Immunological aspects of atherosclerosis

Clinical Science (2013) 125, 221–235 (Printed in Great Britain) doi: 10.1042/CS20120576

Immunological aspects of atherosclerosisKevin J. WOOLLARD

Division of Immunology & Inflammation, Department of Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W120NN, U.K.

AbstractCardiovascular disease is the leading cause of death in several countries. The underlying process isatherosclerosis, a slowly progressing chronic disorder that can lead to intravascular thrombosis. There isoverwhelming evidence for the underlying importance of our immune system in atherosclerosis. Monocytes, whichcomprise part of the innate immune system, can be recruited to inflamed endothelium and this recruitment hasbeen shown to be proportional to the extent of atherosclerotic disease. Monocytes undergo migration into thevasculature, they differentiate into macrophage phenotypes, which are highly phagocytic and can scavenge modifiedlipids, leading to foam cell formation and development of the lipid-rich atheroma core. This increased influx leads toa highly inflammatory environment and along with other immune cells can increase the risk in the development ofthe unstable atherosclerotic plaque phenotype. The present review provides an overview and description of theimmunological aspect of innate and adaptive immune cell subsets in atherosclerosis, by defining their interactionwith the vascular environment, modified lipids and other cellular exchanges. There is a particular focus onmonocytes and macrophages, but shorter descriptions of dendritic cells, lymphocyte populations, neutrophils, mastcells and platelets are also included.

Key words: atherosclerosis, immunology, leucocyte biology, monocyte, macrophage

INTRODUCTION

Cardiovascular disease is the leading cause of death in manycountries, resulting in approximately 16–17 million deaths eachyear [1,2]. The underlying pathological process is atherosclerosis,a slowly progressing chronic disorder of large and medium-sizedarteries that can lead to intravascular thrombosis [3]. The patho-logy is characterized by a chronic inflammatory process at the ar-terial wall, occurring at predilection sites with disturbed laminarflow [4]. It can be initiated by endothelial dysfunction and struc-tural alterations, including the absence of a confluent luminalelastin layer and the exposure of proteoglycans, which permitsubendothelial accumulation of LDLs (low-density lipoproteins)[5,6]. Interactions of modified LDLs within the luminal endothe-lium, as well as with resident cells located in the subendothelialspace, initiate the formation of chemotactic gradients that attractfurther leucocytes (e.g. monocytes, neutrophils, lymphocytes andpossibly circulating stem cells) into the arterial wall from theluminal blood [5,7,8]. A complex cascade of mechanisms, in-cluding the recruitment and migration of leucocytes through the

Abbreviations: ABC, ATP-binding cassette; APC, antigen-presenting cell; ApoE, apolipoprotein E; CCL, CC chemokine ligand; CCR, CC chemokine receptor; DC, dendritic cell; GM-CSF,granulocyte-macrophage colony-stimulating factor; HDL, high-density lipoprotein; HSC, haemopoietic stem cell; HSPC, haemopoietic stem cell progenitor; IFNγ , interferon γ ; IL,interleukin; iNOS, inducible nitric oxide synthase; LDL, low-density lipoprotein; LDLR, LDL receptor; LPS, lipopolysaccharide; MI, myocardial infarction; NET, neutrophil extracellular trap;NK, natural killer; NKT, natural killer T; oxLDL, oxidized LDL; PLTP, phospholipid transfer protein; PSGL-1, P-selectin glycoprotein ligand-1; TGF, transforming growth factor; Th, T-helper;TLR, Toll-like receptor; TNF, tumour necrosis factor; Treg, T-regulatory.

Correspondence: Dr Kevin Woolard (email [email protected]).

activated endothelium in predisposed regions of the intima, aswell as receptor-dependent accumulation of lipids within intimalcells, leads to the formation of foam cells (Figure 1). This cul-minates in the appearance of fatty streaks in the arteries, alteredintima and formation of a lipid core [5,6,9]. The atheroscleroticplaque develops, in which the lipid necrotic core becomes sep-arated from the arterial lumen by a fibrous cap. The plaque thenprogresses into a complicated lesion with a lipid necrotic coreat the center and large numbers of leucocytes at the shouldersof the lesion, including neovascularization of capillaries that mayoriginate from the adventitial vasa vasorum [5]. As the plaquematures, there is an increased possibility of breakdown, which isaccompanied by the formation of thrombi on the plaque’s dam-aged surface [10]. Consequently as the damage occurs, there isan increased risk of thrombi breaking off and travelling to thelung, heart or brain [10].

In the early 19th century, the term arteriosclerosis was in-troduced by Jean Lobstein, and the emergence that inflamma-tion plays a role in atherosclerosis dates to original observationsby Virchow and von Rokitansky [11,12]. In the 1970s, Ross

www.clinsci.org 221

Clin

ical

Sci

ence

ww

w.c

linsc

i.org

K. J. Woollard

Figure 1 Summary of plaque milieuMonocytes (mainly Gr1high/CD14+ ) are recruited into the intima and may differentiate into heterogeneous macrophagesthat accumulate haemolysed debris or cholesterol lipids and form foam cells in the sub-intima. Whether they differentiateinto specific ‘M1-like’ or ‘M2-like’ macrophages from specific recruited monocyte subsets and/or proliferate is unknown.Monocytes may also give rise to DCs in the plaque. Use of vasa vasorum and neovascularization within the plaque leadsto increased trafficking of leucocytes. Egress (cell efflux) of leucocytes from plaques has been observed. Deposition ofmatrix components and recruitment of smooth muscle cells give rise to the fibroproliferative progression of the plaques.Apoptosis of macrophages/foam cells creates a necrotic core. Thinning and erosion of the fibrous cap in unstable plaquesthrough matrix degradation by proteases ultimately results in plaque rupture and platelet thrombus. Further recruitmentof lymphocyte subsets, platelets, neutrophils and other inflammatory leucocytes may help development of the vulnerableplaque. Circulating lipids (LDL) can become modified and potentially bind autoantibodies to oxLDL, which may also occurin the plaque milieu.

222 C© The Authors Journal compilation C© 2013 Biochemical Society

Immunological aspects of atherosclerosis

demonstrated that leucocyte adhesion to the endothelial surfacewas an early feature of atherosclerosis [13]. These observations,along with knowledge that modified LDL contributes to foamcell formation and atherosclerotic disease activity, focused earlyreports on monocytes and macrophages, major players in innateimmunity, which express functional scavenger receptors [14,15].Scavenger receptors recognize modification-specific epitopes of(among others) SRA-1 and SRA-2, SR-B1, LOX-1, MARCO(macrophage receptor with collagenous structure), CD36 andPSOX21 [16]. Although these receptors serve a vital role as medi-ators of intracellular cholesterol accumulation, their detailed im-portance in atherosclerosis remains unclear, and gene-knockoutstudies of hypercholesterolaemic mice have provided contradict-ory results [15,16]. In 1973, Steinman and Cohn [17] provided anew scientific specialty with their discovery of the DC (dendriticcell), a vital antigen-presenting cell in the immune system. Sincethen, DCs have proved to be critical sentinels for atherosclerosis[18].

Components of adaptive immunity are also present in hu-man atherosclerotic lesions, and several studies have indicateda central role for antigen-specific adaptive immune responses inatherogenesis [19]. Studies using mouse experimental models ofatherosclerosis, such as ApoE (apolipoprotein E)− / − or LDLR(LDL receptor) − / − mice, in combination with mice deficientin both B-cells and T-cells, have demonstrated a key role foradaptive immunity in atherosclerosis [19]. Lymphocyte-deficientanimals, for example, lacking recombination-activating gene-1 or-2, or mice with severe combined immunodeficiency under ather-osclerotic settings, have been shown to aggregate atherosclerosisin some cases and inhibit it in others, discussed in [20].

Activated endothelium is characterized by adhesion moleculeexpression and reduced barrier function that can mediate the re-cruitment of leucocytes into atherosclerotic-prone sites in the ar-terial wall [21]. These include expression of adhesion moleculesat sites of turbulent flow that enable recruitment of inflammatorycells [4]. Interestingly, in vivo reports using intravital microscopyindicate that neutrophils are responsible for the majority of transi-ent interactions between leucocytes and endothelial cells coveringatherosclerotic lesions [22,23]. Along with the detection of leu-cocytes in atherosclerotic lesions, a crucial question is how thesecells enter atherosclerotic plaques. Observations from intravitalfluorescence microscopy showing adhesion of neutrophils to ath-erosclerotic lesions and subsequent migration suggest a translu-minal route [24]. However, further work indicates that monocytesmay infiltrate large arteries via adventitial or intimal microvessels[25]. There are relatively fewer studies investigating molecularmechanisms of in vivo arterial leucocyte infiltration, so that thevast majority of in vivo concepts of recruitment in atherosclerosisare extrapolated from microvascular recruitment models, whichpoints towards the need for development of cellular resolutiontechniques to investigate in vivo leucocyte recruitment relateddirectly to atherosclerosis.

Altogether there is overwhelming evidence for the underlyingimportance of immunology in atherosclerosis. A previous re-view has provided a concise understanding of the immunologicalroles of immune cell susbets in atherosclerosis, their recruitmentand metabolic functions [26]. The present review provides an

overview and description of the immune cell response in ath-erosclerosis, specifically monocytes and macrophages, but alsoneutrophils, lymphocytes, mast cells and platelets, by definingtheir response to the vascular environment, modified lipids andother cellular interactions.

MONOCYTES, MACROPHAGESAND DENDRITIC CELLS

MonocytesDuring inflammation, blood monocytes can migrate from bloodto lymphoid and non-lymphoid tissues in response to tissue-derived signals caused, for example, by infection or tissue dam-age [27]. They can phagocytose other cells and toxic molecules[such as oxLDL (oxidized LDL)], produce inflammatory cy-tokines, and can differentiate into inflammatory dentritic cells,macrophages or ultimately foam cells (directly relevant to ath-erogenesis) [28,29]. During atherogenesis, blood monocytes arerecruited into the intima and subintima [30,31]. Upon encoun-tering fatty deposits, via scavenger or other lipid receptors, thesecells can take up modified LDLs (and other lipid species), result-ing in activation, macrophage polarization and accumulation inthe forming lesion [16]. At an early stage of the process, mono-cytes differentiate into foam cells to form early plaques, termedfatty streaks, in the intima [32] (Figure 1). Interestingly, this pro-cess occurs in atherosclerosis-prone areas of the arterial tree andis likely to involve cellular responses to changes in fluid dynamics[4].

Depletion of monocytes in experimental mouse models de-creases the development of atherosclerosis, but wholesale abla-tion of monocytes and macrophages is not a viable therapeuticoption, because of their essential role in immunity [33]. Studieshave documented heterogeneity among human monocytes [34],but the discovery and characterization of monocyte subsets in themouse progressed investigation into the relevance of monocyteheterogeneity in atherosclerosis [35,36].

A number of studies have used histology to identify the cel-lular components of plaques, but adoptive transfer and fate-mapping strategies to study monocyte recruitment (and egress)from plaques, and laser microdissection to directly investigate thefunctions of plaque macrophages, have been reported [37–39].However, the relationship between blood monocytes and plaquemacrophages is an unresolved issue that is difficult to addressdue to their plasticity and swift response to their tissue microen-vironment [40].

Morphologically, monocytes are larger than lymphocytes andcontain a horseshoe or kidney-like nucleus. They are phagocyticand can develop into macrophages and DCs in vitro and in vivo.In humans and mice, at least two monocyte subsets have beendescribed. In humans they are usually classified according toCD14 and CD16 expression levels (see extended discussion be-low). In mice, monocytes can be defined by their expression ofLy6c, which is an epitope of Gr-1. Ly6chigh (Gr-1+ ) monocytesare CCR (CC chemokine receptor) 2high CX3CR1low, CD62L+ ,whereas Ly6clow (Gr-1− ) monocytes are CCR2low, CX3CR1high

and CD62L− [34,40].

www.clinsci.org 223

K. J. Woollard

Including recent nomencaulture labelling of M1/M2 macro-phages (see the Macrophages section below), originally, Ly6chigh

monocytes were called ‘inflammatory’ as they accumulate in theperitoneum in response to inflammatory signals. Ly6clow mono-cytes are called ‘resident’ because of their accumulation in repor-ted tissues in the steady state, or termed ‘patrolling’ because oftheir ability to crawl along the endothelial interface in the steadystate [41,42]. Others have proposed to call Ly6chigh monocytes,in orthology to human monocytes, ‘classical’ and Ly6clow mono-cytes ‘non-classical’, a nomenclature that, perhaps convenientfor some, offers no functional insight and most likely just addsconfusion [43].

