (data not shown). B,
Morphometric analysis revealed compo-
sitional differences between the WT- and
IL-10 Tgrecipient groups lesions. A
representative WT section is shown,
with the necrotic core (NC) area bor-
dered in yellow and the cellular area (C)
bordered in green. L indicates lumen; A,
adventitia. In these analyses, n indicates
multiple representative cross-sections
analyzed for each mouse in both
groups. WT lesions (n21 representative
aortic cross-sections) had a signicantly
larger area of necrotic core (P0.0001)
vs the IL-10 Tg group (n24). Further-
more, the WT lesions (n30) had signi-
cantly less cellularity (P0.004) com-
pared with the IL-10 Tg lesions
measured (n24). Data presented as
meanSE. C, Immunostaining for acti-
vated caspase-3 revealed a marked
increase in apoptotic cells within the WT
lesions. A representative, typical fatty-
streak type lesion is shown for both the
WT and IL-10 Tg BMrecipient groups.
Differences were also observed in more
advanced lesions, although to a lesser
degree.
Figure 7. Intracellular cytokine staining reveals a marked
decrease in monocyte activation in the IL-10 Tg group. Periph-
eral blood lymphocytes were analyzed from mice in each group
at the time of euthanasia, and representative plots are shown.
Monocytes were identied by size, granularity, negative staining
for NK1.1 (data not shown), and positive staining for CD11b.
Intracellular IFN- was identied as described in Figure 4.
Monocytes from the IL-10 Tg BMrecipient mice produced
9-fold less IFN- compared with WT controls.
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findings that IFN- receptordeficient mice develop de-
creased atherosclerosis,
25
and IFN- administration increased
atherosclerosis formation in murine models.
26
Recent work
investigating the impact of pentoxifylline on atherosclerosis
in mice also suggested decreased lesion progression associ-
ated with a Th2 phenotype; mice with decreased lymphocyte
IFN- did not progress significantly beyond the fatty-streak
stage compared with the advanced lesions seen in the control
group.
27
Overexpression of lymphocyte IL-10 altered B cell IgG
class in response to oxidized phospholipids. The Th2 pheno-
type in the IL-10 Tg group influenced B lymphocytes in these
mice to produce a preponderance of IgG
1
antiphospholipid
antibodies. Previous studies have not only documented spe-
cific antibodies in atherosclerosis, but correlated immuno-
globulin subclass with extent of disease in both animal
models and humans.
8,24
Immunoglobulins of the IgG
2a
sub-
class specific for oxidized epitopes have been quantitated in
atherosclerosis, and IgG subclass switching has been demon-
strated during the progression of atherosclerosis.
8,28
These
immunoglobulins most likely modulate atherogenesis. Mice
inoculated with oxidized lipids develop specific antibodies,
which may have a protective effect in vivo
15
; moreover,
treatment of apolipoprotein Edeficient mice with intrave-
nous immunoglobulins significantly decreased early and late
lesion formation.
29
Thus, the T-cell effect on atherosclerosis
in our studies may be executed partially through alterations in
B-lymphocyte function.
The central cell type in atherosclerosis is the macrophage.
30
Importantly, we present evidence that macrophage function
was affected by the Th2 phenotypic alterations we created.
Compared with animals fed a chow diet, circulating mono-
cytes in fat-fed animals are activated as manifested by
expression of cytokines, adhesion molecules, and other
surface-activation markers.
31
In the present studies, we dem-
onstrated a major difference in monocyte activation between
the two groups as measured by IFN- expression. Overall, a
9-fold decrease in IFN- was observed in the monocytes of
the IL-10 Tg group, with most monocytes producing no
detectable IFN-. Previous reports regarding macrophage
function in atherosclerosis
24,32,33
suggest that a dramatic
decrease in monocyte activation would have significant
impact on atherosclerotic development. Furthermore, the
significant reduction in specific bioactive phospholipids
within the aortas of the IL-10overexpressing group suggests
that modulation of macrophage function may lead to alter-
ations in lipid processing and oxidation within the vessel
wall.
T lymphocytes are present in significant numbers within
atherosclerotic lesions of both humans and apolipoprotein
Edeficient mice, as well as prominent in early lesions of
LDL-R
/
mice.
16
Previous studies have shown a 2.5-fold
increase in T-cell infiltration into early atherosclerotic lesions
in IL-10deficient mice, as well as an increase in IFN- in
the more advanced lesions of these mice.
