The Immune Response In

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Atherosclerosis is an inflammatory disease characterizedby intense immunological activity, which increasinglythreatens human health worldwide1. Atherosclerosisinvolves the formation in the arteries of lesions thatare characterized by inflammation, lipid accumula-tion, cell death and fibrosis. Over time, these lesions,which are known as atherosclerotic plaques, mature andgain new characteristics. Although clinical complica-tions of atherosclerosis can arise from plaques causingflow-limiting stenoses, the most severe clinical eventsfollow the rupture of a plaque, which exposes the pro-thrombotic material in the plaque to the blood andcauses sudden thrombotic occlusion of the artery atthe site of disruption. In the heart, atherosclerosis canlead to myocardial infarctionand heart failure; whereas inthe arteries that perfuse the brain, it can cause ischaemicstrokeand transient ischaemic attacks. If atherosclero-sis affects other arterial branches, it can result in renalimpairment, hypertension, abdominal aortic aneurysms

and critical limb ischaemia. As our knowledge of thisdisease increases, we increasingly recognize that there isno simple answer to the question of whether the immuneresponse promotes or retards atherogenesis. Indeed, thetwo arms of the immune response can either promoteor attenuate aspects of atherosclerosis and its complica-tions. This Review summarizes our current understand-ing of the role of adaptive immunity in atherosclerosisand, in particular, weighs the evidence regarding theyin and yang of the immune response at various placesand times in the evolution of this lengthy and complexdisease. We do not discuss the arteriosclerosis of allo-grafted transplants, which is a distinct disease with a

unique pathogenesis, although it might represent anextreme case of immune-driven arteriopathy.

Immunological features of atherosclerosis

In humans, atherosclerotic plaques contain blood-borneinflammatory and immune cells (mainly macrophagesand T cells), as well as vascular endothelial cells, smoothmuscle cells, extracellular matrix, lipids and acellularlipid-rich debris2. These lesions typically present asasymmetrical focal thickenings of the intima, which isthe innermost layer of the artery (FIG. 1). Accumulationof immune cells and lipid droplets in the intima occursduring the f irst stage of plaque formation. Lipid-ladenmacrophages, known as foam cells, outnumber othercells in early plaques (which are known as fatty streaks),but these nascent plaques also contain T cells. Fattystreaks are prevalent in young individuals, never causesymptoms, and can progress into mature atheroscleroticplaques or disappear with time.

Mature plaques (also known as atheromas) have amore complex structure than fatty streaks (FIG. 1). Inthe centre of a plaque, foam cells and extracellular lipiddroplets form a core region that is surrounded by a capof smooth muscle cells and a collagen-rich matrix2.Other cell types present in plaques include dendritic cells(DCs)3, mast cells4, a few B cells2and probably naturalkiller T (NKT) cells. The shoulder region of the plaque,which is where it grows, and the interface between thecap and the core have particularly abundant accumu-lations of T cells and macrophages2. Many of theseimmune cells show signs of activation and produce pro-inflammatory cytokines such as interferon-(IFN) and

*Center for Molecular

Medicine, Department

of Medicine, Karolinska

University Hospital,

Karolinska Institute,

Stockholm, SE-17176,

Sweden.Leducq Transatlantic

Network of Excellence in

Cardiovascular Research,

Brigham and Womens

Hospital and Harvard

Medical School, Boston,Massachusetts, USA.Donald W. Reynolds

Cardiovascular Clinical

Research Center, Department

of Medicine, Brigham

and Womens Hospital and

Harvard Medical School,

Boston, Massachusetts

02115, USA.

Correspondence to P.L.

e-mail:

[email protected]

doi:10.1038/nri1882

Published online

16 June 2006

Plaque

An atherosclerotic lesion

consisting of a fibrotic cap

surrounding a lipid-rich core.

The lesion is the site of

inflammation, lipid

accumulation and cell death.Also known as an atheroma.

The immune response inatherosclerosis: a double-edged swordGran K. Hansson*and Peter Libby

Abstract | Immune responses participate in every phase of atherosclerosis. There is

increasing evidence that both adaptive and innate immunity tightly regulate atherogenesis.

Although improved treatment of hyperlipidaemia reduces the risk for cardiac and cerebral

complications of atherosclerosis, these remain among the most prevalent of diseases

and will probably become the most common cause of death globally within 15 years.This Review focuses on the role of immune mechanisms in the formation and activation of

atherosclerotic plaques, and also includes a discussion of the use of inflammatory markers

for predicting cardiovascular events. We also outline possible future targets for prevention,

diagnosis and treatment of atherosclerosis.

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2006Nature Publishing Group


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Cellular debrisand cholesterol

Shoulder

Media

Blood-vessel lumen

Endothelial cell

Normal artery

Elastic laminaIntima

Media

Foam cell T cellMacrophage Mast cell MonocyteCholesterol Dendritic cellDead cellSmoothmuscle cell

Myocardial infarction

An episode of acute cardiac

ischaemia that leads to death

of heart muscle cells. It is

usually caused by a thrombotic

atherosclerotic plaque.

Ischaemic stroke

An episode of acute regional

ischaemia in the brain leading

to nerve-cell death. It is usually

caused by thrombi or emboli

from atherosclerotic plaques.

Aneurysm

The local dilatation of an artery

caused by weakening of the

artery wall. Some, but not all,

aneurysms are caused by

atherosclerosis.

