terapia il-10 inhibe aterosclerosis

8/12/2019 Terapia IL-10 Inhibe Aterosclerosis

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Nobuhiko Kubo, Hilde Cheroutre, Linda K. Curtiss, Judith A. Berliner and William A. BoisvertLaura J. Pinderski, Michael P. Fischbein, Ganesamoorthy Subbanagounder, Michael C. Fishbein,

Deficient Mice by Altering Lymphocyte and Macrophage Phenotypes!LDL ReceptorOverexpression of Interleukin-10 by Activated T Lymphocytes Inhibits Atherosclerosis in

Print ISSN: 0009-7330. Online ISSN: 1524-4571Copyright 2002 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

doi: 10.1161/01.RES.0000018941.10726.FA2002;90:1064-1071; originally published online April 18, 2002;Circ Res.

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Overexpression of Interleukin-10 by ActivatedT Lymphocytes Inhibits Atherosclerosis in LDL

ReceptorDeficient Mice by Altering Lymphocyteand 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 mechanismof the IL-10 mediated 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 IgG1 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 chronicinflammatory 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 andImmunology (M.P.F.), University Of California Los Angeles; Department of Immunology (N.K., L.K.C., W.A.B.), The Scripps Research Institute, LaJolla; 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 [email protected]

2002 American Heart Association, Inc.Circulation Researchis available at http://www.circresaha.org DOI: 10.1161/01.RES.0000018941.10726.FA

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Molecular Medicine

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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 receptor deficient (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 ModelsLDL-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-10transgenic 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 underinstitutional review board approval.

Polymerase Chain ReactionConfirmation 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 MorphologyAt 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 datasupplement).

Lipid AnalysisFor details concerning liquid analysis, please see the expanded

Materials and Methods in the online data supplement.

IL-10 ELISATotal 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 AnalysisThese 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 DeterminationTotal IgG, IgG2a, IgG1, and malondialdehyde-specific13 IgG2a and

IgG1 in the plasma of the mice was quantitated with a sandwich

ELISA technique as described.8

ImmunostainingImmunohistochemistry was performed on 5-m thick cryostat sec-tions, as discussed in the expanded Materials and Methods in the

online data supplement.

Statistical AnalysisAll statistical analysis was performed using 1-way ANOVA with

StatView (Abacus Concepts, Inc). Data presented are meanSE.

Results

Confirmation of BMT EngraftmentTo 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 ProfilesTotal 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 DevelopmentAnalysis 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 144 955

81 m2

versusIL-10 Tg 77 12956 m2; P0.0001; Figure 3).

Pinderski et al IL-10 Inhibits Atherosclerosis by Altering T Cells 1065



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IL-10 Overexpression Decreases Aortic

Accumulation of Lipids in LDL-R/ MiceAnalysis 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 MicePlasma 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 timepoint (data not shown).

Figure 1. PCR confirms bone marrow engraftment in eachgroup. DNA was extracted from circulating white blood cells ineach mouse in both groups 4 weeks after BMT. PCR was per-formed with primers specific for the IL-10 transgenic construct.PCR products were run on a 1.5% agarose gel, and thetransgene-derived 507-bp product was visualized in all the IL-10

transgenic BMT recipients. Despite equal initial amounts ofDNA, the WT recipients did not display the specific product. Arepresentative gel is shown.

Figure 2. Lipoprotein profiles do not differ between the WT andIL-10 Tg group. Plasma was obtained from all mice at 0 and 20weeks on the atherogenic diet. Total cholesterol and triglycer-ides did not differ significantly between the two groups at anytime point. HDL cholesterol was not significantly different atbaseline, but was mildly decreased in the IL-10 Tg BMrecipientgroup at the time of euthanasia. *Significant change from base-line to time of euthanasia; significant difference between WTand IL-10 Tg group. Data presented as meanSE; WT n10,IL-10 Tg n9. Solid bar indicates WT group; hatched bar, IL-10Tg recipient group.

1066 Circulation Research May 31, 2002



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Intracellular Cytokine Staining/Flow CytometryReveals a Th2 Lymphocytic Shift in the IL-10Tg GroupBecause 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 SubclassesReveals Opposite CD4 T-Helper Influence on BLymphocytes in the IL-10 Tg Group ComparedWith the WT GroupDuring atherogenesis, antibodies against malondialdehyde-

LDL (antiMDA-LDL) are formed15 and have been demon-

strated to undergo a class shift from IgG 2a to IgG1 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 (IgG2a) 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 ComponentsAlthough the atherosclerotic lesions of both groups of mice

contained lipid-laden cells, fibrous caps, and at least somenecrotic cores, the lesions of WT BMrecipient mice were

Figure 3. Atherosclerotic lesions are significantly decreased inthe IL-10 Tg BMrecipient group. Aortas were harvested at thetime of euthanasia, sectioned, and stained with Oil Red O. Prox-

imal aortic atherosclerotic lesion area was quantitated andfound to be approximately 47% decreased in the IL-10 Tggroup (P0.001). Data presented as meanSE; WT n10, IL-10Tg n9.

