chemokine review

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proximal aortic lesions than their control littermates.

Similarly, targeted disruption of the macrophage scav-

enger receptor-A gene in mice, in this case rendering

monocytes deficient in their ability to uptake modified

LDL lipids, resulted in a reduction in the size of ather-

osclerotic lesions in animals deficient in ApoE. Pow-

erful data from this line of investigaton in mouse mod-els therefore suggests a key role for monoyctes in

lesion formation.

The role of T cells in the pathogenesis of athero-

sclerosis is somewhat less clear than the role of

monocytes. It is well recognized that T cells can

accumulate in lesions during the earliest stages of

atherogenesis, perhaps even preceding the monocyte.

Although the exact role of T cells in atherogenesis

remains incompletely understood, by virtue of their

early appearance, persistence, and localization at sites

of lesion growth and rupture, a number of groups have

suggested that they may orchestrate important aspects

of atherogenesis. The effect of total lymphocyte defi-ciency on atherogenesis has been investigated by

crossing ApoE-deficient mice with mice deficient in

RAG-1, a gene essential for normal lymphocyte devel-

opment. RAG-1/ApoE compound mice show a 40%

decrease in atherosclerotic lesions as compared with

ApoE null mice. It must be noted, however, that results

seen in comparable experiments that employ RAG-

2–deficient mice (a mouse that shares a “lymphocyte-

depleted” phenotype similar to that of the RAG-1

knockout) are less striking. Although the latter results

may be explained by differences in genetic back-

ground, diet, or technical differences in lesion assess-

ment, taken together these results suggest a less robusteffect of lymphocyte depletion on atherogenesis as

compared with monocyte depletion.

Further data addressing potential causal interactions

between leukocytes and the formation of atherosclerot-

ic lesions have been generated by studies of the

immune mediator CD40 and its ligand CD40L (also

known as CD154 or gp39). Several groups have recent-

ly demonstrated that cells within human and mouse

atherosclerotic lesions express CD40 and its ligand.

CD40L-positive T cells accumulate in atheroma, and

ligation of CD40 on atheroma-associated cells in vitro

induces proinflammatory cytokines, matrix metallopro-

teinases, adhesion molecules, and tissue factor, com-

ponents previously implicated in atherogenesis. Inhi-

bition of CD40 signaling either by treatment with

antibody against CD40L or by using compound mutant

mouse (ApoE and CD40L null mice) showed a reduc-

tion in atherosclerosis lesion formation. Whether the

CD40 pathway is relevant only to T cells or includes

effects on other atheroma-associated cells that may

express this molecule remains to be clarified.

MONOCYTES AND ACUTE CORONARY SYNDROMES

Not only have leukocytes been implicated in the grad-

ual augmentation of vascular pathology as described

above, but they may also play an important role in the

conversion of stable lesions to unstable, ruptured

plaque. This conversion is the hallmark of the acute

coronary syndromes such as unstable angina and acutemyocardial infarction. Acute coronary events are

thought to result from thrombosis triggered by dis-

ruption of atherosclerotic plaques. Histochemical analy-

sis of human atherectomy specimens suggests that

macrophage-rich areas are more frequently found in

patients with unstable angina and myocardial infarction

than in atherosclerotic tissue from patients with stable

angina. These findings suggest that macrophages are

markers of unstable atherosclerotic lesions. Monocytes

may play a significant role in the pathophysiology of

acute coronary syndromes, possibly by the release of

lytic enzymes that degrade the collagen skeleton of the

fibrous cap and subsequently expose the thrombogeniclipid core to the bloodstream. These observational stud-

ies, which support the role of macrophages in convert-

ing a stable, quiescent plaque to a ruptured, fissuring

lesion, must be validated in other models.

MOLECULAR SIGNALS RESPONSIBLE FOR MONONU-

CLEAR CELL RECRUITMENT: CHEMOATTRACTANT

CYTOKINES

The molecular signals that regulate the trafficking of

mononuclear leukocytes to sites of vascular injury such

as atherosclerotic lesions are unquestionably complex.