Intima

The innermost layer of anartery, which consists of loose

connective tissue and is

covered by a monolayer of

endothelium. Atherosclerotic

plaques form in the intima.

Fibrous cap

A structure composed of a

dense collagen-rich

extracellular matrix with

occasional smooth muscle

cells, macrophages and T cells

that typically overlies the

characteristic central lipid core

of plaques.

tumour-necrosis factor (TNF)5. With time, the plaquecan progress into an even more complex lesion, thelipid core of which has become a paucicellular pool ofcholesterol deposits surrounded by a fibrous capof vary-ing thickness. The fibrous cap prevents contact betweenthe blood and the pro-thrombotic material in the lesion(FIG. 1). Disruption of the cap can lead to thrombosis andmany of the adverse clinical outcomes associated withatherosclerosis.

Models of atherogenesis in mutant mice

Direct analysis of the early phases of human atherosclerosispresents obvious obstacles. Therefore, systematic inves-tigation of the mechanisms that initiate atherosclerosisrelies on animal models of the disease. The availableobservations indicate that there is substantial overlapbetween disease development in these animal models andthe human disease. Two strains of genetically altered micehave been particularly fruitful in this regard. Apoe/

mice lack apolipoprotein E (APOE; which is a keycomponent in cholesterol metabolism), and developspontaneous hypercholesterolaemia and atheroscleroticdisease (which is exacerbated by an atherogenic diet)that progresses to myocardial infarction and stroke 6,7.Low-density-lipoprotein receptor (LDLR)-deficient micerespond to being fed with fat by developing hypercholes-terolaemia and atherosclerotic plaques8. The crossbreed-ing of these mice with mice that carry deletions in genesencoding crucial components of the immune systemhas provided important information on the role of theimmune system in the pathogenesis of atherosclerosis. Inaddition, bone-marrow transplantation of, and spleen-cell

transfer to,Apoe/or Ldlr/mice has offered insights intothe role of specific populations of bone-marrow-derivedcells in disease development.

Immune-cell recruitment initiates atherosclerotic-plaque formation.In experimental animals, endothelialcells in the arteries express leukocyte adhesion mol-ecules, in particular vascular cell-adhesion molecule 1(VCAM1), as part of the initial vascular response tocholesterol accumulation in the intima9(FIG. 2a). Thepatchy distribution of adhesion-molecule expressioncorresponds to the subsequent position at which fattystreaks form10. This patchy pattern of expression prob-ably reflects haemodynamic factors, because the shearstresses and disturbed fluid flows vary over the arterialbed in a similar way to the predilection sites for athero-sclerosis. Interestingly, exposing cultured endothelialcells to oscillatory shear stress that mimics arterialblood flow increases the expression of several leukocyte

adhesion molecules11.Shortly after VCAM1 induction, monocytes and

T cells enter the arterial intima (FIG. 2a). Under the influ-ence of macrophage colony-stimulating factor (M-CSF)produced by endothelial cells and smooth muscle cells12,the monocytes differentiate into macrophages13(FIG. 2b)and T cells can undergo antigen-dependent activation(FIG. 2c;see later). Interestingly, VCAM1 expression bythe endothelium ceases after a few weeks, but smoothmuscle cells begin to express this adhesion molecule14.Expression of VCAM1 and other adhesion molecules bysmooth muscle cells might promote the recruitment andretention of mononuclear cells in the arterial intima.

Figure 1 | Cellular composition of atherosclerotic plaques. The atherosclerotic plaque has a core containing lipids

(which include esterified cholesterol and cholesterol crystals) and debris from dead cells. Surrounding it, a fibrous cap

containing smooth muscle cells and collagen fibres stabilizes the plaque. Immune cells including macrophages, T cells

and mast cells populate the plaque, and are frequently in an activated state. They produce cytokines, proteases, pro-

thrombotic molecules and vasoactive substances, all of which can affect plaque inflammation and vascular function.

Until complications occur, an intact endothelium covers the plaque.

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Endothelialcell

MHC class II

TCR

APC

Scavengerreceptor

VLA4VCAM1

Blood-vessellumen

LDL

oxLDL

Monocyte

MacrophageMonocyte

Foam cell

Chemokinereceptor Chemokine

M-CSF

T cell

TLR T cell Endothelial cell

LPS, HSP60or oxLDL

Pro-inflammatory cytokinesProteasesProcoagulantsPro-apoptotic factors

Increased adhesion moleculesIncreased permeabilityIncreased propensity forthrombus formation

Decreased collagen productionDecreased proliferation

CD4+T cell

TH1 cell

IL-12IL-15IL-18

b

c

oxLDL

TH1 cell

Smoothmuscle cell

MHC class IITCR

IFNTNF

CD40L CD40

Macrophage

ProteasesPro-inflammatorymediators

Decreasedinflammation

TH1 cell

TH2 cell orregulatoryT cell

Smoothmuscle cell

TGF

TGF

IL-10

e

Macrophage

a d

T cell Endothelial cellT cell Endothelial cell

T cell Endothelial cell

Figure 2 | Recruitment and activation of immune cells in atherosclerotic plaques. a |Low-density lipoprotein (LDL)

diffuses from the blood into the innermost layer of the artery, where LDL particles can associate with proteoglycans of the

extracellular matrix. The LDL of this extracellular pool is modified by enzymes and oxygen radicals to form molecules such

as oxidized LDL (oxLDL). Biologically active lipids are released and induce endothelial cells to express leukocyte adhesion

molecules, such as vascular cell-adhesion molecule 1 (VCAM1). Monocytes and T cells bind to VCAM1-expressing

endothelial cells through very late antigen 4 (VLA4) and respond to locally produced chemokines by migrating into the

arterial tissue. b | Monocytes differentiate into macrophages in response to local macrophage colony-stimulating factor