Figure 4. Intracellular cytokine staining reveals a Th2 phenotypein the IL-10 Tg BMrecipient group. Peripheral blood lympho-cytes (A) and splenocytes (B) were analyzed from mice in eachgroup at the time of euthanasia, and representative plots areshown. T lymphocytes were identified by size, granularity, andpositive staining for CD4 (quadrants 2 and 4). After partial cellmembrane permeabilization, phycoerythrin-conjugated antibod-ies against either IL-10 or IFN-were introduced, and cells werethen subjected to 2-color flow cytometry. Cells with positivestaining for cytokines are in quadrants 1 and 2; cells with dou-

ble positive staining cells are in quadrant 2. The WT groupmade 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 TgBMrecipient 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 ofmice 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 cell derived IL-10 in this BMT mouse model of

atherosclerosis, in terms of both macrophage apoptosis andultimate lesion development, despite the absence of T lym-

phocytes within the advanced lesions.

Intracellular Cytokine Staining/Flow CytometryIndicates Marked Activation of CirculatingMonocytes Within the WT-Reconstituted MiceCompared With Minimal Activation in the IL-10Tg BMReconstituted Group of MiceIntracellular 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 themeasurement 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 initiation1,2 may play a role in this

observed decrease in advanced lesions, our present findingssuggest that IL-10 also affects processes associated with

Figure 5. Immunoglobulin subclass analysis reveals a Th2 influ-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 didnot differ between the WT and IL-10 Tg groups (data notshown). The ratio of antiMDA-LDL IgG1to IgG2a, however, sug-gests a Th2 influence on the B lymphocytes in the IL-10 TgBMrecipient group (n7) at both time points measured, com-pared with the WT (n8) group. Differences between WT andIL-10 Tg groups are statistically significant.




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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 andoccurs 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 therapeuticimplications.

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 thelesions in the WT and IL-10 Tg groups.

A, Trichrome staining of lesions in theproximal aorta revealed a higher cellular-ity in the lesions of the IL-10 Tg group,contrasted with more extensive lipid-

filled necrotic cores within the WTlesions. Nearly all intralesional cells wereMOMA-2 positive (data not shown). B,Morphometric analysis revealed compo-sitional differences between the WT- andIL-10 Tgrecipient groups lesions. Arepresentative 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 indicatesmultiple representative cross-sectionsanalyzed for each mouse in bothgroups. WT lesions (n21 representativeaortic cross-sections) had a significantlylarger area of necrotic core (P0.0001)

vs the IL-10 Tg group (n24). Further-more, the WT lesions (n30) had signifi-cantly less cellularity (P0.004) com-pared with the IL-10 Tg lesionsmeasured (n24). Data presented asmeanSE. C, Immunostaining for acti-vated caspase-3 revealed a markedincrease in apoptotic cells within the WTlesions. A representative, typical fatty-streak type lesion is shown for both theWT and IL-10 Tg BMrecipient groups.Differences were also observed in moreadvanced lesions, although to a lesserdegree.

Figure 7. Intracellular cytokine staining reveals a markeddecrease in monocyte activation in the IL-10 Tg group. Periph-eral blood lymphocytes were analyzed from mice in each groupat the time of euthanasia, and representative plots are shown.Monocytes were identified by size, granularity, negative stainingfor NK1.1 (data not shown), and positive staining for CD11b.Intracellular IFN-was identified as described in Figure 4.

Monocytes from the IL-10 Tg BMrecipient mice produced9-fold less IFN-compared with WT controls.


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findings that IFN- receptor deficient 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-streakstage 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 IgG1 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 IgG2a 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

Theseimmunoglobulins most likely modulate atherogenesis. Mice

inoculated with oxidized lipids develop specific antibodies,

which may have a protective effect in vivo15; moreover,

treatment of apolipoprotein E deficient 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 byexpression 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 atherosclerosis24,32,33 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-10 overexpressing 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

E deficient 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-10 deficient 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 descriptions16 suggest

they were most likely present within the early lesions, playinga pivotal role at that time. T lymphocyte derived 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 inthe 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 byincreases 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.

AcknowledgmentsL.J.P. was supported by NIH University of California Los AngelesCardiovascular Scientist Training Program grant T32-HL-07895.L.K.C. is supported by NIH grant HL35297; J.A.B. by NIH grantHL30568; 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 JosephWitztum, University of California San Diego.

References1. 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:e17 e24.


http://circres.ahajournals.org/http://circres.ahajournals.org/http://circres.ahajournals.org/http://circres.ahajournals.org/http://circres.ahajournals.org/http://circres.ahajournals.org/http://circres.ahajournals.org/


9/9

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. ArteriosclerThromb Vasc Biol. 1999;19:611 616.

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;145:33 43.

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 receptor deficient 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 96 89.

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 E knockout 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;92:821 825.

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.


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