Chemokines and atherosclerosis

Atherosclerosis 147 (1999) 213 – 225 www.elsevier.com/locate/atherosclerosis

Review article

Chemokines and atherosclerosis Theresa J. Reape *, Pieter H.E. Groot Department of Vascular Biology, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park North, Coldharbour Road, Harlow, Essex CM19 5AD, UK Received 22 February 1999; received in revised form 21 July 1999; accepted 20 August 1999

Abstract Chemokines or chemotactic cytokines represent an expanding family of structurally related small molecular weight proteins, recognised as being responsible for leukocyte trafficking and activation. Soon after the discovery of this class of cytokines, about a decade ago, monocyte chemoattractant protein-1 (MCP-1) was found to be highly expressed in human atherosclerotic lesions and postulated to be central in monocyte recruitment into the arterial wall and developing lesions. In this review, we will discuss our present knowledge about MCP-1 and its receptor CCR2 and their role in atherogenesis. Although less well established, other chemokines such as RANTES, MIP-1a and MIP-1b have also been implicated in atherosclerotic lesion formation as are a number of more recently discovered chemokines like MCP-4, ELC and PARC. The role of these chemokines in the progression of atherosclerosis will be discussed as well as the emerging role of IL-8, mostly know for its effects on neutrophils. Particular attention will be given not only to the involvement of chemokines in the inflammatory recruitment of monocytes/macrophages, but also to their role in the related local immune responses and vascular remodelling which occur during the formation of unstable atherosclerotic plaques. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chemokine; Atherosclerosis; Inflammation; Macrophage; T-cell; Smooth muscle cell; Endothelial cell

1. General introduction

1.1. The chemokine family of ligands Abbre6iations: BCA-1, B cell chemoattractant 1; DARC, Duffy antigen receptor for chemokines; DC-CK1, dendritic-cell chemokine 1; ELC, EBI1-ligand chemokine; ENA 78, epithelial-cell-derived neutrophil-activating peptide 78; GCP, granulocyte chemotactic protein; GRO, growth regulated oncogene; IL-8, interleukin 8; IP-10, interferon inducible protein 10; LARC, liver and activation-regulated chemokine; MCP, monocyte chemoattractant protein; MDC, monocyte-derived chemokine; MGSA, melanoma growth-stimulating activity; MIG, monokine induced by interferon-g; MIP, macrophage inflammatory protein; NAP-2, neutrophil-activating peptide 2; PARC pulmonary and activation-regulated chemokine; PF-4, platelet factor 4; RANTES, regulated on activation normal T expressed and secreted chemokine; SDF-1, stromal-cell-derived factor 1; SLC, secondary lymphoid-tissue chemokine; TARC, thymus and activation-regulated chemokine. * Corresponding author. Tel.: + 44-1279-627051; fax: +44-1279627049. E-mail addresses: theresa – [email protected] (T.J. Reape), pieter – h – [email protected] (P.H.E. Groot)

Chemokines are a superfamily of structurally related small (most being 8–10 kDa) chemotactic cytokines involved in leukocyte trafficking and activation. Based on the arrangement of the first four conserved cysteines four classes can be distinguished [1–3]. The largest class is that of the CC chemokines (prototype, MCP-1 [4]), where the first two cysteines are adjacent while in the second largest class of CXC chemokines (prototype, IL-8 [5]), the first two cysteines are separated by a single amino acid residue. Only one C chemokine, lymphotactin [6] and one CX3C chemokine, fractalkine [7] has as yet been described. Lymphotactin lacks two of the four conserved cysteines and in fractalkine the first two cysteines are separated by three amino acid residues. Fractalkine has other unusual structural features in that it is encoded as a membrane bound

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molecule with the chemokine domain attached to a mucin-like stalk [7]. CXC chemokines can be further divided into two functional groups depending on the presence of the ELR (Glu-Leu-Arg) motif in their NH2 terminus [8]. This motif was found to be important in CXC chemokine receptor binding on neutrophils [8] and the ELR+ and ELR− CXC chemokines have been found to be angiogenic and angiostatic factors, respectively [9]. In general, the genes for the CXC and CC chemokines are clustered on chromosome 4 (q12 – 21) and 17 (q11 –21), respectively [10]. Some exceptions have become apparent, such as four of a group of five recently described CC chemokines with a specificity for attracting lymphocytes: PARC/DC-CK1 [11,12], LARC [13], ELC [14], SLC [15] and TARC [16]. PARC maps to the traditional CC chemokine cluster on chromosome 17 [11] but LARC maps at chromosome 2, ELC and SLC to chromosome 9 and TARC to chromosome 16 ([13– 15,17], respectively). Another exception is the CXC chemokine SDF, which is localised on chromosome 10 [18].