1
Although T cells
were not detectable in the lesions presently studied after 20
weeks on the atherogenic diet, previous descriptions
16
suggest
they were most likely present within the early lesions, playing
a pivotal role at that time. T lymphocytederived cytokines
could also be acting outside of the lesion to exert critical
effects on the monocytes. In arthritis, another chronic inflam-
matory disease, IL-10 overexpression localized to one joint
area had profound effects on monocyte-mediated inflamma-
tion at distant joints, even without alterations in free-
circulating IL-10.
34
In our disease model, the altered character of the lesions in
the IL-10 Tg group may be even more important than the
decreased lesion burden. Interruption of the CD40-CD40L
system has limited impact on total atherosclerotic lesion area,
but dramatically changes lesion composition.
35,36
Arguably
lesion composition, even more than alteration in total lesion
size, has implications for atherosclerotic complications in
humans and may therefore be a more appropriate therapeutic
target. Although IL-10 may exert some of its influences
through CD40-CD40L,
37
the changes in lesions that we
observed in IL-10 Tg BMrecipient mice (predominantly a
decrease in apoptosis and lipid cores, accompanied by in-
creased cellularity) were not identical to those seen with
direct CD40-CD40L interruption (largely represented by
increases in fibrous changes and a decrease in monocyte
content).
35,36
In summary, this study demonstrates 4 impor-
tant novel findings regarding lymphocyte overproduction of
IL-10 and atherosclerosis: (1) T-cell IL-10 decreased lesion
development even at 20 weeks of cholesterol feeding in the
LDL-R
/
mouse, which develops advanced complex lesions;
(2) overexpression of IL-10 by T lymphocytes created a Th2
phenotype as indicated by their cytokine expression profile
even during the inflammatory stress of atherosclerosis; (3)
B-lymphocyte phenotype, as manifested by immunoglobulin
subclass, was also altered toward a Th2 phenotype; and (4)
T-cell IL-10 inhibited macrophage activation and susceptibil-
ity to apoptosis. These IL-10mediated alterations in lym-
phocytes and macrophages, as well as alterations in matrix
deposition previously documented,
1
likely contribute to both
initiation and progression of atherosclerosis, suggesting a
potential regulatory role for IL-10 in all stages of atherogen-
esis. Because of the therapeutic potential of these observa-
tions to the treatment of atherosclerosis, it will be important
in the future to determine whether lymphocyte IL-10 over-
expression occurring exclusively during lesion progression or
at late stages of lesion remodeling can also influence lesion
development and character. Taken together, these and past
studies suggest that lymphocyte-derived as well as circulating
IL-10 may promise therapeutic strategies for the treatment of
atherosclerosis.
Acknowledgments
L.J.P. was supported by NIH University of California Los Angeles
Cardiovascular Scientist Training Program grant T32-HL-07895.
L.K.C. is supported by NIH grant HL35297; J.A.B. by NIH grant
HL30568; and W.A.B. by NIH grant HL61731. The authors ac-
knowledge technical assistance from Julia Ozbolt and Allis Ip.
Antibodies against MDA-LDL were a kind gift from Dr Joseph
Witztum, University of California San Diego.
References
1. Mallat Z, Besnard S, Duriez M, Deleuze V, Emmanuel F, Bureau MF,
Soubrier F, Esposito B, Duez H, Fievet C, Staels B, Duverger N,
Scherman D, Tedgui A. Protective role of interleukin-10 in atheroscle-
rosis. Circ Res. 1999;85:e17e24.
1070 Circulation Research May 31, 2002
by guest on March 6, 2014 http://circres.ahajournals.org/ Downloaded from
2. 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;19:28472853.
3. Berliner JA, Navab M, Fogelman AM, Frank JS, Demer LL, Edwards PA,
Watson AD, Lusis AJ. Atherosclerosis, basic mechanisms: oxidation,
inflammation, and genetics. Circulation. 1995;91:24882496.
4. Mallat Z, Heymes C, Ohan J, Faggin E, Leseche G, Tedgui A. Expression
of interleukin-10 in advanced human atherosclerotic plaques: relation to
inducible nitric oxide synthase expression and cell death. Arterioscler
Thromb Vasc Biol. 1999;19:611616.