Much recent attention has been focused on the

chemoattractant cytokines, or chemokines, and theirrole in the development of atherosclerosis. This super-

family of leukocyte agonists now approaches 50 mem-

bers. Chemokines are relatively small secreted basic

proteins (8 to 10 kd) that are subdivided into four fam-

ilies based on the relative position of their cysteine

residues in the amino acid backbone (CC, CXC, C,

CXXXC). The CXC chemokine branch can be further

subdivided by structure and function into proteins that

contain the amino acid motif ELRCXC (Glu-Leu-Arg)

and those that do not have this ELR motif amino

terminal to CXC. It is important to note that structural

distinctions of the different branches of the super-

family have been shown to parallel general distinctions

in the biologic activities of chemokines. However, these

distinctions are not without exceptions. Most ELR

CXC chemokines are chemoattractants for neutrophils

but not monocytes or lymphocytes. Non-ELR CXC

chemokines are chemoattractants for lymphocytes but

not neutrophils or monocytes. CC chemokines gener-

ally attract monocytes and lymphocytes but not neu-

trophils.

88 Gerszten et alJ Lab Clin Med

August 2000



Chemokines induce cell activation by binding to

specific G-protein–coupled cell surface receptors on

target cells. Five human CXC chemokine receptors(CXCR1-5) and 9 human CC chemokine receptors

(CCR1-9) have been identified. Most receptors recog-

nize more than one chemokine, and several chemokines

can bind to more than one receptor. However, there is

receptor-ligand specificity within chemokine subfami-

lies, with α-chemokines binding exclusively to CXC

receptors and β-chemokines binding to CC receptors.

CHEMOKINES IN ATHEROSCLEROTIC LESIONS

Both in animal models and in human specimens,

chemokine expression is associated with atherosclerot-

ic lesion development (Fig 1). Expression of multiple

chemokines—including the CC chemokines MCP-1,

MCP-4, RANTES, pulmonary and activation regulated

chemokine, EBI1-ligand chemokine, and the EBI1-lig-

and chemokine–CXC chemokine IL-8—is increased in

human atheroma-associated cells as compared with

normal vessels (Table I). The expression of chemokines

in atherosclerotic lesions is highest in the area border-

ing the necrotic lipid core, near where the fibrous cap

has been shown to rupture in acute coronary syndromes.

In vitro and in vivo, chemokines are induced by a host

of stimuli associated with the atherosclerotic process,

including oxidized lipids, direct vascular injury, growth

factors such as platelet-derived growth factor, and

cytokines such as tumor necrosis factor-α, IL-1β, and

Gerszten et al 89J Lab Clin Med

Volume 136, Number 2

Table I. Chemokines and chemokine receptors

detected in human atherosclerotic lesions

In situ localization Reference

CC chemokines

MCP-1 Mφ, SMC, EC? 8,9,11

MCP-4 Mφ, EC 6

RANTES T cells, EC? 14

PARC Mφ 12

ELC Mφ, SMC 12

CXC chemokines

IL-8 Mφ 7,13

SDF-1 Mφ, SMC, EC 5

IP-10 Mφ, SMC, EC 10

MIG Mφ, EC 10

I-TAC Mφ, EC 10

PARC , Pulmonary and activation regulated chemokine; ELC ,

EBI1-ligand chemokine; MIG , monokine induced by IFN-γ ; I-TAC ,interferon-inducible T cell alpha chemoattractant.

Fig 1. Noxious stimuli, such as cholesterol, injure the vascular endothelium, inducing the elaboration of a num-

ber of chemokines. Endothelial cell elaborated chemokines trigger G-protein–coupled seven-transmembrane-

spanning chemokine receptors on circulating monocytes and memory T cells. Activated chemokine receptors

induce leukocyte adhesion to the endothelium and subsequent extravasation into the underlying developing

lesion. Leukocyte accumulation potentates the development of the atherosclerotic lesion and in some instances

hastens its transformation to an unstable lesion. Recent data suggest that specific chemokines (eg, SDF-1) may

also serve as novel platelet agonists expressed in atherosclerotic lesions.



IFN-γ . We have recently shown that the three

IFN-γ –inducible non-ELR CXC chemokines—IP-10,

monokine induced by IFN-γ , and interferon-inducible

T cell alpha chemoattractant—are highly expressed in

human atheroma as compared with normal vessels. We

have also verified the expression of their receptor,

CXCR3, on all T lymphocytes within human athero-

sclerotic lesions in situ. Thus these specific chemokinesmay partially mediate the potentiating effects of IFN-γ on the atherosclerotic process.