In the steady state, the proportion of circulating Ly6chigh andLy6clow monocytes is similar [41]. In murine models of ath-erosclerosis, mice deficient in ApoE develop large and com-plex lesions when fed a high-fat diet. Hypercholesterolaemiacan induce monocytosis in the bone marrow, blood and spleen[44,45]. Ly6chigh cells increase in number preferentially (whetherthere is an intermediate functional subset in mice requires fur-ther investigation). Recent work has shown that hypercholes-terolaemia induces proliferation of HSPCs [HSC (haemopoieticstem cell) progenitors] involving cholesterol efflux via ApoE andABC (ATP-binding cassette) (ABCA1 and ABCG1) transporters[46]. HSPCs secrete ApoE that can bind to proteoglycans on thecell surface and mediate cholesterol efflux using ABCA1 andABCG1 linking directly to HDLs (high-density lipoproteins).ApoE deficiency impairs cholesterol efflux, increases membranecholesterol content as well as the surface expression of theGM-CSF (granulocyte-macrophage colony-stimulating factor)receptor, leading to accelerated atherosclerosis [46,47]. Ly6chigh

monocytes have been reported to accumulate in the growing ath-eroma preferentially via CCR2 and CX3CR1 [44,48,49]. Ly6clow

monocytes have been reported to infiltrate atherosclerotic lesions(less frequently) via CCR5, but subset-specific differences inatherosclerotic recruitment still require further studies [50]. Thebone marrow contains specialized haemopoietic niches; however,monocyte subsets can also be found in the spleen as part of a reser-voir that can be mobilized in response to systemic inflammatorystimuli such as from MI (myocardial infarction) and recentlyshown to be crucial in accelerated atherosclerosis post-MI [51].

Human peripheral blood monocytes do show heterogeneitysimilar to the existence of subsets in mice [34,41,52]. Hu-man monocytes were identified by their expression of CD14.Subsequent identification of differentially expressed antigenicmarkers further identified CD16 [53]. Similar to mouse peri-pheral blood, at least two principle subsets can be defined.CD14highCD16low monocytes typically represent ∼85–95 % ofthe monocytes in healthy individuals against CD14lowCD16high

monocytes (referenced as CD14dim). These subsets, like thosein mice, differ in many respects, including their expres-sion of adhesion molecules and chemokine receptors [54–56].CD14highCD16low monocytes express (among others) CCR2,CD62L and CD64 and have consequently been associated withthe inflammatory subset in the mouse. CD14dim monocytes lackCCR2 and have higher levels of major histocompatibility com-plex II and CD32. Both subsets express the fractalkine receptor,CX3CR1, but CD14dim monocytes express much higher levels. It

is, however, important to note that there is considerable heterogen-eity in the CD16+ population, with a minor population express-ing both CD14 and CD16 [56]. Therefore in human blood it wouldseem there are three key populations of circulating monocytes:CD14high(CD16low), CD14highCD16high and CD14dim(CD16high).

By performing principal component analysis and by compar-ing the three subsets to each other and to murine subsets, wehave concluded that CD14high and CD14highCD16high monocytescluster with the murine Ly6chigh monocytes, whereas CD14dim

monocytes cluster with Ly6clow monocytes [57]. That study con-tested the importance of using CD16 as a discrimination markerand may suggest using CD14 as an improved marker for de-scribing human monocyte subsets [58]; however, a consensus ofmonocyte subset referencing is needed.

Regardless of nomenclature, human blood monocyte popula-tions describe subset-specific effector functions. In response toLPS (lipopolysaccharide) CD14highCD16high, monocytes secreteTNF (tumour necrosis factor) α, IL (interleukin)-1β and IL-6, whereas CD14highCD16low cells preferentially produce CCL(CC chemokine ligand) 2, IL-10, IL-8, reactive oxygen spe-cies and high levels of IL-6. CD14dim monocytes, which ex-press low levels of CCR2, but higher levels of CX3CR1 exhibitpatrolling behaviour in vivo and resemble murine Ly6clow cells.Interestingly, stimulation of CD14dim monocytes with TLR (Toll-like receptor) 7 and TLR8 agonists selectively up-regulated cy-tokine expression, through specific signalling pathways involvingMEK1 (mitogen-activated kinase/extracellular-signal-regulatedkinase kinase 1) [57]. Overall these observations argue against de-scribing monocyte subsets by phenotype or nomenclature ratherthan function, as each monocyte subset responds specifically toits environment in vivo and in vitro. The description of subset-specific functions opens a plethora of questions regarding rolesin atherosclerosis; for example, do patrolling CD14dim mono-cytes mediate distinct functions in atherosclerosis? Recent ima-ging studies do show Ly6clow monocytes directly interacting withLDL in atherosclerotic plaques [59]. Most importantly; is there asubset-specific fate of blood monocytes in atherosclerosis? Thesequestions will be explored as the area of monocyte heterogeneityexpands.

Specific changes in blood monocyte numbers could be a use-ful clinical biomarker in cardiovascular disease. Monocytosishas been found to predict cardiovascular events in some stud-ies [60–63], but not in others [64,65]. Positive correlation ofCD14dim cells with plasma cholesterol and triacylglycerol levelswas shown in hypercholesterolaemic patients with coronary heartdisease [66]. Circulating numbers of CD16high monocytes seemto correlate with body mass index, insulin resistance (diabetes)and intima-media thickness [67]. Weight loss after gastric bypasssurgery in morbidly obese patients is associated with a significantreduction of CD16high cells [67]. Interestingly CD14highCD16high

monocytes, but not total monocyte counts, predict cardiovascu-lar events in patients with chronic kidney disease and end-stagerenal disease on dialysis, a patient population at increased riskof atherosclerotic complications [68,69]. Additionally, in patientswith symptomatic coronary artery disease compared with healthycontrols, the percentage of CD16high monocytes was increased[70]. Treatment therapies with statins decreased the percentage



of CD16high monocytes [71,72]. Finally, CD16high monocytosisafter stent placement in patients with MI positively correlatedwith restenosis [73]. In these examples (not all of them havebeen described) it would appear CD16high monocytes, in par-ticular CD14highCD16high, correlate with inflammatory disease.Whether this subset is indeed a blood biomarker that can be usedfor clinical diagnosis, will require careful analysis.

Monocytes as a drug target in atherosclerosisInterest has focused on targeting Ly6chigh monocytes, which areregarded as the primary inflammatory monocytes; indeed, tar-geting functionally distinct monocyte subsets may provide at-tractive atherosclerotic targets without affecting immunologicalsurveillance and innate immunity [41]. Leucocytes, includingsome monocytes, accumulate to sites of vascular inflamma-tion through a well-described series of distinct adhesion steps,which can be targeted [74]. Initially, chemokines attract mono-cytes via chemokine receptors. Antagonizing the chemokine re-ceptor CCR2 via nanoparticle-mediated transfer of short inter-fering RNA decreases Ly6chigh monocytes in peripheral tissuesand reduces inflammation associated with atherosclerosis [75].Selectins and their ligands can then mediate leucocyte captureand rolling, a crucial step to be blocked by antibodies [21].PSGL-1 (P-selectin glycoprotein ligand-1) on leucocytes inter-acts with E- and P-selectins on activated endothelium and plate-lets (P-selectin) [76]. It is worth noting that Ly6chigh monocytesexpress PSGL-1 at higher levels compared with Ly6clow mono-cytes, providing a hypothesis for specific accumulation in thegrowing lesion [34,41]. A soluble version of the PSGL-1 lig-and (soluble P-selctin), has also been reported to be raised inplasma from vascular disease patients, which may be involvedwith leucocyte activation, recruitment and thrombotic events[77,78]. This complicates the idea of blocking either PSGL-1or the membrane-bound receptor P-selectin [76]. Pharmaceut-ically inhibiting Mac-1:CD40L, VLA-4:VCAM-1 (vascular celladhesion molecule 1) or LFA-1:ICAM-1 (intercellular adhesionmolecule 1) reduces monocyte adhesion and macrophage lesionnumber [79,80]. However, this concept of a single sequenceof events leading to recruitment, firm adhesion and migrationof monocytes is currently restricted to Ly6chigh monocytes (hu-man CD14high), as the mechanisms for patrolling Ly6clow (humanCD14dim) endothelial interactions have yet to be fully explored,but do involve LFA-1 integrin [42,57].

MacrophagesAlong with monocytes, macrophages are phenotypically andmorphologically heterogeneous, a contributing factor to the de-bate on how to define them [81]. Similar to monocyte hetero-geneity, macrophage populations participate in many vital biolo-gical processes. From the perspective of monocyte fate mapping,one must ask whether specific subsets are restricted to give riseto specific macrophage populations with defined functions, orwhether the subset differences vanish once monocytes accumu-late in tissue. Functionally distinct human macrophages can bederived from monocytes in vitro. Briefly, classical macrophageactivation, which involves culture of cells with GM-CSF, LPSor IFNγ (interferon γ ) yields M1 named macrophages which

are TNFα, IL-1β, IL-6, IL-12 and iNOS (inducible nitric ox-ide synthase) producers [52]. Alternative activation involves cul-ture with IL-4, IL-10 or IL-13 and generates M2 named mac-rophages which are defined by their high expression of anti-inflammatory arginase 1, IL-10 and CD206 (see further de-scription below) [52]. A major limitation to studying monocyteand macrophage biology is a lack of homology between spe-cies; human cells lack F4/80 and Ly6c antigens and, althoughM1/M2 definitions are convenient for in vitro studies, shouldbe used with caution in the in vivo setting. In the steady state,most organs contain their own particular macrophages, many ofwhich may not derive from blood monocytes [34,81–83] andhave specific gene signatures which separate them from eachother and other myeloid populations [84]. Precisely how mono-cyte subsets contribute to macrophage and DC populations in thesteady state or pathological settings is largely unknown. Thereare descriptions of Ly6chigh monocytes that extravasate into tis-sue, differentiate into TNF- and iNOS-producing DCs (Tip-DC),M1-like (classically activated) macrophages, and phagocytosepathogens, produce antibacterial products, and mediate inflam-mation and proteolysis [52]. Likewise, Ly6clow cells in responseto inflammation can extravasate into tissue and have beenshown to differentiate into M2-like macrophages, involved inwound repair, tissue remodelling and expression of chemokines[56,85].

In relation to atherosclerosis, whether both Ly6chigh andLy6clow differentiated monocytes specifically mediate macro-phage foam cell formation in response to lipids requires in-vestigation. Simplistically, human macrophages in atheroscler-otic lesions can be divided into two types, M1 and M2, that ex-press (for example) MCP-1 (monocyte chemoattractant protein1) and mannose receptor respectively [86]. As discussed above,M1 macrophages require IFNγ for initial activation and LPS,CpG or TNF and IL-1 for secondary activation [85,87,88]. M1macrophages can release IL-6 and TNF and express high MHC IIand CD80/86 to direct Th (T-helper) 1 activation [89]. In reportedopposition to M1 macrophages, M2 macrophages are thought tobe anti-inflammatory cells that are critical in the resolution of in-jury [52,87,90]. Furthermore M2 macrophages have been furthersubdivided into three groups: M2a, M2b and M2c [87]. M2a arenon-cytotoxic cells activated by IL-4 and IL-13 and participate intissue repair and extracellular matrix deposition [85]. Arginaseexpression is enhanced in M2a activated mouse (not human) mac-rophages, and in contrast with M1 activated macrophages, the ex-pression of iNOS and production of nitric oxide is reduced [87].M2B-cells are activated by LPS and release anti-inflammatory IL-10, as well as inflammatory cytokines [90,91]. IL-10, TGF (trans-forming growth factor) and glucocorticoids have been shown toactivate M2c cells, which produce high levels of IL-10. There-fore M2b and M2c cells have been described as regulatorymacrophages due to their high secretion of IL-10 [87,92]. Again itmust be repeated that these classifications are based on in vitro ac-tivation and thus would most likely not apply to in vivo functionalbiology. Finally, the haptoglobin–haemoglobin scavenger re-ceptor CD163 has also been associated with an anti-inflammatorymacrophage. In separate studies, macrophages within intimallesions showed strong positivity for CD163, whereas foamy

www.clinsci.org 225

K. J. Woollard

plaque macrophages were CD163low [93]. Distinct populationsof CD163+ macrophages have also been identified in haemor-rhaged atherosclerotic plaques, which were DRlow and were un-like classical lipid necrotic core macrophages [94]. These types ofmacrophages are thought to suppress the impact of haemorrhageon atherosclerotic progression [95].