5. Uyemura K, Demer LL, Castle SC, Jullien D, Berliner JA, Gately MK,
Warrier RR, Pham N, Fogelman AM, Modlin RL. Cross-regulatory roles
of interleukin (IL)-12 and IL-10 in atherosclerosis. J Clin Invest. 1996;
97:21302138.
6. Smith DA, Irving SD, Sheldon J, Cole D, Kaski JC. Serum levels of the
antiinflammatory cytokine interleukin-10 are decreased in patients with
unstable angina. Circulation. 2001;104:746749.
7. Hagenbaugh A, Sharma S, Dubinett SM, Wei SH, Aranda R, Cheroutre
H, Fowell DJ, Binder S, Tsao B, Locksley RM, Moore KW, Kronenberg
M. Altered immune responses in interleukin 10 transgenic mice. J Exp
Med. 1997;185:21012110.
8. Zhou X, Paulsson G, Stemme S, Hansson GK. Hypercholesterolemia is
associated with a T helper (Th) 1/Th2 switch of the autoimmune response
in atherosclerotic apo E-knockout mice. J Clin Invest. 1998;101:
17171725.
9. Frostegard J, Ulfgren AK, Nyberg P, Hedin U, Swedenborg J, Andersson
U, Hansson GK. Cytokine expression in advanced human atherosclerotic
plaques: dominance of pro-inflammatory (Th1) and macrophage-
stimulating cytokines. Atherosclerosis. 1999;145:3343.
10. Boisvert WA, Santiago R, Curtiss LK, Terkeltaub RA. A leukocyte
homologue of the IL-8 receptor CXCR-2 mediates the accumulation of
macrophages in atherosclerotic lesions of LDL receptordeficient mice.
J Clin Invest. 1998;101:353363.
11. Boisvert WA, Spangenberg J, Curtiss LK. Role of leukocyte-specific
LDL receptors on plasma lipoprotein cholesterol and atherosclerosis in
mice. Arterioscler Thromb Vasc Biol. 1997;17:340347.
12. Stinn JL, Taylor MK, Becker G, Nagano H, Hasegawa S, Furakawa Y,
Shimizu K, Libby P, Mitchell RN. Interferon-secreting T-cell popu-
lations in rejecting murine cardiac allografts: assessment by flow
cytometry. Am J Pathol. 1998;153:13831392.
13. Palinski W, Yla-Herttuala S, Rosenfeld ME, Butler SW, Socher SA,
Parthasarathy S, Curtiss LK, Witztum JL. Antisera and monoclonal anti-
bodies specific for epitopes generated during oxidative modification of
low density lipoprotein. Arteriosclerosis. 1990;10:325335.
14. Watson AD, Leitinger N, Navab M, Faull KF, Horkko S, Witztum JL,
Palinski W, Schwenke D, Salomon RG, Sha W, Subbanagounder G,
Fogelman AM, Berliner JA. Structural identification by mass spec-
trometry of oxidized phospholipids in minimally oxidized low density
lipoprotein that induce monocyte/endothelial interactions and evidence
for their presence in vivo. J Biol Chem. 1997;272:1359713607.
15. Witztum JL, Horkko S. The role of oxidized LDL in atherogenesis:
immunological response and anti-phospholipid antibodies. Ann N Y Acad
Sci. 1997;811:8896; discussion 9689.
16. 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;16:10131018.
17. Munder M, Mallo M, Eichmann K, Modolell M. Murine macrophages
secrete interferon- upon combined stimulation with interleukin (IL)-12
and IL-18: a novel pathway of autocrine macrophage activation. J Exp
Med. 1998;187:21032108.
18. Wang J, Wakeham J, Harkness R, Xing Z. Macrophages are a significant
source of type 1 cytokines during mycobacterial infection. J Clin Invest.
1999;103:10231029.
19. Schiller NK, Kubo N, Boisvert WA, Curtiss LK. Effect of -irradiation
and bone marrow transplantation on atherosclerosis in LDL receptor-
deficient mice. Arterioscler Thromb Vasc Biol. 2001;21:16741680.
20. Geng YJ, Henderson LE, Levesque EB, Muszynski M, Libby P. Fas is
expressed in human atherosclerotic intima and promotes apoptosis of
cytokine-primed human vascular smooth muscle cells. Arterioscler
Thromb Vasc Biol. 1997;17:22002208.
