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REVIEW ARTICLE

Chemokines, leukocytes, and atherosclerosis


ROBERT E. GERSZTEN, FRANOIS MACH, ALAIN SAUTY, ANTHONY ROSENZWEIG, and ANDREW D. LUSTER
BOSTON, MASSACHUSETTS, and GENEVA, SWITZERLAND

Abbreviations: ApoE = apolipoprotein E; IFN = interferon; IL = interleukin; IP-10 = IFN-inducible protein 10; LDL = low-density lipoprotein; MCP = monocyte chemoattractant protein; MIP = macrophage inflammatory protein; RAG-1 = recombinase-activating gene-1; RANTES = regulated on activation normal T-cell expressed and secreted; SDF-1 = stroma-derived factor-1

he overwhelming body of evidence suggests that atherosclerosis is a multifactorial process involving the interplay of lipid metabolism, blood cells, coagulation proteins, cytokines, hemodynamic forces, extracellular matrix, and even behavioral risk factors. This process commences as early as childhood and clinically manifests itself later in life. Atherosclerosis is also increasingly viewed as an inflammatory disease of the vascular system. Immunohistochemical analysis of atherosclerotic lesions reveals a prominent leukocyte component. Of the leukocytes present in atheroma, approximately 80% are monocytes or monocytederived macrophages. Lymphocytes, on the other hand, constitute 5% to 20% of this cell population and are predominantly CD4+CD45RO+ (memory T cells). We will first review the evidence implicating monocytes and lymphocytes in the pathogenesis of atherosclerotic lesions. We will then explore the role of the chemoattractant cytokines, or chemokines, as important trafficking signals guiding mononuclear leukocytes

to sites of injury in the vasculature. A better understanding of these molecular signals will lay the foundation for the development of new clinical markers and therapeutics and help establish the timing of interventions.
MONOYCTES, LYMPHOCYTES, AND THE DEVELOPMENT OF ATHEROSCLEROTIC LESIONS

From the Cardiology and Infectious Disease Divisions, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston; and the Cardiology Division, Foundation for Medical Research, University Hospital, Geneva. Submitted for publication November 15, 1999; revision submitted March 4, 2000; accepted March 13, 2000. Reprint requests: Andrew D. Luster, MD, PhD, Infectious Disease Unit, AIDS Research Center, Building 149 13th St, Charlestown, MA 02119. J Lab Clin Med 2000;136:87-92. Copyright 2000 by Mosby, Inc. 0022-2143/2000 $12.00 + 0 5/1/108154

The association between leukocytes and atherosclerotic lesions, both in animal models and in human beings, has long been recognized. However, only recently have investigators extended these observational studies to explore direct causal relationships between leukocytes and atherosclerotic lesion formation. The development of murine models with hyperlipidemia and a propensity to develop atherosclerosis-like lesions, such as LDL receptor or ApoE knockout mice, has been crucial to this line of investigation. These mice develop extensive fatty streaks throughout their aorta and recapitulate much of the phenotype seen in human patients with atherosclerotic disease. Investigators have targeted inflammatory cells or signals by disrupting genes important in leukocyte development and function, then breeding these mice onto strains prone to atherosclerosis. A causal role for monocytes in atherogenesis was first explored in such models. To develop a murine model system to test the role of monocyte-derived macrophages in atherosclerosis, the osteopetrotic (op) mutation in the macrophage colony-stimulating factor gene was bred onto the ApoE-deficient background. The op/ApoE compound deficient mice have decreased blood peripheral blood monocytes and have smaller
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proximal aortic lesions than their control littermates. Similarly, targeted disruption of the macrophage scavenger 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 atherosclerotic lesions in animals deficient in ApoE. Powerful data from this line of investigaton in mouse models therefore suggests a key role for monoyctes in lesion formation. The role of T cells in the pathogenesis of atherosclerosis 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 deficiency on atherogenesis has been investigated by crossing ApoE-deficient mice with mice deficient in RAG-1, a gene essential for normal lymphocyte development. 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 RAG2deficient mice (a mouse that shares a lymphocytedepleted phenotype similar to that of the RAG-1 knockout) are less striking. Although the latter results may be explained by differences in genetic background, diet, or technical differences in lesion assessment, taken together these results suggest a less robust effect of lymphocyte depletion on atherogenesis as compared with monocyte depletion. Further data addressing potential causal interactions between leukocytes and the formation of atherosclerotic lesions have been generated by studies of the immune mediator CD40 and its ligand CD40L (also known as CD154 or gp39). Several groups have recently 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 metalloproteinases, adhesion molecules, and tissue factor, components previously implicated in atherogenesis. Inhibition of CD40 signaling either by treatment with antibody against CD40L or by using compound mutant mouse (ApoE and CD40L null mice) showed a reduction 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 gradual 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 acute myocardial infarction. Acute coronary events are thought to result from thrombosis triggered by disruption of atherosclerotic plaques. Histochemical analysis 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 thrombogenic lipid core to the bloodstream. These observational studies, which support the role of macrophages in converting a stable, quiescent plaque to a ruptured, fissuring lesion, must be validated in other models.
MOLECULAR SIGNALS RESPONSIBLE FOR MONONUCLEAR 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 their role in the development of atherosclerosis. This superfamily of leukocyte agonists now approaches 50 members. Chemokines are relatively small secreted basic proteins (8 to 10 kd) that are subdivided into four families 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 superfamily 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 generally attract monocytes and lymphocytes but not neutrophils.