Overall, macrophages are a key feature of all stages of athero-genesis where they have a significant impact on lesion progression(Figure 1). Fundamentally, as part of their innate immune role,macrophages infiltrate developing lesions and respond to spe-cific TLR ligands and can phagocytose modified LDL [9]. Thiscan result in the secretion of cytokines, chemokines and toxicoxygen and nitrogen radicals that not only direct and amplifythe local immune response, but may lead to localized tissue in-jury. Ultimately macrophages may cause plaque destabilization,rupture and thrombosis, highlighting their destructive role [96].However, as macrophages are heterogeneous, they could developfunctions that facilitate atherosclerotic tissue repair, remodellingand restoration of normal tissue homoeostasis [85]. As it has beendifficult to distinguish the specific activation phenotypes at thesingle cell level in vivo, the extent of macrophage heterogeneityand polarization signals in atherosclerotic plaques still requirecareful investigation.

Macrophages are thought to originate from HSCs, butsome macrophages develop in the embryo before the ap-pearance of definitive HSCs, these yolk-sac-derived macro-phages populate several tissues and depend on specific tran-scription factors for their development and survival [81,97,98].Recently Schulz et al. [82], using fate mapping and con-ditional knockout approaches, supported the concept oftwo lineages of macrophages in mice: (i) derived fromthe bone marrow, phenotyping F4/80lowCD11bhi macrophages(and DCs); and (ii) derived from the yolk sac, pheno-typing F4/80hi macrophages. The fate and functions of theseyolk-sac tissue-resident macrophages in the adult, such as in ath-erosclerotic aortic tissue, remain to be investigated.

Dendritic cellsAntigen-presenting DCs are an essential part of the immune sys-tem because they provide sentinels of tissues for foreign antigens(and danger signals), including delivering of antigen to localizedpopulations of lymphocytes and co-ordinating the immune re-sponse [99–101]. Both the adaptive and the innate immune sys-tems require DC participation [101]. DCs act as professionalAPCs (antigen-presenting cells) that recognize foreign antigensand display them bound to MHC molecules on their surface[100,102,103]. DCs isolated from the mouse aorta are just aseffective at presenting foreign substances and stimulating killercells as those in other immune organs, providing evidence thatDCs in the arterial wall possess the same antigen-presenting prop-erties [104,105]. In the walls of healthy arteries, DCs reside in thesubendothelial space of the tunica intima and the tunica adventi-tia where they can mediate early recognition of danger signals[7,105–107]. DCs also reside in carotid arteries, coronary ar-teries, aortic root, arch and descending aorta of experimentalanimals; these findings facilitated studies of the functional signi-ficance of DCs in atherosclerosis [103–105,108–111] (Figure 1).

DCs belong to the myeloid lineage of blood cells [34]. De-pending on specific markers, phenotypic differences betweenmacrophages and DCs are not simple [112]. CD11c has beena reported marker of (mouse) DCs, but it is also up-regulatedby monocytes and macrophages [50,113]. A previous study hasshown that surface markers for DCs, including co-stimulatorymolecules (CD80, CD86), can be expressed by tissue macro-phages [114]. However, in mice, DCs are currently typically char-acterized by CD11c expression, and inducible overexpression ofMHC II, CD80, CD86 and CD40 [18,114]. Phenotypically DCshave a morphologically distinct dendritic shape. DCs may rep-resent a unique and central subset of professional APCs capableof activating naive lymphocytes. In secondary lymphoid organs ofmice, specific phenotypes of DCs have been defined and are re-viewed elsewhere [18,115]

The involvement of DC apoptosis in atherosclerosis has beeninvestigated using mice overexpressing an apoptosis inhibitor(Bcl-2) under the control of the CD11c cell-specific promoter[116]. This model achieved expansion of DCs and enhanced T-cell activation in vivo with a shift toward Th1 cells and increases inanti-oxLDL antibody sera (IgG2c type). Th1 activity is expectedto promote atherosclerosis; however, this model described noincreased atheroma when transplanted into either LDLR− / − orApoE− / − mice. This paradoxical result was possibly due to areduction in cholesterol with DC expansion. Thus DCs may havea role in the clearance of cholesterol.

Local proliferation of DCs has also been demonstrated in theaorta and in secondary lymphoid organs [117,118]. Along withproliferation, arterial DC numbers are also affected by egress.Monocyte-derived DCs can emigrate from the arterial wall andatherosclerotic plaques at the early stages of atherosclerosis;however, their emigration from developed atherosclerotic lesionsis significantly impaired in mice with dyslipidaemia [119,120].Defective egress of DCs from the aorta, altered trafficking towardlymph nodes and/or proliferation in situ could be another mechan-ism of excessive accumulation of DCs in atherosclerotic lesions.

Overall the functions of DCs in atherosclerosis is still notwell characterized. However, data using mice lacking CX3CR1,CCL2 and CCR5 support the importance of DCs in atheroscler-osis with correlative reduced atherosclerosis and DC numbers[49,121,122]. CX3CR1 deficiency also decreases the survival ofGr1low monocytes, which could correlate with decreased macro-phage and DC aortic content and decreased foam cell formation[123]. Deletion of molecules involved in antigen presentation andDC migration also reduces plaque mass [124,125]. Finally, a re-cent study by Choi et al. [126] demonstrated a pivotal inhibitoryDC population; namely CD103+ classical DCs which were asso-ciated with atherosclerosis protection. Along with elegant in vivoDC imaging studies, these studies point to the importance of DCpopulations in atherosclerosis [127].

In summary monocytes, macrophages and DCs have com-plex roles in mediating atherosclerotic plaque formation, progres-sion and phenotype (Figure 1). Their emerging heterogeneity andsubset-specific interaction with atherogenic lipids, cellular debrisand other immune cells make them ideal targets for immunother-apy which may be utilized for novel anti-atherosclerotic drugdevelopment. Indeed, data inhibiting subset-specific monocyte



recruitment looks promising in experimental atherosclerosis. Fi-nally, modulating macrophage apoptosis or proliferation may alsoprove to be an effective therapeutic target [128].

LYMPHOCYTES

B-cellsSimplistically, the main function of B-cells is to secrete sera anti-bodies of various isotypes. B-1 cells are derived from committedprecursors [129]. They are found predominantly in peritonealand pleural cavities. B-1 cells participate in systemic immunityby producing IgM antibodies of the serum. B-2 cells are the mainpopulation produced in the bone marrow [129]. B-2 cells canproduce IgG antibodies after activation by specific antigens anddifferentiate into plasma cells; other functions include antigenpresentation, cytokine production and lymphoid tissue organiza-tion [129]. B-cells are found in atherosclerotic plaques of miceand humans, localized in the aortic adventitia [130–132]. Ad-ventitial B-cells have been described to form small lymphoidfollicles that may contribute to atherosclerosis [133,134]. BothIgG and IgM antibodies against oxLDL have been described (seefurther description below) [135,136]. A significant decrease inthe number of B-cells following splenectomy in mice or inter-ference with B-1 cell IgM production accelerated atheroscler-otic lesion development, which suggests a protective role for B-cells in plaque formation [135,137,138]. However, descriptionsof pro-atherogenic roles of B-cells have been discussed previ-ously [139,140].

IgM and IgG antibodies are present within atheroscleroticplaques [141]. Specific auto-antibodies against oxLDL have beenfound in the circulation of mice and humans [142,143], whereasIgM antibodies have been shown to protect against atheroscler-osis in mice and rabbits [144]. Injection of IgG1 antibodiesagainst oxLDL epitopes has reduced atherosclerosis in exper-imental mouse models [145]. Indeed, bone marrow chimaerasof B-cell-deficient bone marrow into LDR− / − mice increasedatherosclerosis [137]. In addition, splenectomy aggravates ath-erosclerosis in ApoE− / − mice and transfer of spleen B-cellsinto splenectomized mice reduces atherosclerosis [135]. How-ever, splenectomy does not only affect B-cell biology (see theMonocytes section above). The situation is maybe more complexin humans, with studies showing a positive or negative correlationor no correlation between auto anti-LDL titres and atheroscler-osis or its clinical correlates [146–149]. Regardless, solid phaseIgG and IgM seem to activate macrophage populations in vitro[150]. Much more work is needed to elucidate the role and de-position of automimmune antibodies to oxidized and modifiedLDL in atherosclerosis.

B-cells also exhibit functions that directly modulate immuneresponses in atherosclerotic plaques. B-cells can phagocytoseand internalize their antigen [151,152]. Indeed, compared withmatched wild-type mice, expression of CD80 and CD86 on B-cells was higher in atherosclerotic plaques of young ApoE− / −

mice [153]. Moreover, a positive correlation between the levelof CD80+ B-cells and the severity of carotid atherosclerosiswas found in humans [154]. Furthermore, B-cells can modulate

immune responses through immunomodulatory cytokine produc-tion [155].

T-cellsInitial studies of the role of T-cells in the development of ather-osclerosis have focused on the role of Th1 cells, which produceIFNγ , and that of Th2 cells, which can produce IL-4 [156]. Im-portantly, IFNγ is present in human lesions which can inducehigher expression of MHC II, enhanced protease and chemokinesecretion, up-regulation of adhesion molecules, induction of pro-inflammatory cytokines, and enhanced activation of macrophagesand endothelial cells [157]. Mice deficient in IFNγ or its receptorhave a lower lesion burden, and mice that receive IFNγ had an in-creased lesional size [158–160]. The roles of other T-cell subsets,including those of Treg (T-regulatory) cells have been addressed(see below) [161]. Th2 and Th17 cells have also been discussedto influence atherosclerotic changes [162]. Clonal expansion ofmemory effector and CD8+ T-cells has been demonstrated inlesions from humans and ApoE− / − mice [163]. Indeed lympho-cyte counts seem to be inversely correlated with coronary heartdisease and its complications [164].

Treg cellsSeveral studies have demonstrated a protective effect of vari-ous subsets of Treg cells in models of atherosclerosis. Foxp3+

expressed on Treg cells has been found in the plaques of miceand humans [165]. Transfer of Foxp3+ T-cells has also beenshown to be protective against atherosclerosis [166]. IL-10 orTGFβ (cytokines partly responsible for the protective functionsof Treg cells), have been shown to be anti-atherogenic. Geneticdepletion or blockade of IL-10 or TGFβ with neutralizing an-tibodies accelerated lesion development and aggravated vascu-lar inflammation. Reportedly pathogenic Th1 and Th2 responsesare exacerbated to the disadvantage of Treg cell responses [167].Moreover, T-cell restricted inactivation of the TGFβ signallingpathway specifically led to uncontrolled T-cell proliferation, in-flammation, autoimmune disease and increased atherosclerosis[167]. Treg cells are detected in much lower amounts in athero-sclerotic plaques than in other chronically inflamed tissues, suchas in the skin of patients with psoriasis, where Treg cells canrepresent up to approximately 25 % of T-cells [168]. These find-ings indicate an impairment of local tolerance against antigensin atherosclerotic plaques. Moreover, ApoE− / − mice exhibit alower number of Treg cells in the spleen compared with matchedcontrol mice [169]. Although Treg cells can inhibit T-cell ac-tivation and thereby modulate atherosclerosis, they might alsoprovide substantial protection against atherosclerosis by interact-ing with APCs. Treg cells inhibited foam-cell formation in vitroand promoted macrophage differentiation towards M2-like cells,dependent on TGFβ and IL-10 [170].

NKT (natural killer T)-cellsRecent research into atherogenesis has further elucid-ated the role of NKT-cells [171]. NKT cells are anintermediate of the innate and adaptive immune systems whoshare similarities with both NK (natural killer) cells (see below)

www.clinsci.org 227

K. J. Woollard

and T-cells [171]. NKT cells are modulatory to CD1d, a moleculewith antigen-presenting properties [171]. NKT cells express theNK1.1 marker protein in mice and CD161 and CD56 in humans[171]. They also seem to express CD4 [171]. NKT cells havebeen found in atherosclerotic plaques, albeit in small numbers[171]. In plaques, NKT cells can be activated by signals, such asoxLDL, and can release cytokines of both Th1 and Th2 [171].This is supported by studies in which NKT− / − mice displayedfewer atherosclerotic lesions [171].