(M-CSF) and other stimuli. Expression of many pattern-recognition receptors increases, including scavenger receptors

and Toll-like receptors (TLRs). Scavenger receptors mediate macrophage uptake of oxLDL particles, which leads tointracellular cholesterol accumulation and the formation of foam cells. TLRs bind lipopolysaccharide (LPS), heat-shock

protein 60 (HSP60), oxLDL and other ligands, which instigates the production of many pro-inflammatory molecules by

macrophages.c | T cells undergo activation after interacting with antigen-presenting cells (APCs), such as macrophages

or dendritic cells, both of which process and present local antigens including oxLDL, HSP60 and possibly components

of local microorganisms. A T helper 1 (TH1)-cell-dominated response ensues, possibly owing to the local production of

interleukin-12 (IL-12), IL-18 and other cytokines. Antigen presentation and TH1-cell differentiation might also occur in

regional lymph nodes. d | TH1 cells produce inflammatory cytokines including interferon-(IFN ) and tumour-necrosis

factor (TNF) and express CD40 ligand (CD40L). These messengers prompt macrophage activation, production of

proteases and other pro-inflammatory mediators, activate endothelial cells, increase adhesion-molecule expression

and the propensity for thrombus formation, and inhibit smooth-muscle-cell proliferation and collagen production.

e | Plaque inflammation might be attenuated in response to the anti-inflammatory cytokines IL-10 and transforming

growth factor-(TGF), which are produced by several cell types including regulatory T cells, macrophages, and for TGF,also vascular cells and platelets. TCR, T-cell receptor.

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Vasa vasorum

Small nutrient vessels in the

normal adventitia and outer

media of the artery wall,

which can also give rise to

microvessels in the plaque.

cells2. Small populations of mast cells, B cells and DCsoccur in plaques and, together with T cells, monocytesand macrophages, might traffic between the blood inthe arterial lumina, the lesioned artery wall, the vasavasorum microvessels that penetrate the artery andthe regional lymph nodes.

The ratio of CD4+ to CD8+T cells in advancedplaques resembles that found in peripheral blood2. MostT cells are T cells,but there is also a small propor-tion of T cells. Lesions of early stages of experimentalatherosclerosis contain oligoclonal expansions of CD4+cells expressing an T-cell receptor (TCR)37, indi-cating activation in response to a limited set of localantigens (FIG. 2c). The CD4+T cells that are isolatedfrom human plaques are mostly CD45RO-expressingmemory and/or effector T cells38. An initial round ofT-cell activation in response to athero-antigens mightoccur in the regional lymph nodes, possibly after anti-gen presentation by DCs trafficking from the plaqueto the lymph node39. After entering the blood, previ-ously activated memory and/or effector T cells bind

cell-surface adhesion molecules that are expressed byendothelial cells at the plaque surface and/or in the vasa

vasorum, and then enter the plaque. Macrophages inthe plaque expressing MHC class II molecules mightthen present antigen to these T cells, leading to furtherrounds of activation.

Very recently,Ath1, which is an atherosclerosis sus-ceptibility locus on mouse chromosome 1, was mappedto the gene encoding OX40 ligand (OX40L; also knownas TNFSF4), which is a co-stimulatory factor for T-cellactivation40. Reduced expression of this protein was asso-ciated with reduced atherosclerosis in inbred strains thatdiffer in their Tnfsf4 alleles and also in mice carrying atargeted deletion of this gene40. Polymorphisms in humanTNFSF4were found to be associated with coronaryatherosclerosis and with an increased risk for myocar-dial infarction in a human genetic epidemiology study 40.These data re-emphasize the importance ofimmuneactivation in atherosclerosis and its complications41.

Plaque antigens activate local cellular adaptive immu-

nity.Cloning T cells from surgically removed humanplaques has identified several cell-mediated, local adap-tive immune reactions. CD4+T-cell clones derived fromplaques recognize oxLDL, with other clones recognizingHSP60 or other antigens derived from certain pathogenicmicroorganisms, such as Chlamydia pneumoniae 42,43

(FIG. 2c). In all of these cases, antigen recognition wasrestricted by HLA-DR and involved TCR+CD4+T cells42,44,45.

Antigen-presenting cells selectively internalizeoxLDL particles through the scavenger-receptor path-way. After proteolytic processing, fragments of theprotein component of LDL, APOB, bind nascent MHCclass II molecules and traffic to the cell surface. Indeed,APOB fragments are among the peptides displayed mostfrequently by HLA-DR molecules in cultured humanlymphoblastoid cells46. Therefore, receptor-mediatedendocytosis and the antigen-presentation pathway facili-tate MHC class II presentation of LDL-derived peptides

to CD4+T cells. As expected, no T cells react with nativeLDL components. However, oxidative modificationof LDL breaks tolerance and oxLDL-reactive T cells local-ize in plaques, lymph nodes, and in the blood of patientswith atherosclerosis and experimental animals42,47.OxLDL-reactive CD4+ T cells probably recognizeAPOB-derived oligopeptides carrying adducts formedduring oxidation48; whereas oxLDL-specific antibodiesreact with oxidized phospholipids such as phosphoryl-choline49,50, as well as aldehyde-peptide epitopes includingmalondialdehyde-lysine42,44,45.