21. Geng Y, Shane RB, Berencsi K, Gonczol E, Zaki MH, Margolis DJ,
Trinchieri G, Rook AH. Chlamydia pneumoniae inhibits apoptosis in
human peripheral blood mononuclear cells through induction of IL-10.
J Immunol. 2000;164:55225529.
22. Hashimoto S, Yamada M, Motoyoshi K, Akagawa KS. Enhancement of
macrophage colony-stimulating factor-induced growth and differentiation
of human monocytes by interleukin-10. Blood. 1997;89:315321.
23. Libby P, Geng YJ, Sukhova GK, Simon DI, Lee RT. Molecular deter-
minants of atherosclerotic plaque vulnerability. Ann N Y Acad Sci. 1997;
811:134142; discussion 142145.
24. 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;19:734742.
25. Gupta S, Pablo AM, Jiang X, Wang N, Tall AR, Schindler C. IFN-
potentiates atherosclerosis in ApoE knock-out mice. J Clin Invest. 1997;
99:27522761.
26. Whitman SC, Ravisankar P, Elam H, Daugherty A. Exogenous
interferon- enhances atherosclerosis in apolipoprotein E/mice. Am J
Pathol. 2000;157:18191824.
27. Laurat E, Poirier B, Tupin E, Caligiuri G, Hansson GK, Bariety J,
Nicoletti A. In vivo downregulation of T helper cell 1 immune responses
reduces atherogenesis in apolipoprotein Eknockout mice. Circulation.
2001;104:197202.
28. Palinski W, Miller E, Witztum JL. Immunization of low density
lipoprotein (LDL) receptor-deficient rabbits with homologous
malondialdehyde-modified LDL reduces atherogenesis. Proc Natl Acad
Sci U S A. 1995;92:821825.
29. Nicoletti A, Kaveri S, Caligiuri G, Bariety J, Hansson GK. Immuno-
globulin treatment reduces atherosclerosis in apo E knockout mice. J Clin
Invest. 1998;102:910918.
30. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med. 1999;
340:115126.
31. Han KH, Tangirala RK, Green SR, Quehenberger O. Chemokine receptor
CCR2 expression and monocyte chemoattractant protein-1mediated che-
motaxis in human monocytes: a regulatory role for plasma LDL. Arte-
rioscler Thromb Vasc Biol. 1998;18:19831991.
32. Mach F, Schonbeck U, Bonnefoy JY, Pober JS, Libby P. Activation of
monocyte/macrophage functions related to acute atheroma complication
by ligation of CD40: induction of collagenase, stromelysin, and tissue
factor. Circulation. 1997;96:396399.
33. Qiao JH, Tripathi J, Mishra NK, Cai Y, Tripathi S, Wang XP, Imes S,
Fishbein MC, Clinton SK, Libby P, Lusis AJ, Rajavashisth TB. Role of
macrophage colony-stimulating factor in atherosclerosis: studies of osteo-
petrotic mice. Am J Pathol. 1997;150:16871699.
34. Whalen JD, Lechman EL, Carlos CA, Weiss K, Kovesdi I, Glorioso JC,
Robbins PD, Evans CH. Adenoviral transfer of the viral IL-10 gene
periarticularly to mouse paws suppresses development of collagen-
induced arthritis in both injected and uninjected paws. J Immunol. 1999;
162:36253632.
35. Lutgens E, Cleutjens KB, Heeneman S, Koteliansky VE, Burkly LC,
Daemen MJ. Both early and delayed anti-CD40L antibody treatment
induces a stable plaque phenotype. Proc Natl Acad Sci U S A. 2000;97:
74647469.
36. Schonbeck U, Sukhova GK, Shimizu K, Mach F, Libby P. Inhibition of
CD40 signaling limits evolution of established atherosclerosis in mice.
Proc Natl Acad Sci U S A. 2000;97:74587463.
37. Suttles J, Milhorn DM, Miller RW, Poe JC, Wahl LM, Stout RD. CD40
signaling of monocyte inflammatory cytokine synthesis through an
ERK1/2-dependent pathway: a target of interleukin (IL)-4 and IL-10
anti-inflammatory action. J Biol Chem. 1999;274:58355842.
Pinderski et al IL-10 Inhibits Atherosclerosis by Altering T Cells 1071
by guest on March 6, 2014 http://circres.ahajournals.org/ Downloaded from