NK cellsNK cells are a subset of cytotoxic lymphocytes found in humanatherosclerotic lesions [171]. Interestingly, defective cytolysis byNK cells in perforin− / − mice does not affect atherosclerosisin a double knockout model using LDLR− / − [172]. Moreover,LDLR− / − Lystbeige mutant mice, that have defective protein re-lease from cytoplasmic granules, display reduced atherosclerosis.In contrast, combined LDLR− / − Lystbeige with Rag1− / − mutantmice demonstrate increased atherogenesis and lipid sera levels[172]. NK cells are still present in these models, therefore otherNK cell functions may be involved. In another bone marrow chi-maera model, LDLR− / − mice using cells from transgenic miceexpressing Ly49A under the control of the granzyme-A promoter,the absence of fully functional NK cells resulted in 70 % reduc-tion of atherosclerotic lesion formation [173]. However, effectsfrom contaminating cellular populations in these experimentalmodels cannot be excluded.

In summary, lymphocytes make up a heterogeneous popula-tion of effector cells that are involved in atherosclerosis (Fig-ure 1). They can be recruited to evolving plaques and havecytotoxic, regulatory and inflammatory functions. Importantly,B-cells can secrete autoantibodies to modified LDLs, which mayhave roles in promoting or inhibiting atherosclerosis.

NEUTROPHILS

Neutrophils have been detected in aortic lesions of primates,humans and mice and have been correlated with cardiovascularincidence and prognosis [14,22,174]. However, it is worth not-ing that mice have a myeloid-biased immune system comparedwith humans, denoting another significant difference betweenmice and human immune physiology, which should always betaken into consideration [175]. Neutrophils represent approxim-ately 2 % of plaque leucocytes and accumulate in regions withhigh monocyte density (in mice) [22]. Neutrophils use the CCL5receptors (CCR1 and CCR5) to enter atherosclerotic plaques,which correlates significantly with plaque size [24]. Disrupt-ing CXCR4–CXCL12 interaction increases peripheral neutro-phil counts and modulates lesion formation, whereas neutrophildepletion predominantly reduces early plaque size [24,174]. Adisturbed lipid balance facilitates neutrophil recruitment and in-filtration to lesions via TLR signalling and pro-inflammatorygene expression, upon specific activating signals [176]. Hyper-lipidaemia can trigger neutrophilia by stimulating granulopoiesisand bone marrow egress, involving CXCL1 plasma overexpres-

sion [24]. However, direct roles for neutrophil lipid scavengingand metabolism have been limited; there are reports of HDLsinhibiting neutrophil activation and adhesion [177], although thedirect role of neutrophil activation by proatherogenic lipid speciesrequires further investigation.

Interestingly, neutrophil depletion specifically decreasesLy6chigh monocyte recruitment [178]. Neutrophils can rapidlybecome apoptotic in the intima, attracting macrophages for scav-enging [174]. Signals released by damaged neutrophils may helpresolve inflammation, where RNA may enhance TLR3 signal-ing to delay inflammatory lesion development, through increasedatherosclerosis in TLR3− / − ApoE− / − mice [179]. Furthermore,neutrophils activated through TLRs, Fc receptors or cytokinereceptors can release nuclear content that forms a scaffold con-taining antimicrobial proteins, named NETs (neutrophil extracel-lular traps) [180]. NETs have been identified in association withhuman and murine atherosclerotic lesions [181]. Finally, neut-rophils may link coagulation and inflammation; evidence showsthat neutrophil serine proteases with externalized nucleosomespromote intravascular thrombus growth in vivo via tissue factor-and factor XII-dependent coagulation, perhaps contributing toplaque thrombosis [182].

In summary, neutrophils respond rapidly to their microenvir-onment, releasing cytotoxic reagents, including NET formation.Their role in atherosclerosis is becoming apparent, demonstratingneutrophil-specific responses in inflammatory plaque phenotypes(Figure 1). More research is needed to define further neutrophilinteractions with other known pro-atherosclerotic agents, includ-ing responses to modified lipid species.

MAST CELLS AND PLATELETS

Mast cellsMast cells are derived from bone marrow cells and circulate inthe blood as mast cell precursors to be recruited to particulartissues and organs, such as lung and skin, where they mature intotissue mast cells [183]. In the 1950s the first paper to describe arole for mast cells in cardiovascular diseases was published [184].Studies showing the colocalization of mast cells expressing thepro-angiogenic factor bFGF (basic fibroblast growth factor) withintraplaque neovessels emerged [185]. Human coronary arteryspecimen analysis reported subendothelial mast cells in proxim-ity to microthrombi [186]. Interestingly, mast cell degranulationhas been shown to mediate in vivo uptake of LDL by macro-phages in the peritoneal cavity of rats [187]. Indeed, mast cellsystemic activation aggravates atherosclerotic lesion formationin the brachiocephalic artery in ApoE− / − mice [188].

Mast cells operate in the first line of host defence againstpathogens such as parasites and bacteria: various pathogenshave been detected in human atherosclerotic lesions, such asChlamydia pneumoniae and Aggregatibacter actinomycetem-comitans that can activate mast cells in vitro [189]. Moreover,levels of IgE, a significant mast cell activator, are elevated inpatients with unstable angina pectoris or dyslipidaemia and thismay have a direct link to atherosclerosis by mediating arterial cell



apoptosis, although the pathogenesis is yet to be fully elucidated[190,191].

Interestingly, mast-cell-derived chymase degrades cholesterylester transfer protein activity, indicating a novel role for mast cellproducts in lipid metabolism [192]. Specifically, PLTP (phos-pholipid transfer protein) transfers phospholipids from liposomedonor particles to acceptor HDL particles and facilitates pre-HDLformation. Chymase degrades PLTP fragments, thereby reducingPLTP pre-HDL activity and inhibiting cholesterol efflux frommacrophages [193].

PlateletsAnucleated cells of 1–2 μm have been described since the mid-19th century for their primary physiological role in sensing dam-aged vessel endothelium and accumulating at sites of injury toinitiate blood clotting [194]. The mechanism of thrombus form-ation can be divided into four steps: platelet tethering, activ-ation and firm adhesion, aggregation and platelet recruitment,and thrombus establishment [195]. The recognition that plate-lets modulate immune and inflammatory responses is now wellestablished [196]. It is not known whether platelets exert theirpossible atherosclerotic inflammatory functions by continuouslyinteracting with the endothelium and leucocytes or, over time,cause asymptomatic thrombi promoting leucocyte recruitment.Platelets are well equipped to facilitate leucocyte recruitment tosites of injury (and inflammation), and platelet expression of com-plement receptors and interactions with bacterial pathogens havebeen described previously [197,198]. Platelets store and releaseantibacterial proteins and, upon activation, produce significantamounts of cytokines and chemokines which are released fromthe α-granules [199]. Proteomic studies indicate that thrombin-stimulated platelets release more than 300 distinct proteins [200].Several of these secreted proteins have been identified in ather-osclerotic lesions [194], albeit not necessarily correlated withplatelet expression.

In summary, mast cells operate as the first line of host de-fense against pathogens. These pathogens are found in athero-sclerotic plaques where mast cell functions are poorly described(Figure 1). Mast cell activators including IgE are known to mod-ulate experimental atherosclerosis; however, their role in humanatherosclerosis still remains to be elucidated. Platelet roles in ath-erosclerosis have been described for a number of years; ‘athero-thrombosis’ is an established theme in atherosclerotic research(Figure 1). Emerging platelet chemokine, cytokine and micro-particle formation will undoubtedly mediate novel interactionsin atherosclerosis biology and require further investigation.

CONCLUSION

Inflammatory cells have largely been considered detrimental inthe pathogenesis of atherosclerosis and formation of the vul-nerable plaque (summary of plaque milieu shown in Figure 1).The argument for the role of monocyte-derived macrophagesis considerable. They express scavenger receptors, modifyingand oxidizing lipoproteins and can accumulate cholesteryl esters.

Macrophages express high levels of matrix metalloproteinases,pro-inflammatory cytokines and chemoattractants and can pro-duce tissue factors. This data originated from studies of mice de-ficient in functional macrophages crossed with atherosclerosis-prone mice, resulting in significantly inhibited atherosclerosis.This implies that differentiation of monocytes into macrophagesis critical for atherogenesis. However, as the knowledge of im-munology has advanced, it has become clear that atherosclerosisinvolves activation of both cellular innate and humoral immunity,which balances the inflammatory response to lipid deposition inthe arterial wall. Moreover, the publication on the heterogeneousphenotypes of monocytes and macrophages with independenteffector functions have now complicated the concept that mac-rophages are not just ‘bad’ in the pathogenesis of atheroscler-osis. Immunological research will help to identify these novelsubset-specific effector functions in atherosclerosis. This in turnwill help develop pharmaceutical targets for inhibiting vulner-able atherosclerosis and its pathological end points. Indeed wenow have the tools, at least in experimental models, to alter thecourse of atherosclerosis through immune modulation. Despitethese exciting advances, the best strategies for the treatment ofestablished atherosclerosis remain a challenge and many immun-ological therapeutic interventions have not been tested in humans;this must be addressed in order to bring immune-related modu-lation of atherosclerosis into clinical reality.

FUNDING

K.J.W. is currently funded by a British Heart Foundation fellowship.

REFERENCES

1 Dahlof, B. (2010) Cardiovascular disease risk factors:epidemiology and risk assessment. Am. J. Cardiol. 105, 3A–9A

2 Lloyd-Jones, D. M. (2010) Cardiovascular risk prediction: basicconcepts, current status, and future directions. Circulation121, 1768–1777

3 Hansson, G. K. (2005) Inflammation, atherosclerosis, andcoronary artery disease. N. Engl. J. Med. 352, 1685–1695

4 Hahn, C. and Schwartz, M. A. (2009) Mechanotransduction invascular physiology and atherogenesis. Nat. Rev. Mol. Cell Biol.10, 53–62

5 Ross, R. (1993) The pathogenesis of atherosclerosis: aperspective for the 1990s. Nature 362, 801–809

6 Kwon, G. P., Schroeder, J. L., Amar, M. J., Remaley, A. T. andBalaban, R. S. (2008) Contribution of macromolecular structureto the retention of low-density lipoprotein at arterial branchpoints. Circulation 117, 2919–2927

7 Millonig, G., Schwentner, C., Mueller, P., Mayerl, C. and Wick, G.(2001) The vascular-associated lymphoid tissue: a new site oflocal immunity. Curr. Opin. Lipidol. 12, 547–553

8 Wick, G., Knoflach, M. and Xu, Q. (2004) Autoimmune andinflammatory mechanisms in atherosclerosis. Annu. Rev.Immunol. 22, 361–403

9 Moore, K. J. and Tabas, I. (2011) Macrophages in thepathogenesis of atherosclerosis. Cell 145, 341–355

10 Stoll, G. and Bendszus, M. (2006) Inflammation andatherosclerosis: novel insights into plaque formation anddestabilization. Stroke 37, 1923–1932

www.clinsci.org 229

K. J. Woollard

11 Mayerl, C., Lukasser, M., Sedivy, R., Niederegger, H., Seiler, R.and Wick, G. (2006) Atherosclerosis research from past topresent–on the track of two pathologists with opposing views,Carl von Rokitansky and Rudolf Virchow. Virchows Arch. 449,96–103

12 Methe, H. and Weis, M. (2007) Atherogenesis andinflammation–was Virchow right? Nephrol. Dial. Transplant. 22,1823–1827

13 Ross, R. (1999) Atherosclerosis–an inflammatory disease. N.Engl. J. Med. 340, 115–126

14 Weber, C., Zernecke, A. and Libby, P. (2008) The multifacetedcontributions of leukocyte subsets to atherosclerosis: lessonsfrom mouse models. Nat. Rev. Immunol. 8, 802–815

15 Kzhyshkowska, J., Neyen, C. and Gordon, S. (2012) Role ofmacrophage scavenger receptors in atherosclerosis.Immunobiology 217, 492–502

16 Greaves, D. R. and Gordon, S. (2009) The macrophagescavenger receptor at 30 years of age: current knowledge andfuture challenges. J. Lipid Res. 50 (Suppl.), S282–S286

17 Steinman, R. M. and Cohn, Z. A. (1973) Identification of a novelcell type in peripheral lymphoid organs of mice. I. Morphology,quantitation, tissue distribution. J. Exp. Med. 137, 1142–1162