Most oxLDL-reactive CD4+T cells have a T helper 1(T

H1)-cell phenotype42,47. Because T

H1 cytokines (such as

IFN) generally stimulate pro-atherosclerotic processes(see later), these T cells probably promote atherogenesis,a conclusion supported by adoptive-transfer studies insevere combined immunodeficient (SCID) mice lack-ing APOE51. As expected, these mice show substantiallyreduced atherosclerotic plaques compared with immuno-competentApoe/mice. Transfer of CD4+T cells fromimmunocompetentApoe/mice to SCID mice lacking

ApoE increases the atherogenesis found in immuno-deficient mice, to almost the same level as that foundin fully immunocompetentApoe/mice. Therefore, thenet effect of CD4+T cells is to increase atherogenesis inmice susceptible to atherosclerotic disease. Obviously,this finding does not preclude the existence of T-cellsubsets that might mitigate disease.

Recent studies have identified transcripts encodingV14J281-containing TCR -chains in plaques ofhypercholesterolaemic mice, indicating the presenceof NKT cells52. The abundance of CD1 molecules inplaques53indicates that CD1-mediated NKT-cell acti-

vation takes place, but the absence of specific markersfor NKT cells has hampered direct immunohistologicaldemonstration of NKT cells in plaques. However, admin-istration of ligands that specifically activate NKT cells toApoe/mice shows that NKT-cell activation increasesearly atherosclerotic plaque development concomitantlywith increased local expression of pro-inflammatorycytokines, whereas abrogation of CD1-mediated antigenpresentation reduces disease52,54. Therefore, NKT cellscontribute to atherosclerosis, probably by antigen-specific activation in response to lipid antigens presentin plaques.

A role for TH

1/TH

2-cytokine balance?Analyses ofcell-surface-marker expression and cytokine secretion

indicate activation of a remarkably large proportionof T cells in plaques38. T

H1-type cytokines dominate

in mouse models of atherosclerosis and in humanplaques. For example, human plaques contain cellsproducing IFN, interleukin-12 (IL-12), IL-15, IL-18and TNF, but few cells producing the T

H2-type cytokine

IL-4 (REFS 42,55,56). Together with the histopatho-logical features of accumulation of macrophages andT cells, the predominance of T

H1-type cytokines indi-

cates that atherosclerosis is a TH

1-cell-driven disease(FIG. 2d). This hypothesis is supported by studies ingenetically altered mice that show that there is reducedatherosclerosis in hypercholesterolaemic mice lacking

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Tissue factor

A procoagulant that stimulates

thrombus formation, when in

contact with blood, by

accelerating the action of

factors VIIa and Xa.

IFNor its receptor57,58, IL-12 (REF. 59), IL-18 (REF. 60),TNF61or the T

H1-cell-inducing transcription factor

T-bet62. Administration of recombinant IFN63orthe T

H1-cell-inhibiting drug pentoxifyllin64to hyper-

cholesterolaemic mice led to increased and decreasedatherosclerosis, respectively, lending further supportto this hypothesis64.

If TH

1 cytokines stimulate plaque formation andT

H2 cytokines inhibit T

H1-cell responses, can T

H2-cell

responses protect against atherosclerosis? In supportof this proposition, C57BL/6 mice (which are proneto T

H1-type immune responses) develop fatty streaks

if fed a high-cholesterol diet, whereas BALB/c mice(which are prone to T

H2-type immune responses)

are resistant to atherogenesis65,66. Targeted deletionof the gene encoding signal transducer and activa-tor of transcription 6 (STAT6), a transcription factorthat is essential for the differentiation of T

H2 cells,

renders BALB/c mice susceptible to atherogenesis andthis occurs in parallel with a switch from T

H2-cell to

TH1-cell responses66.

Although these studies of the development offatty streaks indicate opposing roles for T

H1 and T

H2

cells in disease development, mice that develop moreadvanced plaques show a more complicated picture.Pharmacological inhibition of T

H1 cells using pentoxi-

fyllin or IL-18-binding protein inhibits atherosclerosisinApoe/mice64,67, and administration of recombinantT

H1 cytokines (recombinant IL-18 and IFN) exacer-

bates disease63,68. However, data for IL-4, which isthe prototypic T

H2 cytokine, are inconclusive. Some

studies have shown that IL-4 has a protective effect,whereas others found reduced disease in the absenceof IL-4 (REFS 59,69). These divergent findings, underdifferent experimental conditions, might reflect thecomplex range of biological activities found for IL-4,including stimulation of scavenger-receptor expres-sion and the induction of elastin degrading MMP12,which can lead to aneurysm formation70. Defining therole of T

H2 cells in atherosclerosis, therefore, requires

further study.

Pro-atherosclerotic action of TH1 cells.How can T

H1

cells promote disease development? IL-12 and IL-18,which are produced by macrophages and smooth mus-cle cells in plaques, can indirectly affect the develop-ment of plaques by promoting T

H1-cell differentiation.