1.2. Chemokine receptors The biological activities of chemokines are mediated by specific seven-transmembrane-domain G protein coupled receptors expressed on the surface of their target cells [19,20]. To date, nine receptors for CC chemokines (CCR1-9/10) and five for CXC chemokines (CXCR1–5) have been cloned and characterised [21– 23] as are the receptors for fractalkine (CX3CR1) and lymphotactin (CR1) [24,25]. Figure 1 shows a compilation of these chemokine receptors and their ligands. Looking at Fig. 1, it is clear that chemokine receptors often accept more than one chemokine as their ligand while many chemokines are also able to interact with multiple chemokine receptors. Although the network of chemokines and chemokine receptors is complex, different chemokine classes tend to display distinct specificity towards one or more classes of leukocytes and this is associated with differences in chemokine receptor expression profiles between different leukocytes. In general, CXC chemokines attract neutrophils while CC chemokines attract one or more classes of mononuclear cells (monocytes, T and B lymphocytes), basophils or eosinophils. Lymphotactin attracts T-cells while fractalkine attracts monocytes and T-cells [3]. However, this segregation is slightly over-simplified, for instance the CXC chemokines, IP-10 and MIG are chemotactic for lymphocytes not neutrophils [26]. Also, in addition to it’s neutrophil attracting properties, IL-8 has been reported by some laboratories to be a potent T-cell chemoattractant [27]. The ability of the chemokines to attract different classes and sub-classes of leukocytes and the complexity of the network of chemokine recep-

tors and chemokines is thought to be central in finetuning inflammatory responses to the requirements of the organism for tissue repair or combating disease. Interestingly, there is now increasing information on the expression of chemokine receptors on cells other that leukocytes such as endothelial cells (EC) [28,29] and vascular smooth muscle cells (VSMC) [30] — cells which are themselves intimately involved in the inflammatory/immune responses which occur during atherosclerosis. Most chemokines possess a heparinbinding site and can interact with proteoglycans on cell surfaces and in the extracellular matrix. This interaction is thought to be responsible for the establishment of chemokine gradients over endothelial cells which is important for initial leukocyte recruitment and subsequent migration through the tissue [31,32].

1.3. Atherosclerosis: an inflammatory disease The attraction of leukocytes to a specific area of the vasculature and migration through the underlying tissue is central in the generation of an inflammatory response, e.g. in wound healing, infections, etc. Many diseases with an inflammatory component are the result of this beneficial recruitment process getting out of control. It is generally recognised now that atherosclerosis is among the group of chronic diseases in which over-recruitment of leukocytes — in this case monocytes and to a lesser extent T-cells — is at the root of the pathology [33]. Recruitment of leukocytes requires the expression, on the endothelium and the leukocyte, of various classes of adhesion molecules, e.g. selectins, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and the presence of counter receptor molecules on the leukocyte/endothelial cell, in addition to the establishment of a chemotactic gradient to guide leukocytes to the source of the inflammatory signal [34,35]. In atherosclerosis, the site of origin of the inflammatory signal is the vessel wall itself. In the following sections of this review we will discuss the current ideas on what triggers this initial inflammatory response and focus on the role of chemokines and their receptors in the aetiology of lesion formation and progression.

1.4. The initial trigger in lesion formation Atherosclerosis is a multifactorial disease and over the years numerous risk factors associated with its manifestation have been identified. Among these, elevated levels of plasma cholesterol, in particular lowdensity lipoprotein (LDL), is recognised as a major risk factor, as are hypertension, diabetes mellitus and smoking [36]. Studies employing animal models, but now also increasingly in humans, have shown that both early and advanced atherosclerosis is associated with

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arterial luminal endothelium dysfunction [36,37]. Among other possible triggers, modified LDL has been identified as an inducer of endothelial dysfunction.

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High concentrations of plasma LDL leads to higher concentrations in the sub-endothelial space where LDL can become oxidatively modified by reactive oxygen

Fig. 1. Chemokine receptors and their ligands. Fig. 2. PARC mRNA expression in a human atherosclerotic plaque. Serial sections of a carotid endarterectomy specimen showing PARC mRNA expression in a macrophage-rich area of the specimen. (A) Dark-field photomicrograph showing in situ hybridisation for PARC mRNA using a specific 35S-labeled antisense riboprobe. (B) Immunohistochemical staining for macrophages using CD68. (C) Dark-field view of nonhybridising PARC sense riboprobe. Bar represents 200 mm.