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Fig 1. Noxious stimuli, such as cholesterol, injure the vascular endothelium, inducing the elaboration of a number of chemokines. Endothelial cell elaborated chemokines trigger G-proteincoupled seven-transmembranespanning 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.

Chemokines induce cell activation by binding to specific G-proteincoupled 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 recognize more than one chemokine, and several chemokines can bind to more than one receptor. However, there is receptor-ligand specificity within chemokine subfamilies, with -chemokines binding exclusively to CXC receptors and -chemokines binding to CC receptors.
CHEMOKINES IN ATHEROSCLEROTIC LESIONS

Table I. Chemokines and chemokine receptors detected in human atherosclerotic lesions


In situ localization Reference

CC chemokines MCP-1 MCP-4 RANTES PARC ELC CXC chemokines IL-8 SDF-1 IP-10 MIG I-TAC

M, SMC, EC? M, EC T cells, EC? M M, SMC M M, M, M, M,

8,9,11 6 14 12 12 7,13 5 10 10 10

Both in animal models and in human specimens, chemokine expression is associated with atherosclerotic lesion development (Fig 1). Expression of multiple chemokinesincluding the CC chemokines MCP-1, MCP-4, RANTES, pulmonary and activation regulated chemokine, EBI1-ligand chemokine, and the EBI1-ligand chemokineCXC chemokine IL-8is 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 bordering the necrotic lipid core, near where the fibrous cap has been shown to rupture in acute coronary syndromes.

SMC, EC SMC, EC EC EC

PARC, Pulmonary and activation regulated chemokine; ELC, EBI1-ligand chemokine; MIG, monokine induced by IFN-; I-TAC, interferon-inducible T cell alpha chemoattractant.

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-, IL-1, and

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Table II. Chemokines and chemokine repairs implicated in the pathogenesis of murine models of atherosclerosis
Targeted gene deletion

Murine athero model

Pathologic findings

CXCR2 CCR2 MCP-1

LDL receptor knockout ApoE knockout LDL receptor knockout ApoB transgenic

50% 50% 80% 70%

M M M M

and and and and

lesions lesions lesions lesions

IFN- . We have recently shown that the three IFN- inducible non-ELR CXC chemokinesIP-10, monokine induced by IFN-, and interferon-inducible T cell alpha chemoattractantare 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 atherosclerotic lesions in situ. Thus these specific chemokines may partially mediate the potentiating effects of IFN- on the atherosclerotic process. As seen in Table I, chemokine expression in atherosclerotic lesions is noted in endothelial cells, leukocytes, 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 signals. 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 chemotactic 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 atherosclerotic process. Up-regulation of chemokines is also seen in other forms of vascular pathology associated with the atherosclerotic process. The expression of MCP-1 and IP10, for example, is increased in the arterial wall in response to balloon injury and may contribute to the restenotic 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

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 levels are likely to be predictors of overall atherosclerotic burden and remain the subject of future investigation. Finally, elevated circulating levels of the C-C chemokines MCP-1, MIP-1 , and RANTES were recently reported in patients with congestive heart failure, and levels were inversely correlated with left ventricular 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 explained by the presence of activated leukocytes and endothelial cells in pathologic tissues, more recent investigation suggests a direct role for chemokines in the atherosclerotic 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, mice lacking 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 atheroslcerotic process remains a subject for future investigation.
MECHANISMS OF CHEMOKINE FUNCTION

In addition to analyzing atherosclerotic tissue for chemokine expression, investigators have more recent-

In vitro, chemokines can induce impressive leukocyte chemotaxis across synthetic membranes or cultured cell monolayers. However, the molecular mechanism by which chemokines modulate monocyte-

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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 recombinant adenoviruses encoding specific endothelial adhesion molecules to transduce human endothelial cells. We found that specific chemokines such as MCP-1 and IL-8 rapidly convert initial monocyte tethering (rolling) on transduced monolayers to firm adhesion via the activation 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 integrinmediated firm arrest, is not predicted by a given chemokines effect in simpler, surrogate in vitro assays, such as the measurement of calcium transients or chemotaxis.
NOVEL ROLES FOR CHEMOKINES: PLATELETS

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. Experiments in animal models of glomerulonephritis, for example, have used a naturally occurring chemokine receptor antagonist encoded by the Kaposis sarcomaassociated herpesvirus (human herpesvirus 8), vMIP-2. Administration of vMIP2 ameliorates damage seen in a murine model of crescentic nephritis. In animal 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 antagonists. Although the bulk of the clinical work to date has focused on kidney and rheumatologic diseases, the increasing recognition of the inflammatory component of atherogenesis will no doubt make it a focus of this line of investigation in the near future.
CONCLUSION

Not only do platelets play a well-recognized role in hemostasis and acute thrombus formation, they are also thought to have proinflammatory and growthregulatory 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 contain numerous other chemokines, previous work has not focused on the platelet as a target for chemokines. Because platelets are in contact with cells that produce 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 protein 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 inflammatory cells and in the formation of acute thrombus after plaque rupture, they merit future validation in vivo.
CHEMOKINES AS POTENTIAL TARGETS FOR FUTURE INTERVENTION

Although the association between leukocytes and atherosclerosis 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 activators, 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-endothelial 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 mononuclear cell recruitment has provided insights into the cellular and molecular mechanisms of atherogenesis. Most importantly, it has identified new potential targets for therapeutic intervention.

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