18 Koltsova, E. K. and Ley, K. (2011) How dendritic cells shapeatherosclerosis. Trends Immunol. 32, 540–547

19 Andersson, J., Libby, P. and Hansson, G. K. (2010) Adaptiveimmunity and atherosclerosis. Clin. Immunol. 134, 33–46

20 Hansson, G. K. (2002) The B cell: a good guy in vasculardisease? Arterioscler. Thromb. Vasc. Biol. 22, 523–524

21 Woollard, K. J. and Chin-Dusting, J. (2007) Therapeutictargeting of p-selectin in atherosclerosis. Inflamm. Allergy DrugTargets 6, 69–74

22 Rotzius, P., Thams, S., Soehnlein, O., Kenne, E., Tseng, C. N.,Bjorkstrom, N. K., Malmberg, K. J., Lindbom, L. and Eriksson,E. E. (2010) Distinct infiltration of neutrophils in lesionshoulders in ApoE− / − mice. Am. J. Pathol. 177, 493–500

23 Eriksson, E. E., Xie, X., Werr, J., Thoren, P. and Lindbom, L.(2001) Direct viewing of atherosclerosis in vivo: plaque invasionby leukocytes is initiated by the endothelial selectins. FASEB J.15, 1149–1157

24 Drechsler, M., Megens, R. T., van Zandvoort, M., Weber, C. andSoehnlein, O. (2010) Hyperlipidemia-triggered neutrophiliapromotes early atherosclerosis. Circulation 122, 1837–1845

25 Eriksson, E. E. (2011) Intravital microscopy on atherosclerosisin apolipoprotein E-deficient mice establishes microvessels asmajor entry pathways for leukocytes to advanced lesions.Circulation 124, 2129–2138

26 Glass, C. K. and Olefsky, J. M. (2012) Inflammation and lipidsignaling in the etiology of insulin resistance. Cell Metab. 15,635–645

27 Geissmann, F., Auffray, C., Palframan, R., Wirrig, C., Ciocca, A.,Campisi, L., Narni-Mancinelli, E. and Lauvau, G. (2008) Bloodmonocytes: distinct subsets, how they relate to dendritic cells,and their possible roles in the regulation of T-cell responses.Immunol. Cell Biol. 86, 398–408

28 Tacke, F. and Randolph, G. J. (2006) Migratory fate anddifferentiation of blood monocyte subsets. Immunobiology 211,609–618

29 Varol, C., Yona, S. and Jung, S. (2009) Origins andtissue-context-dependent fates of blood monocytes. Immunol.Cell Biol. 87, 30–38

30 Swirski, F. K., Pittet, M. J., Kircher, M. F., Aikawa, E., Jaffer, F. A.,Libby, P. and Weissleder, R. (2006) Monocyte accumulation inmouse atherogenesis is progressive and proportional to extentof disease. Proc. Natl. Acad. Sci. U.S.A. 103, 10340–10345

31 Patel, S. S., Thiagarajan, R., Willerson, J. T. and Yeh, E. T.(1998) Inhibition of α4 integrin and ICAM-1 markedly attenuatemacrophage homing to atherosclerotic plaques inApoE-deficient mice. Circulation 97, 75–81

32 Galkina, E. and Ley, K. (2009) Immune and inflammatorymechanisms of atherosclerosis (∗). Annu. Rev. Immunol. 27,165–197

33 Stoneman, V., Braganza, D., Figg, N., Mercer, J., Lang, R.,Goddard, M. and Bennett, M. (2007) Monocyte/macrophagesuppression in CD11b diphtheria toxin receptor transgenic micedifferentially affects atherogenesis and established plaques.Circ. Res. 100, 884–893

34 Geissmann, F., Manz, M. G., Jung, S., Sieweke, M. H., Merad,M. and Ley, K. (2010) Development of monocytes,macrophages, and dendritic cells. Science 327, 656–661

35 Geissmann, F., Jung, S. and Littman, D. R. (2003) Bloodmonocytes consist of two principal subsets with distinctmigratory properties. Immunity 19, 71–82

36 Gorgani, N. N., Ma, Y. and Clark, H. F. (2008) Gene signaturesreflect the marked heterogeneity of tissue-residentmacrophages. Immunol. Cell Biol. 86, 246–254

37 Cybulsky, M. I., Won, D. and Haidari, M. (2004) Leukocyterecruitment to atherosclerotic lesions. Can. J. Cardiol. 20(Suppl. B), 24B–28B

38 Swirski, F. K., Weissleder, R. and Pittet, M. J. (2009)Heterogeneous in vivo behavior of monocyte subsets inatherosclerosis. Arterioscler. Thromb. Vasc. Biol. 29,1424–1432

39 Trogan, E., Choudhury, R. P., Dansky, H. M., Rong, J. X., Breslow,J. L. and Fisher, E. A. (2002) Laser capture microdissectionanalysis of gene expression in macrophages fromatherosclerotic lesions of apolipoprotein E-deficient mice. Proc.Natl. Acad. Sci. U.S.A. 99, 2234–2239

40 Geissmann, F., Gordon, S., Hume, D. A., Mowat, A. M. andRandolph, G. J. (2010) Unravelling mononuclear phagocyteheterogeneity. Nat. Rev. Immunol. 10, 453–460

41 Woollard, K. J. and Geissmann, F. (2010) Monocytes inatherosclerosis: subsets and functions. Nat. Rev. Cardiol. 7,77–86

42 Auffray, C., Fogg, D., Garfa, M., Elain, G., Join-Lambert, O.,Kayal, S., Sarnacki, S., Cumano, A., Lauvau, G. andGeissmann, F. (2007) Monitoring of blood vessels and tissuesby a population of monocytes with patrolling behavior. Science317, 666–670

43 Ziegler-Heitbrock, L., Ancuta, P., Crowe, S., Dalod, M., Grau, V.,Hart, D. N., Leenen, P. J., Liu, Y. J., MacPherson, G., Randolph,G. J. et al. (2010) Nomenclature of monocytes and dendriticcells in blood. Blood 116, e74–e80

44 Swirski, F. K., Libby, P., Aikawa, E., Alcaide, P., Luscinskas, F. W.,Weissleder, R. and Pittet, M. J. (2007) Ly-6Chi monocytesdominate hypercholesterolemia-associated monocytosis andgive rise to macrophages in atheromata. J. Clin. Invest. 117,195–205

45 Tacke, F., Alvarez, D., Kaplan, T. J., Jakubzick, C., Spanbroek,R., Llodra, J., Garin, A., Liu, J., Mack, M., van Rooijen, N. et al.(2007) Monocyte subsets differentially employ CCR2, CCR5,and CX3CR1 to accumulate within atherosclerotic plaques. J.Clin. Invest. 117, 185–194

46 Murphy, A. J., Akhtari, M., Tolani, S., Pagler, T., Bijl, N., Kuo,C. L., Wang, M., Sanson, M., Abramowicz, S., Welch, C. et al.(2011) ApoE regulates hematopoietic stem cell proliferation,monocytosis, and monocyte accumulation in atheroscleroticlesions in mice. J. Clin. Invest. 121, 4138–4149

47 Yvan-Charvet, L., Pagler, T., Gautier, E. L., Avagyan, S., Siry,R. L., Han, S., Welch, C. L., Wang, N., Randolph, G. J., Snoeck,H. W. and Tall, A. R. (2010) ATP-binding cassette transportersand HDL suppress hematopoietic stem cell proliferation.Science 328, 1689–1693

48 Varol, C., Landsman, L., Fogg, D. K., Greenshtein, L., Gildor, B.,Margalit, R., Kalchenko, V., Geissmann, F. and Jung, S. (2007)Monocytes give rise to mucosal, but not splenic, conventionaldendritic cells. J. Exp. Med. 204, 171–180



49 Combadiere, C., Potteaux, S., Rodero, M., Simon, T., Pezard, A.,Esposito, B., Merval, R., Proudfoot, A., Tedgui, A. and Mallat, Z.(2008) Combined inhibition of CCL2, CX3CR1, and CCR5abrogates Ly6C(hi) and Ly6C(lo) monocytosis and almostabolishes atherosclerosis in hypercholesterolemic mice.Circulation 117, 1649–1657

50 Wu, H., Gower, R. M., Wang, H., Perrard, X. Y., Ma, R., Bullard,D. C., Burns, A. R., Paul, A., Smith, C. W., Simon, S. I. andBallantyne, C. M. (2009) Functional role of CD11c+ monocytesin atherogenesis associated with hypercholesterolemia.Circulation 119, 2708–2717

51 Dutta, P., Courties, G., Wei, Y., Leuschner, F., Gorbatov, R.,Robbins, C. S., Iwamoto, Y., Thompson, B., Carlson, A. L.,Heidt, T. et al. (2012) Myocardial infarction acceleratesatherosclerosis. Nature 487, 325–329

52 Gordon, S. and Taylor, P. R. (2005) Monocyte and macrophageheterogeneity. Nat. Rev. Immunol. 5, 953–964

53 Passlick, B., Flieger, D. and Ziegler-Heitbrock, H. W. (1989)Identification and characterization of a novel monocytesubpopulation in human peripheral blood. Blood 74,2527–2534

54 Strauss-Ayali, D., Conrad, S. M. and Mosser, D. M. (2007)Monocyte subpopulations and their differentiation patternsduring infection. J. Leukoc. Biol. 82, 244–252

55 Weber, C., Belge, K. U., von Hundelshausen, P., Draude, G.,Steppich, B., Mack, M., Frankenberger, M., Weber, K. S. andZiegler-Heitbrock, H. W. (2000) Differential chemokine receptorexpression and function in human monocyte subpopulations. J.Leukoc. Biol. 67, 699–704

56 Auffray, C., Sieweke, M. H. and Geissmann, F. (2009) Bloodmonocytes: development, heterogeneity, and relationship withdendritic cells. Annu. Rev. Immunol. 27, 669–692

57 Cros, J., Cagnard, N., Woollard, K., Patey, N., Zhang, S. Y.,Senechal, B., Puel, A., Biswas, S. K., Moshous, D., Picard, C.et al. (2010) Human CD14dim monocytes patrol and sensenucleic acids and viruses via TLR7 and TLR8 receptors.Immunity 33, 375–386

58 Ingersoll, M. A., Spanbroek, R., Lottaz, C., Gautier, E. L.,Frankenberger, M., Hoffmann, R., Lang, R., Haniffa, M., Collin,M., Tacke, F. et al. (2010) Comparison of gene expressionprofiles between human and mouse monocyte subsets. Blood115, e10–e19

59 Haka, A. S., Potteaux, S., Fraser, H., Randolph, G. J. andMaxfield, F. R. (2012) Quantitative analysis of monocytesubpopulations in murine atherosclerotic plaques bymultiphoton microscopy. PLoS ONE 7, e44823

60 Grau, A. J., Boddy, A. W., Dukovic, D. A., Buggle, F., Lichy, C.,Brandt, T. and Hacke, W. (2004) Leukocyte count as anindependent predictor of recurrent ischemic events. Stroke 35,1147–1152

61 Nasir, K., Guallar, E., Navas-Acien, A., Criqui, M. H. and Lima,J. A. (2005) Relationship of monocyte count and peripheralarterial disease: results from the National Health and NutritionExamination Survey 1999–2002. Arterioscler. Thromb. Vasc.Biol. 25, 1966–1971

62 Horne, B. D., Anderson, J. L., John, J. M., Weaver, A., Bair, T. L.,Jensen, K. R., Renlund, D. G. and Muhlestein, J. B. (2005)Which white blood cell subtypes predict increasedcardiovascular risk? J. Am. Coll. Cardiol. 45, 1638–1643

63 Dragu, R., Khoury, S., Zuckerman, R., Suleiman, M., Mutlak, D.,Agmon, Y., Kapeliovich, M., Beyar, R., Markiewicz, W.,Hammerman, H. and Aronson, D. (2008) Predictive value ofwhite blood cell subtypes for long-term outcome followingmyocardial infarction. Atherosclerosis 196, 405–412

64 Gurm, H. S., Bhatt, D. L., Lincoff, A. M., Tcheng, J. E.,Kereiakes, D. J., Kleiman, N. S., Jia, G. and Topol, E. J. (2003)Impact of preprocedural white blood cell count on long termmortality after percutaneous coronary intervention: insightsfrom the EPIC, EPILOG, and EPISTENT trials. Heart 89,1200–1204

65 Rana, J. S., Boekholdt, S. M., Ridker, P. M., Jukema, J. W.,Luben, R., Bingham, S. A., Day, N. E., Wareham, N. J.,Kastelein, J. J. and Khaw, K. T. (2007) Differential leucocytecount and the risk of future coronary artery disease in healthymen and women: the EPIC-Norfolk Prospective PopulationStudy. J. Intern. Med. 262, 678–689