By contrast, IFNand TNF directly accelerate disease

through their actions on macrophages and vascularcells (FIG. 2c,d). IFNactivates macrophages, therebyincreasing their production of nitric oxide, pro-inflam-matory cytokines, and pro-thrombotic and vasoactivemediators. Additionally,IFNinhibits endothelial-cellproliferation71, the proliferation and differentiationof vascular smooth muscle cells72, and also decreasescollagen production by these smooth muscle cells73.Decreasing the cell and collagen content of the fibrouscap might reduce the stability of the plaque. Therefore,the combined effects of IFNon cells of the formingplaque promote inflammation and extracellular-matrixdestabilization.

The pro-inflammatory cytokine TNF triggers vascu-lar inflammation through the NF-B pathway, inducingthe production of reactive oxygen and nitrogen species,proteolytic enzymes and pro-thrombotic tissue factorbyendothelial cells, and modulates the fibrinolytic capacityof the cells7476. TNF also has profound metabolic effectsthat include the suppression of lipoprotein lipase, whichleads to the accumulation of triglyceride-rich lipopro-teins in the blood. Such lipoproteins, and the TNF levels,have been associated with heart disease in clinical stud-ies7779. Genetic loss-of-function studies also support theidea that TNF has a pro-atherogenic role61.

CD40 and CD40L: a co-stimulatory dyad with pro-atherogenic action.The cell-surface proteins CD40andCD40 ligand (CD40L; also known as CD154) have sev-eral similarities to soluble pro-inflammatory cytokines.CD40 ligation on cells found in plaques triggers aninflammatory response similar to that elicited by TNF,that is, secretion of other cytokines and MMPs, andexpression of adhesion molecules80. Importantly, CD40

ligation causes expression of the procoagulant tissuefactor by human macrophages, something that solublepro-inflammatory cytokines do not do. Macrophages andT cells express CD40 and CD40L, as do vascular endothe-lial cells, smooth muscle cells and platelets81,82. Therefore,CD40 ligation propagates inflammatory activation in allthe main cell types involved in atherogenesis. Inhibitionof CD40 ligation andinactivation of the gene encodingCD40L reduces atherosclerotic plaques in hypercholes-terolaemic mice83,84. Unfortunately, CD40 blockade inhumans can promote platelet aggregation and thrombosis,which is an obstacle to its clinical application.

Anti-atherogenic immunity

Anti-inflammatory cytokines.Although local cellularimmunity predominantly promotes atherosclerosisthrough the action of cell-surface molecules (such asCD40CD40L) and cytokines (such as IFNand TNF),counterbalancing factors can function to dampen dis-ease activity (FIG. 2e). Two anti-inflammatory cytokines,IL-10 and transforming growth factor-(TGF), provideparticularly important atheroprotective signals.

Two groups have reported previously that IL-10-deficient C57BL/6 mice that consume a fatty diet developan increased quantity of fatty streaks compared withwild-type mice85,86. By contrast, Il10transgenic C57BL/6mice do not develop fatty streaks, thereby providing evi-

dence of a protective role for IL-10 in atherosclerosis85,86.The mouse model used in these early studies mimickedthe initial stage of atherogenesis, but the mice didnot develop lesions similar to human clinical disease.However, subsequent experiments usingApoe/mice,which develop atherosclerotic lesions that are more simi-lar to those found in humans, also show an atheroprotec-tive role for IL-10 (REF. 87). Interestingly, IL-10 promotesarteriopathy in transplanted hearts, indicating a morecomplex picture.

The pluripotent cytokine TGFhas many effects on adiverse range of cell types and can inhibit atherosclerosisat least as well as IL-10. For example, TGFpromotes

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collagen production, which could increase plaquestability. Treatment with tamoxifen, which is a TGF-stimulating oestrogen-receptor agonist, reduces theformation of fatty streaks in C57BL/6 mice fed withfat88, whereas administration of TGF-specific block-ing antibodies or decoy receptors for TGFreducesatherosclerotic plaque formation in Ldlr /mice89,90.However, these studies did not identify the mechanismof action of TGF.

Two more-recent studies show that TGFexerts itsatheroprotective effects by modulating T-cell activation.In the first study, crossbreeding mice carrying dominant-negative TGFreceptors (that were expressed underthe control of the Cd4 promoter) with Apoe/miceled to a fivefold increase in plaque size and advancedplaques were found in the proximal aorta of 12-week-old crossbred mice91. Notably, the plaques showed signsof increased inflammation and had fewer interstitialcollagen fibres, a characteristic of human plaques thatcause thrombosis (see later). In the second study, bonemarrow from mice that expressed a dominant-negative

form of the type II TGFreceptor (that was expressedunder the control of the Cd2promoter) was transplantedto irradiated Ldlr /mice92. Again, plaques showed signsof substantial inflammation and a poorly developedcollagenous matrix. These studies show the importantatheroprotective effects of TGFthat occur through thedampening of T-cell activity.