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species produced by EC, resident macrophages or SMC. Once formed in the sub-endothelial space, oxidised (ox) LDL may injure the endothelium and play a role in the increased adherence of leukocytes and their migration into the vascular wall [36]. It has been shown that oxidised LDL can up-regulate adhesion molecules on EC [38,39] and that LDL, minimally modified by oxidation, can increase MCP-1 mRNA expression in EC and SMC [40]. During the oxidation of LDL, up to 40% of the phosphatidylcholine (PC) of LDL may be converted to lysoPC through phospholipase A2 activity [41]. LysoPC has direct atherogenic effects such as induction of expression of adhesion molecules and MCP-1 on EC [42,43], inhibition of relaxation of EC and is mitogenic for macrophages [42], strongly implying that lysoPC is a main contributor to the atherogenic effects of oxLDL. There is also evidence that enzymatic, non-oxidative degradation of LDL results in a potentially atherogenic LDL molecule [44]. This non-oxidised form of LDL, prepared by treatment in vitro with trypsin, cholesterol esterase and neuraminidase, was found to be immunologically similar to LDL found in atherosclerotic plaques [45] and can activate complement, cause selective adhesion of monocytes and T-cells to the endothelium, stimulate their transmigration to the subendothelium [46] and induce MCP-1 expression and release from macrophages [45]. Similarly, infection of endothelial cells with agents such as cytomegalovirus, herpes simplex virus, or Chlamydia pneumoniae can result in cell lysis, procoagulant activity and increased leukocyte adhesion [47]. Arterial bifurctions, branches and curvatures, which typically characterise lesion prone areas, cause alterations in blood flow patterns in these areas and it has been shown that shear stress can upregulate expression of ICAM-1 [48] and MCP-1 [49] in cultured endothelial cells. As mentioned briefly above, initial attachment of leukocytes to the endothelium or ‘rolling’ is thought to be controlled by a class of adhesion molecules, the selectins, which are expressed on both the leukocyte and the endothelial cell. Spreading and firm attachment of the leukocyte occurs through interaction between integrins on the leukocytes and another class of adhesion molecules on the endothelial cells. Chemokines, sequestered and immobilised on the surface of the endothelium through proteoglycan binding, are thought to be important in enhancing integrin adhesiveness, thus mediating the arrest and firm adhesion of the leukocyte, where it can come into contact with chemokine signals required for migration through the tissue to the site of inflammation [34,35]. Direct evidence in support of this theory comes from the finding that the chemokines SDF-1, ELC, exodus-2/SLC and LARC are capable of triggering almost immediate integrin-dependent arrest of lymphocytes under physiological shear [50]. Monocyte/macrophages make up the bulk of the infiltrated

leukocyte population and are considered to be the main inflammatory mediators in atherosclerosis. T-cells are also prevalent and are most abundant in the fibrous cap where they constitute up to 20% of the cell population [51]. Many are in an activated state and producing interferon (IFN) g, suggesting the involvement of local immune responses during atherosclerosis [51–53].

1.5. Role of chemokines in early plaque formation The presence of chemokines in atherosclerosis is well documented. A considerable number of studies have been directed towards determining the role of chemokines, most notably MCP-1, in the initial stages of plaque formation. MCP-1 has been shown to be expressed mainly by macrophages [54–56] in human lesions, but also by smooth muscle cells [54]. MCP-1 protein expression has also been detected on the luminal endothelium of early human atherosclerotic lesions [56]. Increased MCP-1 expression was found in the carotid arteries of non-human primates in response to dietary hypercholesterolemia and the cells responsible for most of the MCP-1 expression during early lesion development in this model were the medial SMC [57]. These findings led some authors to speculate on early expression of MCP-1 by SMC and/or endothelial cells, in response to hypercholesterolemia or other insults to the vessel wall, being responsible for the initial influx of monocytes into the vessel wall. The infiltrated monocyte/macrophages could then begin to express MCP-1 resulting in a continued influx of monocytes into the plaque — explaining the persistent expression of MCP-1 by advanced plaque macrophages [54–56]. Although models of balloon injury do not represent models for atherosclerosis, support for early MCP-1 expression by SMC comes from a time course study in balloon injured rabbit arteries, where up-regulation of MCP-1 was demonstrated after just 2 h — a time point when the authors did not observe infiltration of leukocytes into the injured/de-nuded vessel (i.e. no endothelium or adventitia) [58]. In a rat vein graft model of intimal hyperplasia, MCP-1 up-regulation precedes maximal macrophage infiltration and consistent with findings in advanced human atherosclerotic lesions, levels of MCP1 did not return to base-line after acute inflammation had subsided in this model [59]. However, studies done on balloon injured pig arteries do not support this theory, but rather demonstrate that MCP-1 is produced by the infiltrating monocyte/macrophages and is not expressed preceding leukocyte invasion into the injured vessel wall [60]. No conclusive data exists, therefore, as to which cells are responsible for early MCP-1 expression or indeed whether MCP-1 is the first signal for leukocyte infiltration in atherosclerosis. However, most of the data strongly suggests that MCP-1 has an intimate involvement in the continued infiltration of mono-