66 Rothe, G., Herr, A. S., Stohr, J., Abletshauser, C., Weidinger, G.and Schmitz, G. (1999) A more mature phenotype of bloodmononuclear phagocytes is induced by fluvastatin treatment inhypercholesterolemic patients with coronary heart disease.Atherosclerosis 144, 251–261

67 Poitou, C., Dalmas, E., Renovato, M., Benhamo, V., Hajduch, F.,Abdennour, M., Kahn, J. F., Veyrie, N., Rizkalla, S., Fridman,W. H. et al. (2011) CD14dimCD16+ and CD14+ CD16+

monocytes in obesity and during weight loss: relationships withfat mass and subclinical atherosclerosis. Arterioscler. Thromb.Vasc. Biol. 31, 2322–2330

68 Rogacev, K. S., Seiler, S., Zawada, A. M., Reichart, B., Herath,E., Roth, D., Ulrich, C., Fliser, D. and Heine, G. H. (2011)CD14+ + CD16+ monocytes and cardiovascular outcome inpatients with chronic kidney disease. Eur. Heart J. 32, 84–92

69 Heine, G. H., Ulrich, C., Seibert, E., Seiler, S., Marell, J.,Reichart, B., Krause, M., Schlitt, A., Kohler, H. and Girndt, M.(2008) CD14+ + CD16+ monocytes but not total monocytenumbers predict cardiovascular events in dialysis patients.Kidney Int. 73, 622–629

70 Schlitt, A., Heine, G. H., Blankenberg, S., Espinola-Klein, C.,Dopheide, J. F., Bickel, C., Lackner, K. J., Iz, M., Meyer, J.,Darius, H. and Rupprecht, H. J. (2004) CD14+ CD16+

monocytes in coronary artery disease and their relationship toserum TNF-α levels. Thromb. Haemost. 92, 419–424

71 Imanishi, T., Ikejima, H., Tsujioka, H., Kuroi, A., Ishibashi, K.,Komukai, K., Tanimoto, T., Ino, Y., Takeshita, T. and Akasaka, T.(2010) Association of monocyte subset counts with coronaryfibrous cap thickness in patients with unstable angina pectoris.Atherosclerosis 212, 628–635

72 Kashiwagi, M., Imanishi, T., Tsujioka, H., Ikejima, H., Kuroi, A.,Ozaki, Y., Ishibashi, K., Komukai, K., Tanimoto, T., Ino, Y. et al.(2010) Association of monocyte subsets with vulnerabilitycharacteristics of coronary plaques as assessed by 64-slicemultidetector computed tomography in patients with stableangina pectoris. Atherosclerosis 212, 171–176

73 Liu, Y., Imanishi, T., Ikejima, H., Tsujioka, H., Ozaki, Y., Kuroi, A.,Okochi, K., Ishibashi, K., Tanimoto, T., Ino, Y. et al. (2010)Association between circulating monocyte subsets and in-stentrestenosis after coronary stent implantation in patients withST-elevation myocardial infarction. Circ. J. 74, 2585–2591

74 Springer, T. A. (1994) Traffic signals for lymphocyte recirculationand leukocyte emigration: the multistep paradigm. Cell 76,301–314

75 Leuschner, F., Dutta, P., Gorbatov, R., Novobrantseva, T. I.,Donahoe, J. S., Courties, G., Lee, K. M., Kim, J. I., Markmann,J. F., Marinelli, B. et al. (2011) Therapeutic siRNA silencing ininflammatory monocytes in mice. Nat. Biotechnol. 29,1005–1010

76 Woollard, K. J. and Chin-Dusting, J. (2010) P-selectinantagonism in inflammatory disease. Curr. Pharm. Des. 16,4113–4118

77 Woollard, K. J., Suhartoyo, A., Harris, E. E., Eisenhardt, S. U.,Jackson, S. P., Peter, K., Dart, A. M., Hickey, M. J. andChin-Dusting, J. P. (2008) Pathophysiological levels of solubleP-selectin mediate adhesion of leukocytes to the endotheliumthrough Mac-1 activation. Circ. Res. 103, 1128–1138

78 Woollard, K. J., Kling, D., Kulkarni, S., Dart, A. M., Jackson, S.and Chin-Dusting, J. (2006) Raised plasma soluble P-selectin inperipheral arterial occlusive disease enhances leukocyteadhesion. Circ. Res. 98, 149–156

www.clinsci.org 231

K. J. Woollard

79 Wolf, D., Hohmann, J. D., Wiedemann, A., Bledzka, K.,Blankenbach, H., Marchini, T., Gutte, K., Zeschky, K., Bassler,N., Hoppe, N. et al. (2011) Binding of CD40L to Mac-1′sI-domain involves the EQLKKSKTL motif and mediates leukocyterecruitment and atherosclerosis–but does not affect immunityand thrombosis in mice. Circ. Res. 109, 1269–1279

80 Huo, Y., Hafezi-Moghadam, A. and Ley, K. (2000) Role ofvascular cell adhesion molecule-1 and fibronectin connectingsegment-1 in monocyte rolling and adhesion on earlyatherosclerotic lesions. Circ. Res. 87, 153–159

81 Hashimoto, D., Miller, J. and Merad, M. (2011) Dendritic celland macrophage heterogeneity in vivo. Immunity 35, 323–335

82 Schulz, C., Gomez Perdiguero, E., Chorro, L., Szabo-Rogers, H.,Cagnard, N., Kierdorf, K., Prinz, M., Wu, B., Jacobsen, S. E.,Pollard, J. W. et al. (2012) A lineage of myeloid cellsindependent of Myb and hematopoietic stem cells. Science336, 86–90

83 Ginhoux, F., Liu, K., Helft, J., Bogunovic, M., Greter, M.,Hashimoto, D., Price, J., Yin, N., Bromberg, J., Lira, S. A. et al.(2009) The origin and development of nonlymphoid tissueCD103+ DCs. J. Exp. Med. 206, 3115–3130

84 Miller, J. C., Brown, B. D., Shay, T., Gautier, E. L., Jojic, V.,Cohain, A., Pandey, G., Leboeuf, M., Elpek, K. G., Helft, J. et al.(2012) Deciphering the transcriptional network of the dendriticcell lineage. Nat. Immunol. 13, 888–899

85 Gordon, S. (2003) Alternative activation of macrophages. Nat.Rev. Immunol. 3, 23–35

86 Bouhlel, M. A., Derudas, B., Rigamonti, E., Dievart, R., Brozek,J., Haulon, S., Zawadzki, C., Jude, B., Torpier, G., Marx, N. et al.(2007) PPARγ activation primes human monocytes intoalternative M2 macrophages with anti-inflammatory properties.Cell Metab. 6, 137–143

87 Martinez, F. O., Sica, A., Mantovani, A. and Locati, M. (2008)Macrophage activation and polarization. Front. Biosci. 13,453–461

88 Mantovani, A., Sica, A., Sozzani, S., Allavena, P., Vecchi, A. andLocati, M. (2004) The chemokine system in diverse forms ofmacrophage activation and polarization. Trends Immunol. 25,677–686

89 Wilson, H. M., Chettibi, S., Jobin, C., Walbaum, D., Rees, A. J.and Kluth, D. C. (2005) Inhibition of macrophage nuclearfactor-κB leads to a dominant anti-inflammatory phenotype thatattenuates glomerular inflammation in vivo. Am. J. Pathol. 167,27–37

90 Martinez, F. O., Helming, L. and Gordon, S. (2009) Alternativeactivation of macrophages: an immunologic functionalperspective. Annu. Rev. Immunol. 27, 451–483

91 Anderson, C. F. and Mosser, D. M. (2002) Cutting edge: biasingimmune responses by directing antigen to macrophage Fcγreceptors. J. Immunol. 168, 3697–3701

92 Mosser, D. M. and Edwards, J. P. (2008) Exploring the fullspectrum of macrophage activation. Nat. Rev. Immunol. 8,958–969

93 Komohara, Y., Hirahara, J., Horikawa, T., Kawamura, K., Kiyota,E., Sakashita, N., Araki, N. and Takeya, M. (2006) AM-3K, ananti-macrophage antibody, recognizes CD163, a moleculeassociated with an anti-inflammatory macrophage phenotype. J.Histochem. Cytochem. 54, 763–771

94 Boyle, J. J., Harrington, H. A., Piper, E., Elderfield, K., Stark, J.,Landis, R. C. and Haskard, D. O. (2009) Coronary intraplaquehemorrhage evokes a novel atheroprotective macrophagephenotype. Am. J. Pathol. 174, 1097–1108

95 Boyle, J. J., Johns, M., Kampfer, T., Nguyen, A. T., Game, L.,Schaer, D. J., Mason, J. C. and Haskard, D. O. (2012) Activatingtranscription factor 1 directs Mhem atheroprotectivemacrophages through coordinated iron handling and foam cellprotection. Circ. Res. 110, 20–33

96 Choudhury, R. P., Lee, J. M. and Greaves, D. R. (2005)Mechanisms of disease: macrophage-derived foam cellsemerging as therapeutic targets in atherosclerosis. Nat. Clin.Pract. Cardiovasc. Med. 2, 309–315

97 Gomez Perdiguero, E., Schulz, C. and Geissmann, F. (2013)Development and homeostasis of “resident” myeloid cells: thecase of the microglia. Glia 61, 112–120

98 Chorro, L. and Geissmann, F. (2010) Development andhomeostasis of ‘resident’ myeloid cells: the case of theLangerhans cell. Trends Immunol. 31, 438–445

99 Lipscomb, M. F. and Masten, B. J. (2002) Dendritic cells:immune regulators in health and disease. Physiol. Rev. 82,97–130

100 Steinman, R. M., Hawiger, D. and Nussenzweig, M. C. (2003)Tolerogenic dendritic cells. Annu. Rev. Immunol. 21, 685–711

101 Heath, W. R., Belz, G. T., Behrens, G. M., Smith, C. M.,Forehan, S. P., Parish, I. A., Davey, G. M., Wilson, N. S.,Carbone, F. R. and Villadangos, J. A. (2004)Cross-presentation, dendritic cell subsets, and the generationof immunity to cellular antigens. Immunol. Rev. 199, 9–26

102 Gelin, C., Sloma, I., Charron, D. and Mooney, N. (2009)Regulation of MHC II and CD1 antigen presentation: fromubiquity to security. J. Leukoc. Biol. 85, 215–224

103 Randolph, G. J. and Potteaux, S. (2010) Vascular dendritic cellsas gatekeepers of lipid accumulation within nascentatherosclerotic plaques. Circ. Res. 106, 227–229

104 Choi, J. H., Do, Y., Cheong, C., Koh, H., Boscardin, S. B., Oh,Y. S., Bozzacco, L., Trumpfheller, C., Park, C. G. and Steinman,R. M. (2009) Identification of antigen-presenting dendritic cellsin mouse aorta and cardiac valves. J. Exp. Med. 206, 497–505

105 Bobryshev, Y. V. and Lord, R. S. (1995) Ultrastructuralrecognition of cells with dendritic cell morphology in humanaortic intima. Contacting interactions of vascular dendritic cellsin athero-resistant and athero-prone areas of the normal aorta.Arch. Histol. Cytol. 58, 307–322

106 Bobryshev, Y. V. (2005) Dendritic cells in atherosclerosis:current status of the problem and clinical relevance. Eur. HeartJ. 26, 1700–1704

107 Niessner, A. and Weyand, C. M. (2010) Dendritic cells inatherosclerotic disease. Clin. Immunol. 134, 25–32

108 Bobryshev, Y. V. and Lord, R. S. (1998) Mapping of vasculardendritic cells in atherosclerotic arteries suggests theirinvolvement in local immune-inflammatory reactions.Cardiovasc. Res. 37, 799–810

109 Bobryshev, Y. V., Taksir, T., Lord, R. S. and Freeman, M. W.(2001) Evidence that dendritic cells infiltrate atheroscleroticlesions in apolipoprotein E-deficient mice. Histol. Histopathol.16, 801–808

110 Ozmen, J., Bobryshev, Y. V., Lord, R. S. and Ashwell, K. W.(2002) Identification of dendritic cells in aortic atheroscleroticlesions in rats with diet-induced hypercholesterolaemia. Histol.Histopathol. 17, 223–237

111 Jongstra-Bilen, J., Haidari, M., Zhu, S. N., Chen, M., Guha, D.and Cybulsky, M. I. (2006) Low-grade chronic inflammation inregions of the normal mouse arterial intima predisposed toatherosclerosis. J. Exp. Med. 203, 2073–2083