Several cell types can produce TGFand IL-10,including platelets, macrophages, endothelial cells,smooth muscle cells and regulatory T cells. Activationof regulatory T cells could therefore offer a means ofantigen-specific atheroprotection (FIG. 2e). A recent studysupports this idea by showing that the transfer of naturalCD4+CD25+regulatory T (T

Reg

) cells reduces athero-sclerosis, whereas depletion of CD25+cells increases dis-ease inApoe/mice93. Depletion of CD25+cells in micelacking functional TGFreceptors on T cells did not alterplaque size, indicating that this cytokine mediates theatheroprotective effect of regulatory T cells93.

Humoral immunity.In addition to innate immunityand T cells, antibodies with different specificities canparticipate in atherosclerosis. Humans and experimen-tal animals with disease have antibodies specific foroxLDL particles44. B-cell epitopes in oxLDL includeamino-acid residues of APOB that are modified bylipid peroxidation products, such as malondialdehyde

and 4-hydroxynonenal. Although some clinical and epi-demiological studies have found positive correlationsbetween the presence of antibodies specific for oxLDLand the progression of atherosclerosis94,95, other studieshave not detected any correlation. Interestingly, anti-bodies specific for oxLDL, mainly of the IgM isotype,also circulate in asymptomatic humans96and crossreactwith apoptotic bodies49. These antibodies bind theoxidized phospholipids in oxLDL and also recognizephosphorylcholine in the cell wall of Streptococcuspneumoniae49. Phosphorylcholine-specific IgM con-sists of germline-encoded antibodies of the T15 typethat are produced by B1 cells49. Therefore, expansion

of B-cell clones that produce T15-type antibodies, forexample during a pneumococcal infection, might affectthe development of plaques. Indeed, immunization ofLdlr /mice with a pneumococcal vaccine reduced theextent of atherosclerosis50.

Molecular mimicry could explain the crossreactiv-ity between the humoral immune responses to oxLDL,apoptotic bodies and pneumococci. This mechanismmight also apply to HSP60, another antigen associatedwith atherosclerosis97. HSP60 is a chaperone moleculethat is involved in protein folding and can be detectedin plaques. Antibodies specific for HSP60 are found inexperimental animals that have atherosclerosis and havebeen correlated with disease progression in a humancohort study98. Present in prokaryotes and eukaryotes,HSP60 has shown remarkable sequence conservation dur-ing evolution. As antibodies specific for HSP60 crossreactbetween microbial and eukaryotic HSP60, antibodiesthat react to human HSP60 can be generated in responseto infection with microbes that express HSP60, such asC. pneumoniae99.

Several further experimental, and some human, stud-ies show that humoral immunity can protect againstatherosclerosis. Splenectomy increases atherosclerosis inbothApoe/mice and humans100. InApoe/mice, trans-fer of splenic B cells from atherosclerotic animals intosplenectomized recipients protects against disease, pos-sibly because of the production of protective antibodiesby B cells100.

Immunization experiments identify oxLDL andHSP60 as important antigens that can induce protective,as well as detrimental, immune responses (TABLE 1; seealso later). A tentative conclusion from these studies is thatT

H1-type immune responses promote disease; whereas

humoral immunity has protective effects, possibly byeliminating antigens before they reach plaques.

Adaptive immunity disrupts plaques

In general, the gravest clinical complications of athero-sclerosis result from the sudden thrombotic occlusion ofan artery101. The sudden onset of myocardial infarction,as well as many strokes and episodes of acute limb ischae-mia, is caused by thrombi that arise from atheroscleroticplaques that do not necessarily tightly narrow the artery.Therefore, many episodes of damage to the heart muscle,brain or lower extremities can occur without warning, alltoo often with devastating consequences.

Physical disruption of a plaque is the most frequent

cause of thrombotic occlusions. Indeed, the most fre-quent patho-anatomical substrate for sudden coronarythrombosis is rupture of the fibrous cap that overlies thelipid core of the plaque101(FIG. 3). Fibres of interstitial col-lagens (types I and III) normally confer biomechanicalstability on the fibrous cap of the plaque. As discussedearlier, the T

H1-cytokine IFNstrongly inhibits the

production of interstitial collagens by vascular smoothmuscle cells, which are the main source in the arterialwall of this extracellular-matrix macromolecule73. IFNcan also inhibit the proliferation of smooth musclecells, thereby reducing the stabilizing and collagen-synthesizing cellular component of the plaque72. Also,

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Angina pectoris

A reversible attack of chest

discomfort, usually caused by

an imbalance between the

oxygen demand of the working

heart muscle and the

insufficient supply through

narrow, atheroscleroticcoronary arteries.

Angioplasty

A percutaneous catheter

procedure that inflates a

balloon in areas of narrowing

(stenosis) in arteries.

Statins

A class of drugs that inhibit the

rate-limiting enzyme (3-

hydroxy-3-methylglutaryl

coenzyme A reductase) in the

pathway of cholesterol

biosynthesis.

proteases elaborated mainly from activated macrophagesin plaques can degrade collagen102,103. In addition, liga-tion of CD40 expressed by macrophages increases theproduction of matrix-degrading proteases that include

the interstitial collagenases of the MMP family, MMP1,MMP8 and MMP13 (REF. 104). Therefore, T

H1 cells

probably have an essential role in regulating the func-tions of smooth muscle cells (collagen-fibre formation)and macrophages (collagen degradation) that cruciallyregulate the integrity of the fibrous cap of the plaqueand therefore its susceptibility to rupture and provokethrombosis.