As seen in Table I, chemokine expression in athero-

sclerotic lesions is noted in endothelial cells, leuko-

cytes, and even stromal elements such as smooth

muscle cells. Furthermore, a number of chemokine

receptors are also expressed on these various cell types,

suggesting a complex interplay of proinflammatory sig-

nals. For example, evidence suggests that MCP-1 can

be expressed by monocytes, endothelial cells, and

smooth muscle cells, as can its target receptor CCR2.

Similarly, the CXC chemokine IP-10 is also expressed

by these three cell types, and in addition to its chemo-tactic activity on activated T cells, it is chemotactic for

smooth muscle cells and inhibits neovascularization

and wound healing in vivo. Therefore chemokines not

only may augment leukocyte recruitment but also may

regulate a number of vascular cell functions related to

the acute and chronic manifestations of the atheroscle-

rotic process.

Up-regulation of chemokines is also seen in other

forms of vascular pathology associated with the ather-

osclerotic process. The expression of MCP-1 and IP-

10, for example, is increased in the arterial wall in

response to balloon injury and may contribute to

the rest enotic process. Furthermore, MCP-1 is also

markedly up-regulated in vessels in a rat model that

recapitulates the accelerated vasculopathy seen in heart

transplant patients.

CHEMOKINES AS MARKERS OF CARDIOVASCULAR

DISEASE

In addition to analyzing atherosclerotic tissue for

chemokine expression, investigators have more recent-

ly begun to assess the potential prognostic implications

of serum chemokine levels. Both MCP-1 and IL-8 have

been observed to be elevated in patients with acute

coronary syndromes. However, no data are presently

available on chemokine levels in patients registered in

large epidemiologic cohorts. Elevated chemokine lev-

els are likely to be predictors of overall atheroscleroticburden and remain the subject of future investiga-

tion. Finally, elevated circulating levels of the C-C

chemokines MCP-1, MIP-1α, and RANTES were

recently reported in patients with congestive heart fail-

ure, and levels were inversely correlated with left ven-

tricular ejection fraction. Elevated chemokine levels

may solely reflect the cytokine milieu in “end-stage”

heart failure or in fact play a role in the pathogenesis

of this condition.

DIRECT EVIDENCE FOR CHEMOKINES IN MONOCYTE

RECRUITMENT AND LESION DEVELOPMENT

Although these correlational studies might be explainedby the presence of activated leukocytes and endothe-

lial cells in pathologic tissues, more recent investiga-

tion suggests a direct role for chemokines in the ath-

erosclerotic process. Using targeted gene deletion,

several groups have recently assessed the role of

chemokines or their receptors in atheroma formation.

Chemokine/chemokine receptor knockouts have been

bred with genetically modified mouse models with

increased susceptibility to atherosclerosis. Data from

these transgenic, hypercholesterolemic models show

that mice lacking the MCP-1 receptor or the MCP-1

ligand are less susceptible to atherosclerosis and have

fewer monocytes in vascular lesions. Furthermore, micelacking the IL-8 receptor are also less susceptible to

atherosclerosis and have fewer monocyte-rich lesions

as well. As seen in Table II, this line of investigation

has now been carried out in three different animal

models that mimic much of the pathology seen in

human atherosclerosis. These compelling experiments

confirm prior animal studies stressing the role of

leukocytes in lesion formation. Furthermore, these

data suggest that chemokines and their receptors are

critical mediators of the monocyte recruitment that

potentiates the atherosclerotic process. Whether mice

that are compound deficient for multiple chemokines

(or receptors) will be completely resistant to the ath-

eroslcerotic process remains a subject for future inves-

tigation.

MECHANISMS OF CHEMOKINE FUNCTION

In vitro, chemokines can induce impressive leuko-

cyte chemotaxis across synthetic membranes or

cultured cell monolayers. However, the molecular

mechanism by which chemokines modulate monocyte-


August 2000

Table II. Chemokines and chemokine repairs impli-

cated in the pathogenesis of murine models of

atherosclerosis

Targeted

gene

deletion Murine athero model Pathologic findings

CXCR2 LDL receptor knockout ≅50% ↓Mφ and lesionsCCR2 ApoE knockout ≅50% ↓Mφ and lesions

MCP-1 LDL receptor knockout ≅80% ↓Mφ and lesions

ApoB transgenic ≅70% ↓Mφ and lesions



endothelium interactions under flow conditions such as

those seen in the vasculature remains incompletely

defined. To investigate the mechanisms of monocyte

adhesion in a vascular model, we have used recombi-

nant adenoviruses encoding specific endothelial adhe-

sion molecules to transduce human endothelial cells.