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Table 1 Relevant functions of chemokines which are expressed in primary human atherosclerosis Chemokine

Function

Cellular Source in lesions

MCP-1 MIP-1a MCP-4 MIP-1 b RANTES PARC ELC IL-8

Monocyte [88], activated T-cell chemoattractant [104]: inducer of adhesion of monocytes [70] Monocyte [62,72,73] activated T-cell chemoattractant [72] Monocyte [88], activated T-cell chemoattractant [107] Monocyte [88], T-cell chemoattractant [107] Monocyte [88,106], activated T-cell chemoattractant[106] Naive T-cell chemoattractant [12] T-cell chemoattractant [14,74]; inducer of adhesion of lymphocytes [50] Monocyte [70],activated T-cell chemoattractant [27]; angiogenic factor [65]; stimulates SMC migration and proliferation [66]; inducer of adhesion of monocytes [70]

Macrophage, SMC, EC [54–56] Macrophage, EC [62] T-cell [61] T-cell [61] Macrophage, T-cell [61] Macrophage [63] Macrophage, SMC [63] Macrophage [67,68]

cytes into lesions and perhaps other downstream chronic modulatory events.

1.6. Chemokines and the more ad6anced atherosclerotic plaque In addition to MCP-1, several other CC chemokines have been found to be associated with advanced atherosclerotic lesions (see Table 1 for summary): MIP1a and MIP-1b are expressed by T-cells in human plaques [61] and the number of T-cells expressing these chemokines correlates with the total number of T-cells found in the plaques. RANTES is also expressed by lesion T-cells but in a smaller population (about 5%) [61]. MCP-4 is expressed in advanced plaques by endothelial cells of the vasa vasorum and in lesional macrophages [62]. We have recently found two lymphocyte specific chemoattractants, PARC/DC-CK1 and ELC, to be highly expressed in human atherosclerotic plaques, PARC exclusively by macrophages and ELC by macrophages and SMC [63]. In contrast to its expression pattern in atherosclerotic plaques, RANTES is highly expressed in human transplant-associated accelerated atherosclerosis by macrophages, lymphocytes, myofibroblasts and endothelial cells [64]. Less attention has been given to the CXC chemokines in the pathogenesis of atherosclerosis due to their traditional specificity for neutrophils. However, as mentioned earlier, IL-8 can function as a chemoattractant for T-cells [27], is an angiogenic factor [65] and can induce vascular SMC proliferation and migration [66]. In support of a role for CXC chemokines in atherosclerosis, IL-8 has been shown to be expressed by plaque macrophages in humans [67,68]. In addition, CXCR2 and its murine homologue, mIL-8RH, were detected in macrophage rich areas of advanced lesions in humans and LDL receptor knockout mice [69]. Interestingly, LDL receptor knockout mice which were irradiated and repopulated with bone marrow cells lacking the murine homologue of CXCR2 had less extensive lesions and fewer macrophages than those mice receiving bone marrow cells expressing the receptor [69]. This strongly

implies a role for CXCR2 in atherosclerotic lesion formation. A recent study has shed light on a mechanism for IL-8 receptor mediated monocyte recruitment during atherosclerosis [70]. Although IL-8 mediated monocyte chemotaxis has not been demonstrated before, these authors found that IL-8 was capable of inducing chemotaxis of freshly isolated, elutriated peripheral blood monocytes. More significant was the finding that both IL-8 and MCP-1 could convert monocyte rolling to firm adhesion on endothelial monolayers, expressing E-selectin under flow conditions. This finding has important implications for the role of CXC chemokines in atherosclerosis, as it highlights a role for CXC chemokines in monocyte recruitment chemokines, not as potent chemoattractants but as potent promoters of firm adhesion of monocytes to the endothelium. Chemokine expression has not only been found in occlusive atherosclerotic plaques, but both MCP-1 and IL-8 have been found to be associated with macrophage rich regions in abdominal aortic aneurysms [71].