112 Hume, D. A. (2008) Macrophages as APC and the dendritic cellmyth. J. Immunol. 181, 5829–5835

113 Gower, R. M., Wu, H., Foster, G. A., Devaraj, S., Jialal, I.,Ballantyne, C. M., Knowlton, A. A. and Simon, S. I. (2011)CD11c/CD18 expression is upregulated on blood monocytesduring hypertriglyceridemia and enhances adhesion to vascularcell adhesion molecule-1. Arterioscler. Thromb. Vasc. Biol. 31,160–166

114 Buono, C. and Lichtman, A. H. (2004) Co-stimulation andplaque-antigen-specific T-cell responses in atherosclerosis.Trends Cardiovasc. Med. 14, 166–172

115 Shortman, K. and Liu, Y. J. (2002) Mouse and human dendriticcell subtypes. Nat. Rev. Immunol. 2, 151–161



116 Gautier, E. L., Huby, T., Saint-Charles, F., Ouzilleau, B., Pirault,J., Deswaerte, V., Ginhoux, F., Miller, E. R., Witztum, J. L.,Chapman, M. J. and Lesnik, P. (2009) Conventional dendriticcells at the crossroads between immunity and cholesterolhomeostasis in atherosclerosis. Circulation 119, 2367–2375

117 Zhu, S. N., Chen, M., Jongstra-Bilen, J. and Cybulsky, M. I.(2009) GM-CSF regulates intimal cell proliferation in nascentatherosclerotic lesions. J. Exp. Med. 206, 2141–2149

118 Kabashima, K., Banks, T. A., Ansel, K. M., Lu, T. T., Ware, C. F.and Cyster, J. G. (2005) Intrinsic lymphotoxin-β receptorrequirement for homeostasis of lymphoid tissue dendritic cells.Immunity 22, 439–450

119 Packard, R. R., Maganto-Garcia, E., Gotsman, I., Tabas, I., Libby,P. and Lichtman, A. H. (2008) CD11c+ dendritic cells maintainantigen processing, presentation capabilities, and CD4+ T-cellpriming efficacy under hypercholesterolemic conditionsassociated with atherosclerosis. Circ. Res. 103, 965–973

120 Llodra, J., Angeli, V., Liu, J., Trogan, E., Fisher, E. A. andRandolph, G. J. (2004) Emigration of monocyte-derived cellsfrom atherosclerotic lesions characterizes regressive, but notprogressive, plaques. Proc. Natl. Acad. Sci. U.S.A. 101,11779–11784

121 Combadiere, C., Potteaux, S., Gao, J. L., Esposito, B.,Casanova, S., Lee, E. J., Debre, P., Tedgui, A., Murphy, P. M. andMallat, Z. (2003) Decreased atherosclerotic lesion formation inCX3CR1/apolipoprotein E double knockout mice. Circulation107, 1009–1016

122 Liu, P., Yu, Y. R., Spencer, J. A., Johnson, A. E., Vallanat, C. T.,Fong, A. M., Patterson, C. and Patel, D. D. (2008) CX3CR1deficiency impairs dendritic cell accumulation in arterial intimaand reduces atherosclerotic burden. Arterioscler. Thromb. Vasc.Biol. 28, 243–250

123 Landsman, L., Bar-On, L., Zernecke, A., Kim, K. W.,Krauthgamer, R., Shagdarsuren, E., Lira, S. A., Weissman, I. L.,Weber, C. and Jung, S. (2009) CX3CR1 is required formonocyte homeostasis and atherogenesis by promoting cellsurvival. Blood 113, 963–972

124 Buono, C., Pang, H., Uchida, Y., Libby, P., Sharpe, A. H. andLichtman, A. H. (2004) B7-1/B7-2 costimulation regulatesplaque antigen-specific T-cell responses and atherogenesis inlow-density lipoprotein receptor-deficient mice. Circulation 109,2009–2015

125 Sun, J., Hartvigsen, K., Chou, M. Y., Zhang, Y., Sukhova, G. K.,Zhang, J., Lopez-Ilasaca, M., Diehl, C. J., Yakov, N., Harats, D.et al. (2010) Deficiency of antigen-presenting cell invariantchain reduces atherosclerosis in mice. Circulation 122,808–820

126 Choi, J. H., Cheong, C., Dandamudi, D. B., Park, C. G.,Rodriguez, A., Mehandru, S., Velinzon, K., Jung, I. H., Yoo, J. Y.,Oh, G. T. and Steinman, R. M. (2011) Flt3 signaling-dependentdendritic cells protect against atherosclerosis. Immunity 35,819–831

127 Koltsova, E. K., Garcia, Z., Chodaczek, G., Landau, M.,McArdle, S., Scott, S. R., von Vietinghoff, S., Galkina, E., Miller,Y. I., Acton, S. T. and Ley, K. (2012) Dynamic T cell-APCinteractions sustain chronic inflammation in atherosclerosis. J.Clin. Invest. 122, 3114–3126

128 Andres, V., Pello, O. M. and Silvestre-Roig, C. (2012)Macrophage proliferation and apoptosis in atherosclerosis.Curr. Opin. Lipidol. 23, 429–438

129 Lahoute, C., Herbin, O., Mallat, Z. and Tedgui, A. (2011)Adaptive immunity in atherosclerosis: mechanisms and futuretherapeutic targets. Nat. Rev. Cardiol. 8, 348–358

130 Aubry, M. C., Riehle, D. L., Edwards, W. D., Maradit-Kremers, H.,Roger, V. L., Sebo, T. J. and Gabriel, S. E. (2004) B-lymphocytesin plaque and adventitia of coronary arteries in two patientswith rheumatoid arthritis and coronary atherosclerosis:preliminary observations. Cardiovasc. Pathol. 13, 233–236

131 Zhou, X. and Hansson, G. K. (1999) Detection of B cells andproinflammatory cytokines in atherosclerotic plaques ofhypercholesterolaemic apolipoprotein E knockout mice. Scand.J. Immunol. 50, 25–30

132 Galkina, E., Kadl, A., Sanders, J., Varughese, D., Sarembock,I. J. and Ley, K. (2006) Lymphocyte recruitment into the aorticwall before and during development of atherosclerosis ispartially L-selectin dependent. J. Exp. Med. 203, 1273–1282

133 Watanabe, M., Sangawa, A., Sasaki, Y., Yamashita, M.,Tanaka-Shintani, M., Shintaku, M. and Ishikawa, Y. (2007)Distribution of inflammatory cells in adventitia changed withadvancing atherosclerosis of human coronary artery. J.Atheroscler. Thromb. 14, 325–331

134 Grabner, R., Lotzer, K., Dopping, S., Hildner, M., Radke, D.,Beer, M., Spanbroek, R., Lippert, B., Reardon, C. A., Getz, G. S.et al. (2009) Lymphotoxin β receptor signaling promotestertiary lymphoid organogenesis in the aorta adventitia of agedApoE− / − mice. J. Exp. Med. 206, 233–248

135 Caligiuri, G., Nicoletti, A., Poirier, B. and Hansson, G. K. (2002)Protective immunity against atherosclerosis carried by B cellsof hypercholesterolemic mice. J. Clin. Invest. 109, 745–753

136 Shaw, P. X., Horkko, S., Chang, M. K., Curtiss, L. K., Palinski,W., Silverman, G. J. and Witztum, J. L. (2000) Naturalantibodies with the T15 idiotype may act in atherosclerosis,apoptotic clearance, and protective immunity. J. Clin. Invest.105, 1731–1740

137 Major, A. S., Fazio, S. and Linton, M. F. (2002) B-lymphocytedeficiency increases atherosclerosis in LDL receptor-null mice.Arterioscler. Thromb. Vasc. Biol. 22, 1892–1898

138 Lewis, M. J., Malik, T. H., Ehrenstein, M. R., Boyle, J. J., Botto,M. and Haskard, D. O. (2009) Immunoglobulin M is required forprotection against atherosclerosis in low-density lipoproteinreceptor-deficient mice. Circulation 120, 417–426

139 Ait-Oufella, H., Herbin, O., Bouaziz, J. D., Binder, C. J.,Uyttenhove, C., Laurans, L., Taleb, S., Van Vre, E., Esposito, B.,Vilar, J. et al. (2010) B cell depletion reduces the developmentof atherosclerosis in mice. J. Exp. Med. 207, 1579–1587

140 Kyaw, T., Tay, C., Khan, A., Dumouchel, V., Cao, A., To, K., Kehry,M., Dunn, R., Agrotis, A., Tipping, P. et al. (2010) ConventionalB2 B cell depletion ameliorates whereas its adoptive transferaggravates atherosclerosis. J. Immunol. 185, 4410–4419

141 van Leeuwen, M., Damoiseaux, J., Duijvestijn, A. and Tervaert,J. W. (2009) The therapeutic potential of targeting B cells andanti-oxLDL antibodies in atherosclerosis. Autoimmun. Rev. 9,53–57

142 Tsimikas, S. (2006) Oxidized low-density lipoprotein biomarkersin atherosclerosis. Curr. Atheroscler. Rep. 8, 55–61

143 Palinski, W., Horkko, S., Miller, E., Steinbrecher, U. P., Powell,H. C., Curtiss, L. K. and Witztum, J. L. (1996) Cloning ofmonoclonal autoantibodies to epitopes of oxidized lipoproteinsfrom apolipoprotein E-deficient mice. Demonstration ofepitopes of oxidized low density lipoprotein in human plasma. J.Clin. Invest. 98, 800–814

144 Binder, C. J., Chou, M. Y., Fogelstrand, L., Hartvigsen, K., Shaw,P. X., Boullier, A. and Witztum, J. L. (2008) Natural antibodies inmurine atherosclerosis. Curr. Drug Targets 9, 190–195

145 Schiopu, A., Frendeus, B., Jansson, B., Soderberg, I.,Ljungcrantz, I., Araya, Z., Shah, P. K., Carlsson, R., Nilsson, J.and Fredrikson, G. N. (2007) Recombinant antibodies to anoxidized low-density lipoprotein epitope induce rapid regressionof atherosclerosis in apobec-1− / − /low-density lipoproteinreceptor− / − mice. J. Am. Coll. Cardiol. 50, 2313–2318

146 Hulthe, J., Wikstrand, J., Lidell, A., Wendelhag, I., Hansson,G. K. and Wiklund, O. (1998) Antibody titers against oxidizedLDL are not elevated in patients with familial hypercholestero-lemia. Arterioscler. Thromb. Vasc. Biol. 18, 1203–1211

147 Tornvall, P., Waeg, G., Nilsson, J., Hamsten, A. and Regnstrom,J. (2003) Autoantibodies against modified low-densitylipoproteins in coronary artery disease. Atherosclerosis 167,347–353

www.clinsci.org 233

K. J. Woollard

148 Fredrikson, G. N., Hedblad, B., Berglund, G., Alm, R., Nilsson,J. A., Schiopu, A., Shah, P. K. and Nilsson, J. (2007)Association between IgM against an aldehyde-modified peptidein apolipoprotein B-100 and progression of carotid disease.Stroke 38, 1495–1500

149 Tsimikas, S., Brilakis, E. S., Lennon, R. J., Miller, E. R.,Witztum, J. L., McConnell, J. P., Kornman, K. S. and Berger, P. B.(2007) Relationship of IgG and IgM autoantibodies to oxidizedlow density lipoprotein with coronary artery disease andcardiovascular events. J. Lipid Res. 48, 425–433

150 Boyle, J. J., Christou, I., Iqbal, M. B., Nguyen, A. T., Leung,V. W., Evans, P. C., Liu, Y., Johns, M., Kirkham, P. and Haskard,D. O. (2012) Solid-phase immunoglobulins IgG and IgM activatemacrophages with solid-phase IgM acting via a novel scavengerreceptor a pathway. Am. J. Pathol. 181, 347–361

151 Cancro, M. P. (2012) Editorial: phagocytic B cells: deja vu allover again? J. Leukoc. Biol. 91, 519–521

152 Allan, L. L., Hoefl, K., Zheng, D. J., Chung, B. K., Kozak, F. K.,Tan, R. and van den Elzen, P. (2009) Apolipoprotein-mediatedlipid antigen presentation in B cells provides a pathway forinnate help by NKT cells. Blood 114, 2411–2416

153 Afek, A., Harats, D., Roth, A., Keren, G. and George, J. (2004)Evidence for the involvement of T cell costimulation through theB-7/CD28 pathway in atherosclerotic plaques fromapolipoprotein E knockout mice. Exp. Mol. Pathol. 76, 219–223