We found that specific chemokines such as MCP-1 andIL-8 rapidly convert initial monocyte tethering (rolling)

on transduced monolayers to firm adhesion via the acti-

vation of leukocyte integrins. These data were the first

to show that chemokines could augment monocyte firm

adhesion under flow conditions and may speak to an

important role for chemokines in the initial step of

monocyte infiltration to the injured endothelium.

Chemokines may therefore play a role in “priming”

monocytes, markedly enhancing their interactions with

surface-expressed adhesion molecules. Interestingly,

this important biologic effect, converting low-affinity

selectin-mediated rolling to higher-affinity integrin-

mediated firm arrest, is not predicted by a givenchemokine’s effect in simpler, “surrogate” in vitro

assays, such as the measurement of calcium transients

or chemotaxis.

NOVEL ROLES FOR CHEMOKINES: PLATELETS

Not only do platelets play a well-recognized role in

hemostasis and acute thrombus formation, they are

also thought to have proinflammatory and growth-

regulatory properties that contribute to the progression

of atherosclerosis. Platelet activation releases multiple

growth factors and inflammatory mediators, including

chemokines, into the microenvironment. In fact,

the first chemokine described, platelet factor 4,was identified as a heparin-binding protein released

from activated platelets and has been used as an in vivo

marker of platelet activation. Although platelets con-

tain numerous other chemokines, previous work has not

focused on the platelet as a target for chemokines.

Because platelets are in contact with cells that pro-

duce chemokines, we have investigated the effect of

chemokines on platelet aggregation and found that

SDF-1, a CXC chemokine and a known chemotactic

factor for lymphocytes and monocytes, induced platelet

activation measured by aggregation and calcium flux.

In addition, we have found high levels of SDF-1 pro-

tein in human atherosclerotic plaques but not in normal

vessels (Fig 1). Although these data suggest a potential

role for SDF-1 both in the recruitment of inflammato-

ry cells and in the formation of acute thrombus after

plaque rupture, they merit future validation in vivo.

CHEMOKINES AS POTENTIAL TARGETS FOR FUTURE

INTERVENTION

The studies detailed above suggest that chemokines

and their receptors play a vital role in the trafficking of

mononuclear cells and pathogenesis of atherosclerosis.

Therefore, targeting this superfamily of molecules and

their receptors represents a possible future strategy for

treating this important human disease. Advances have

already been made in other disease states in which

chemokines have been shown to play key roles. Exper-iments in animal models of glomerulonephritis, for

example, have used a naturally occurring chemokine

receptor antagonist encoded by the Kaposi’s sarco-

ma–associated herpesvirus (human herpesvirus 8),

vMIP-2. Administration of vMIP2 ameliorates damage

seen in a murine model of crescentic nephritis. In ani-

mal models of rheumatoid arthritis, small peptide

antagonists of the MCP-1 receptor are presently being

investigated, because preliminary data suggest that they

diminish inflammation and lesion size. Finally, recent

reports cite the advances in the synthesis of large

combinatorial small molecule libraries, which are

already being screened for potential chemokine antag-onists. Although the bulk of the clinical work to date

has focused on kidney and rheumatologic diseases, the

increasing recognition of the inflammatory compo-

nent of atherogenesis will no doubt make it a focus

of this line of investigation in the near future.

CONCLUSION

Although the association between leukocytes and ath-

erosclerosis has long been recognized, only recently

have genetic models allowed for direct testing of the

role of inflammation in atherogenesis. Monocytes and

T cells are clearly important in the pathogenesis of this

disease. In vitro, chemokines are potent leukocyte acti-vators, and recent attention has been focused on the

mechanism by which they may promote mononuclear

leukocyte recruitment to atherosclerotic lesions in vivo.

They appear to convert low-affinity leukocyte-endothe-

lial interactions to higher-affinity interactions, leading

to firm arrest along the vessel wall and subsequent

extravasation into surrounding tissues. Identification of

chemokines as important vascular signals in mononu-

clear cell recruitment has provided insights into the cel-

lular and molecular mechanisms of atherogenesis. Most

importantly, it has identified new potential targets for

therapeutic intervention.

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August 2000

chemokine review

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