Once coagulation factors in the blood gain accessto the lipid core of the plaque following rupture of thefibrous cap, thrombosis commonly ensues. Tissue factor,the potent procoagulant expressed by a subpopulationof macrophages in the lipid core of the plaque, triggersthese thromboses101. As noted earlier, ligation of CD40expressed by macrophages strongly induces expressionof tissue factor80. Indeed, T cells expressing CD40L local-ize in the vicinity of macrophages that are expressing tis-sue factor in the lipid core of human plaques105. Becauseplatelets can also express CD40L82when activated, positivefeedback can amplify the local inflammatory response,once a thrombus begins to form, because of generation ofthe protease thrombin induced by tissue factor and plate-let activation induced by thrombin. Therefore, althoughT cells could orchestrate the pathophysiology of plaquedisruption, dysregulated antigen-nonspecific pathwaysprobablyamplify and sustain the formation of thrombi.Modulation of immunity in atherosclerosisImmunopharmacological intervention against sympto-matic atherosclerosis.Although thrombi cause most ofthe acute complications of atherosclerosis, the gradualformation of stenoses that impede blood flow causesmany of the chronic symptoms of atherosclerotic disease,such as angina pectoris (chest discomfort precipitatedtypically by physical or emotional stress). Recent decadeshave witnessed important advances in the ability of inter-

ventions, particularly percutaneous procedures, to relievestenoses and reduce ischaemia. Until recently, however,the long-term success of mechanical procedures, suchas the deployment of arterial stents (metal scaffolds to

hold arteries open) and balloon angioplasty (inflationof miniature balloons in blocked segments of arter-ies to expand the arterial lumen), has been limitedby re-growth of intimal tissue which is known as

in-stent stenosis and restenosis, respectively. Thisfibro-proliferative response of the injured artery canre-occlude the lumen within months in a substantialminority of patients.

Recently, the coating of stents with immunosup-pressive agents, for example sirolimus (Rapamycin),has shown striking effectiveness at reducing in-stentstenosis106. This advance has markedly improved clinicaloutcomes in patients undergoing percutaneous interven-tion. Early preclinical studies provided the experimentalbasis for this important therapeutic advance by show-ing that another immunosuppressant, cyclosporin,reduces intimal-cell proliferation in response to arterialinjury107.

The use of statinshas shown striking clinical benefitin preventing atherosclerotic complications during thepast decade. Numerous clinical trials have establishedthat 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (drugs of the statin family)can reduce various atherosclerotic complications108.The lowering of LDL cholesterol concentrations in theblood doubtless accounts for much of this remarkableclinical benefit.However,recent data indicate that partof the clinical benefit of statins occurs because of ananti-inflammatory effect that is apparently not relatedto LDL reduction109. (See REF. 110for a detailed discus-sion of non-LDL-lowering effects of statins).

By blocking HMG-CoA reductase, statins preventthe formation of lipids that control the function ofseveral intracellular proteins111. By acting on the MHCclass II transactivator (CIITA), statins can interfere withthe transcriptional induction of MHC class II mol-ecules, which would decrease immune activation in theplaque112. Statins can also limit the accelerated arterio-sclerosis (sclerosis of the arterial walls)that complicatessolid-organ transplantation, a disease that often occursin the absence of increased concentrations of LDL113.They also seem to reduce disease activity in patients withrheumatoid arthritis114and in mice with experimentalautoimmune encephalomyelitis115. All these results lend

Table 1 | Immunization against atherosclerosis in experimental models

Antigen Route Animal model Effect on atherosclerosis References

MDA-LDL Subcutaneous WHHL rabbits Reduced 124

oxLDL Subcutaneous Fat-fed NZW rabbits Reduced 125

MDA-LDL Subcutaneous Apoe/mice Reduced 47,126

MDA-LDL Subcutaneous Ldlr/mice Reduced 127

APOB-peptides Subcutaneous Apoe/mice Reduced 95,128

MDA-LDL Subcutaneous Cd4/Apoe/mice Reduced 129

HSP65 Subcutaneous Ldlr/mice Increased 130

HSP65 Peroral/nasal Ldlr/mice Reduced 131,132

2-GPI Subcutaneous Ldlr/mice Increased 133

APO, apolipoprotein; GPI, glycoprotein I; HSP65, heat-shock protein 65; LDL, low-density lipoprotein; LDLR, LDL receptor; MDA-LDL,malondialdehyde-modified LDL; NZW, New Zealand white; oxLDL, oxidized LDL; WHHL, Watanabe hereditably hyperlipidaemic.

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Blood-vessel lumen

Endothelial cell

Elasticlamina

Cellular debrisand cholesterol

Rupture

Thrombus

Platelet

Erythrocyte

Fibrin

Foam cell T cellMacrophage Mast cell MonocyteCholesterol Dendritic cellDead cellSmoothmuscle cell

Peroxisome-proliferator-

activated receptors

Nuclear receptors that

participate in the regulation

of cellular metabolism and

differentiation.

Thiazolidinedione

A class of medication, used

to treat diabetes, that binds

peroxisome-proliferator-

activated receptor-.

support to the idea that the immunomodulatory actionsof statins also contribute to their effects in patients withatherosclerosis.