154 Tanigawa, T., Kitamura, A., Yamagishi, K., Sakurai, S., Nakata,A., Yamashita, H., Sato, S., Ohira, T., Imano, H., Shimamoto, T.and Iso, H. (2003) Relationships of differential leukocyte andlymphocyte subpopulations with carotid atherosclerosis inelderly men. J. Clin. Immunol. 23, 469–476

155 Lund, F. E., Garvy, B. A., Randall, T. D. and Harris, D. P. (2005)Regulatory roles for cytokine-producing B cells in infection andautoimmune disease. Curr. Dir. Autoimmun. 8, 25–54

156 Tedgui, A. and Mallat, Z. (2006) Cytokines in atherosclerosis:pathogenic and regulatory pathways. Physiol. Rev. 86, 515–581

157 Hansson, G. K., Robertson, A. K. and Soderberg-Naucler, C.(2006) Inflammation and atherosclerosis. Annu. Rev. Pathol. 1,297–329

158 Gupta, S., Pablo, A. M., Jiang, X., Wang, N., Tall, A. R. andSchindler, C. (1997) IFN-γ potentiates atherosclerosis in ApoEknock-out mice. J. Clin. Invest. 99, 2752–2761

159 Whitman, S. C., Ravisankar, P., Elam, H. and Daugherty, A.(2000) Exogenous interferon-γ enhances atherosclerosis inapolipoprotein E− / − mice. Am. J. Pathol. 157, 1819–1824

160 Buono, C., Come, C. E., Stavrakis, G., Maguire, G. F., Connelly,P. W. and Lichtman, A. H. (2003) Influence of interferon-γ on theextent and phenotype of diet-induced atherosclerosis in theLDLR-deficient mouse. Arterioscler. Thromb. Vasc. Biol. 23,454–460

161 Mallat, Z., Ait-Oufella, H. and Tedgui, A. (2005) Regulatory T cellresponses: potential role in the control of atherosclerosis. Curr.Opin. Lipidol. 16, 518–524

162 Zhu, J. and Paul, W. E. (2010) Peripheral CD4+ T-celldifferentiation regulated by networks of cytokines andtranscription factors. Immunol. Rev. 238, 247–262

163 Paulsson, G., Zhou, X., Tornquist, E. and Hansson, G. K. (2000)Oligoclonal T cell expansions in atherosclerotic lesions ofapolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc.Biol. 20, 10–17

164 Coller, B. S. (2005) Leukocytosis and ischemic vasculardisease morbidity and mortality: is it time to intervene?Arterioscler. Thromb. Vasc. Biol. 25, 658–670

165 Gotsman, I., Grabie, N., Gupta, R., Dacosta, R., MacConmara,M., Lederer, J., Sukhova, G., Witztum, J. L., Sharpe, A. H. andLichtman, A. H. (2006) Impaired regulatory T-cell response andenhanced atherosclerosis in the absence of induciblecostimulatory molecule. Circulation 114, 2047–2055

166 Mallat, Z., Gojova, A., Brun, V., Esposito, B., Fournier, N.,Cottrez, F., Tedgui, A. and Groux, H. (2003) Induction of aregulatory T cell type 1 response reduces the development ofatherosclerosis in apolipoprotein E-knockout mice. Circulation108, 1232–1237

167 Mallat, Z., Ait-Oufella, H. and Tedgui, A. (2007) Regulatory T-cellimmunity in atherosclerosis. Trends Cardiovasc. Med. 17,113–118

168 de Boer, O. J., van der Meer, J. J., Teeling, P., van der Loos, C.M. and van der Wal, A. C. (2007) Low numbers of FOXP3positive regulatory T cells are present in all developmentalstages of human atherosclerotic lesions. PLoS ONE 2, e779

169 Mor, A., Planer, D., Luboshits, G., Afek, A., Metzger, S.,Chajek-Shaul, T., Keren, G. and George, J. (2007) Role ofnaturally occurring CD4+ CD25+ regulatory T cells inexperimental atherosclerosis. Arterioscler. Thromb. Vasc. Biol.27, 893–900

170 Liu, G., Ma, H., Qiu, L., Li, L., Cao, Y., Ma, J. and Zhao, Y.(2011) Phenotypic and functional switch of macrophagesinduced by regulatory CD4+ CD25+ T cells in mice. Immunol.Cell Biol. 89, 130–142

171 Whitman, S. C. and Ramsamy, T. A. (2006) Participatory role ofnatural killer and natural killer T cells in atherosclerosis:lessons learned from in vivo mouse studies. Can. J. Physiol.Pharmacol. 84, 67–75

172 Schiller, N. K., Boisvert, W. A. and Curtiss, L. K. (2002)Inflammation in atherosclerosis: lesion formation in LDLreceptor-deficient mice with perforin and Lystbeige mutations.Arterioscler. Thromb. Vasc. Biol. 22, 1341–1346

173 Whitman, S. C., Rateri, D. L., Szilvassy, S. J., Yokoyama, W. andDaugherty, A. (2004) Depletion of natural killer cell functiondecreases atherosclerosis in low-density lipoprotein receptornull mice. Arterioscler. Thromb. Vasc. Biol. 24, 1049–1054

174 Zernecke, A., Bot, I., Djalali-Talab, Y., Shagdarsuren, E.,Bidzhekov, K., Meiler, S., Krohn, R., Schober, A., Sperandio, M.,Soehnlein, O. et al. (2008) Protective role of CXC receptor4/CXC ligand 12 unveils the importance of neutrophils inatherosclerosis. Circ. Res. 102, 209–217

175 Libby, P., Ridker, P. M. and Hansson, G. K. (2011) Progress andchallenges in translating the biology of atherosclerosis. Nature473, 317–325

176 Yvan-Charvet, L., Welch, C., Pagler, T. A., Ranalletta, M.,Lamkanfi, M., Han, S., Ishibashi, M., Li, R., Wang, N. and Tall,A. R. (2008) Increased inflammatory gene expression in ABCtransporter-deficient macrophages: free cholesterolaccumulation, increased signaling via toll-like receptors, andneutrophil infiltration of atherosclerotic lesions. Circulation118, 1837–1847

177 Murphy, A. J., Woollard, K. J., Suhartoyo, A., Stirzaker, R. A.,Shaw, J., Sviridov, D. and Chin-Dusting, J. P. (2011) Neutrophilactivation is attenuated by high-density lipoprotein andapolipoprotein A-I in in vitro and in vivo models of inflammation.Arterioscler. Thromb. Vasc. Biol. 31, 1333–1341

178 Soehnlein, O., Zernecke, A., Eriksson, E. E., Rothfuchs, A. G.,Pham, C. T., Herwald, H., Bidzhekov, K., Rottenberg, M. E.,Weber, C. and Lindbom, L. (2008) Neutrophil secretionproducts pave the way for inflammatory monocytes. Blood 112,1461–1471

179 Cole, J. E., Navin, T. J., Cross, A. J., Goddard, M. E.,Alexopoulou, L., Mitra, A. T., Davies, A. H., Flavell, R. A.,Feldmann, M. and Monaco, C. (2011) Unexpected protectiverole for Toll-like receptor 3 in the arterial wall. Proc. Natl. Acad.Sci. U.S.A. 108, 2372–2377

180 Papayannopoulos, V. and Zychlinsky, A. (2009) NETs: a newstrategy for using old weapons. Trends Immunol. 30, 513–521

181 Megens, R. T., Vijayan, S., Lievens, D., Doring, Y., vanZandvoort, M. A., Grommes, J., Weber, C. and Soehnlein, O.(2012) Presence of luminal neutrophil extracellular traps inatherosclerosis. Thromb. Haemost. 107, 597–598



182 Massberg, S., Grahl, L., von Bruehl, M. L., Manukyan, D.,Pfeiler, S., Goosmann, C., Brinkmann, V., Lorenz, M., Bidzhekov,K., Khandagale, A. B. et al. (2010) Reciprocal coupling ofcoagulation and innate immunity via neutrophil serineproteases. Nat. Med. 16, 887–896

183 Taylor, M. L. and Metcalfe, D. D. (2001) Mast cells in allergyand host defense. Allergy Asthma Proc. 22, 115–119

184 Cairns, A. and Constantinides, P. (1954) Mast cells in humanatherosclerosis. Science 120, 31–32

185 Lappalainen, H., Laine, P., Pentikainen, M. O., Sajantila, A. andKovanen, P. T. (2004) Mast cells in neovascularized humancoronary plaques store and secrete basic fibroblast growthfactor, a potent angiogenic mediator. Arterioscler. Thromb.Vasc. Biol. 24, 1880–1885

186 Mayranpaa, M. I., Heikkila, H. M., Lindstedt, K. A., Walls, A. F.and Kovanen, P. T. (2006) Desquamation of human coronaryartery endothelium by human mast cell proteases: implicationsfor plaque erosion. Coron. Artery Dis. 17, 611–621

187 Kokkonen, J. O. (1989) Stimulation of rat peritoneal mast cellsenhances uptake of low density lipoproteins by rat peritonealmacrophages in vivo. Atherosclerosis 79, 213–223

188 Bot, I., de Jager, S. C., Zernecke, A., Lindstedt, K. A., vanBerkel, T. J., Weber, C. and Biessen, E. A. (2007) Perivascularmast cells promote atherogenesis and induce plaquedestabilization in apolipoprotein E-deficient mice. Circulation115, 2516–2525

189 Zhang, T., Kurita-Ochiai, T., Hashizume, T., Du, Y., Oguchi, S.and Yamamoto, M. (2010) Aggregatibacteractinomycetemcomitans accelerates atherosclerosis with anincrease in atherogenic factors in spontaneously hyperlipidemicmice. FEMS Immunol. Med. Microbiol. 59, 143–151

190 Kovanen, P. T., Manttari, M., Palosuo, T., Manninen, V. and Aho,K. (1998) Prediction of myocardial infarction in dyslipidemicmen by elevated levels of immunoglobulin classes A, E, and G,but not M. Arch. Intern. Med. 158, 1434–1439

191 Wang, J., Cheng, X., Xiang, M. X., Alanne-Kinnunen, M., Wang,J. A., Chen, H., He, A., Sun, X., Lin, Y., Tang, T. T. et al. (2011)IgE stimulates human and mouse arterial cell apoptosis andcytokine expression and promotes atherogenesis in ApoE− / −

mice. J. Clin. Invest. 121, 3564–3577192 Lee-Rueckert, M., Vikstedt, R., Metso, J., Jauhiainen, M. and

Kovanen, P. T. (2008) Association of cholesteryl estertransfer protein with HDL particles reduces its proteolyticinactivation by mast cell chymase. J. Lipid Res. 49,358–368

193 Lee, M., Metso, J., Jauhiainen, M. and Kovanen, P. T. (2003)Degradation of phospholipid transfer protein (PLTP) andPLTP-generated pre-β -high density lipoprotein by mast cellchymase impairs high affinity efflux of cholesterol frommacrophage foam cells. J. Biol. Chem. 278,13539–13545

194 Lievens, D. and von Hundelshausen, P. (2011) Platelets inatherosclerosis. Thromb. Haemost. 106, 827–838

195 Jackson, S. P. (2011) Arterial thrombosis–insidious,unpredictable and deadly. Nat. Med. 17, 1423–1436

196 Semple, J. W., Italiano, Jr, J. E. and Freedman, J. (2011)Platelets and the immune continuum. Nat. Rev. Immunol. 11,264–274

197 Devi, S., Kuligowski, M. P., Kwan, R. Y., Westein, E., Jackson,S. P., Kitching, A. R. and Hickey, M. J. (2010) Plateletrecruitment to the inflamed glomerulus occurs via anαIIbβ3/GPVI-dependent pathway. Am. J. Pathol. 177,1131–1142

198 Yeaman, M. R. (2010) Platelets in defense against bacterialpathogens. Cell. Mol. Life Sci. 67, 525–544

199 Gawaz, M., Langer, H. and May, A. E. (2005) Platelets ininflammation and atherogenesis. J. Clin. Invest. 115,3378–3384

200 Senzel, L., Gnatenko, D. V. and Bahou, W. F. (2009) The plateletproteome. Curr. Opin. Hematol. 16, 329–333

Received 22 October 2012/11 February 2013; accepted 15 March 2013

Published on the Internet 7 May 2013, doi: 10.1042/CS20120576

www.clinsci.org 235

Documents

Immunological aspects of atherosclerosis