Recent studies have establishedthat another categoryof anti-atherosclerotic drugs, the ligands for a group ofnuclear transcription factors known as peroxisome-proliferator-activated receptors(PPARs), can inhibit T-cellactivation in vitro. Activators of both PPAR

(members

of the fibrate class of drugs) and PPAR(members ofthe thiazolidinedionefamily of drugs) can reduce T-cellactivation, as was shown by decreased production ofIFN, TNF and IL-2 (REF. 116). PPARagonists alsoinhibit inflammatory activation of vascular smooth

muscle cells117. Therefore, activation of PPARor PPARmight also affect atherosclerosis in a beneficial mannerby blunting the adaptive and innate immune responses.

Nonspecific anti-inflammatory therapies, such asnon-steroidal anti-inflammatory drugs (NSAIDs), havenot improved the cardiovascular outcome. Indeed, treat-ment with NSAIDs selective for cyclooxygenase-2 seemsto increase the risk of thrombotic complications118,119.Despite their marked anti-inflammatory properties,glucocorticosteroids themselves probably increase,rather than decrease, atherogenesis, as chronic admin-istration of these agents adversely affects plasma lipopro-teins, promotes insulin resistance and sodium retention,

Figure 3 | Plaque activation, rupture and thrombosis. When activated, immune cells including macrophages, T cells

and mast cells can release pro-inflammatory cytokines, which reduce collagen formation and induce the expression of

tissue factor. Proteases that attack the collagenous cap are also released by activated immune cells. The weakened plaque

might fissure when subjected to the forces of arterial blood pressure. Exposure of subendothelial structures and

procoagulants such as tissue factor promotes platelet aggregation and thrombosis. A thrombus forms and might occlude

the lumen of the artery, leading to acute ischaemia.

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C-reactive protein

An acute-phase reactant

protein, the plasma

concentration of which

increases in inflammatory

states.

and inhibits collagen and elastin formation. It thereforedoes not offer a reasonable therapeutic alternative in thechronic phases of atherosclerosis.

Vaccination against atherosclerosis?Parenteral immu-nization with malondialdehyde-modified LDL (thatis, LDL with a defined oxidative modification) ormalondialdehyde-modified peptides derived from theLDL protein apolipoprotein inhibits atherosclerosis andthis occurs in parallel with increased titres of antibodyspecific for the immunogen (TABLE 1). Interestingly, pro-tection through this route does not require CD4+T-cellhelp120. Therefore, protection seems to depend mostlyon humoral immunity, at least in this model.

By contrast, the outcomes after immunization withHSP60 or its mycobacterial homologue HSP65 arecomplex (TABLE 1). Parenteral immunization in C57BL/6mice fed with fat, as well as Ldlr /mice, aggravatesdisease, whereas oral or nasal immunization elicitsprotective immunity. Induction of mucosal immunityinvolves activation of regulatory T cells that produce

anti-inflammatory cytokines and also high titres ofspecific antibodies. Therefore, the precise mechanismby which mucosal immunization leads to reducedatherosclerosis remains to be clarified.

Although several questions remain, the immuniza-tion experiments with malondialdehyde-modified LDLand HSP60 indicate that it is possible that a vaccina-tion strategy might protect against atherosclerosis andits complications. Obviously, many obstacles remain,rendering the success of this approach unpredictable,particularly in humans.

Conclusion

The evidence reviewed in this article supports theinvolvement of the immune response in atherosclerosisfrom its initiation through to its thrombotic complica-tions. The concept that adaptive immunity pivotallyregulates atherogenesis has already been clinically useful.Markers of the acute-phase response, notably C-reactiveprotein(CRP), predict the prognosis of patients whohave already sustained a cardiovascular event121. Lesserelevations of CRP concentration, measured with a highlysensitive assay and previously considered in the normalrange, can be used to predict cardiovascular events inapparently well populations109,122,123. Markers of height-ened innate immune responses, such as CRP, correlatewith worse outcomes in individuals with acute coronarysyndromes121.

Systemic administration of non-selective immuno-suppressive drugs will probably not be useful for thetreatment of atherosclerosis, at least during its longasymptomatic phase, because of the need for prolongedtherapy and the potential toxicities of such treatments.

However, the immune system in its full complexity offersmuch more subtle targets for therapeutic manipulation.As more details emerge of the specific pathways involvedin the immune response and the inflammation thatoccur in atherosclerosis, more selective interventionsmight prove appropriate for long-term anti-atheroscle-rotic therapy. Also, as our ability to gauge the risk ofacute complications improves, we might be able to targetin a much more selective manner those therapies thatwould otherwise impair host defences if administeredon a long-term basis.

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AcknowledgementsWe regret that we have not been able to cite many important

papers owing to space limitations. Our research is supported

by grants from the Swedish Research Council, Heart-Lung

Foundation, European Community, US National Institutes of

Health and Leducq Foundation.

Competing interests statementThe authors declare no competing financial interests.

DATABASESThe following terms in this article are linked online to:

Entrez Gene:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene

APOE | CCL2 | CD14 | CD40 | CD40L | CD68 | CX3CR1 |

HSP60 | IFN | IL-4 | LDLR | MMP1 | PPAR | PPAR | TLR4 | TNF |VCAM1

FURTHER INFORMATIONPeter Libbys homepage:

http://reynolds.brighamandwomens.org/faculty/libby.asp

Gran K. Hanssons homepage:

http://www.ki.se/medicin/medicine_ks/experimental_

cardiovascular_research_unit/index_en.html

Access to this links box is available online.

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