2. Chemokines and specific cellular interactions In this section, we will focus on the interactions between chemokines and cells involved in atherosclerotic plaque development. Indeed Table 1 specifically highlights chemokine functions relevant to atherosclerosis. However, it is important to remember that many chemokines have multiple target cells and functions, e.g. MCP-1, in addition to being a potent monocyte and T-cell chemoattractant, has activity on basophils [3,10], RANTES and MCP-4 can attract eosinophils [3,10,72,73] and ELC is also a chemoattractant for B-cells [74]. How can inflammatory responses be specific? As discussed in the sections above, leukocyte recruitment into tissues is a complex, multistep process in which chemokines, while playing a central role, do not act alone. It is this multistep process from initial leukocyte-EC recognition to stable attachment, involving adhesion molecules, chemokines and integrins, that

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requires multiple receptor – ligand pairs and control points. This is the basis of the model for diversity and specificity in leukocyte-EC recognition which implies that the use of different combinations of receptor–ligand pairs allows for an individual receptor – ligand pair to be involved in more than one leukocyte trafficking event [75,76].

2.1. Endothelial cells As mentioned earlier, the luminal endothelium is the first point of contact for circulating leukocytes in the artery wall and thus plays a pivotal role in the initiation of atherosclerosis lesions. There are many examples in vitro of upregulation of adhesion molecule and chemokine expression in endothelial cells by modified lipoproteins [38–40,42 – 44,46]. It is well documented that inflammatory cytokines have potent effects on chemokine production by EC. Human umbilical vein EC stimulated with interferon (IFN) g and tumour necrosis factor (TNF) a results in strong RANTES production and this occurs at the mRNA level [77]. Cultured lung microvascular EC stimulated with the appropriate cytokine or combination of cytokines can express increased levels of IL-8 and MCP-1 [78]. Expression of inflammatory cytokines, capable of regulating local chemokine production, in atherosclerotic lesions is very prevalent [79,80] and indeed, as mentioned above, MCP-1 has been shown to be expressed by endothelial cells in early atherosclerotic lesions and MCP-4 expression has been observed on the luminal endothelium of coronary arteries [62]. Fractalkine, the only CX3C chemokine discovered to date [7], displays properties of both traditional chemokines and of adhesion molecules [7,24]. This new chemokine is of potential interest in atherosclerosis as it is a potent chemoattractant for both monocytes and T-cells and is up-regulated on inflammatory cytokine stimulated EC [7]. In addition, it has been found that adhesion via fractalkine and it’s receptor CX3CR1 appears to be independent of activation of integrins and chemotaxis [24]. The mucin stalk domain is thought to function primarily as a presentation molecule for the chemokine domain. Interestingly, if another CC chemokine, TARC [16], is linked to the mucin domain of fractalkine, it is capable of inducing adhesion of cells expressing it’s receptor CCR4. It remains to be seen, therefore, whether chemokines in general, presented on molecules such as proteoglycans, can directly mediate adhesion of leukocytes in addition to enhancing adhesiveness of integrins. It will be of great interest to determine whether this chemokine is expressed on the endothelium of blood vessels. A further process in which chemokines play an important role, which may have important implications for atherosclerosis and related conditions, is neovascu-

larisation/angiogenesis [81]. Neovascularisation in atherosclerosis, restenosis and aneurysm disease is well documented [82,83] and has been shown to be associated with plaque haemorrhage [84]. Areas of arterial atherosclerosis are associated with a marked increase in the density of microvessels and although endothelial cell proliferation within plaques is quite variable, in some cases it is remarkably elevated [82]. Neovascularisation is an important component of inflammatory responses and associated repair/remodelling [85]. Indeed, areas of neovascularisation in atherosclerotic plaques and aneurysms are strongly associated with macrophages [83]. As mentioned earlier in this review, CXC chemokines containing the functional ELR motif (IL-8; GRO a, b, g, GCP-2; NAP-2) are angiogenic, and those which do not are angiostatic (IP-10; MIG; PF4) [9]. Interestingly, IL-8, which is expressed by macrophages in both atherosclerosis and aneurysms, is a potent angiogenic factor [65], equivalent on a molar basis to traditional angiogenic factors such as basic fibroblast growth factor and vascular endothelial growth factor [86]. Although there is no evidence as of yet for expression of CXCRs on plaque microvessel EC, CXCR1 mRNA has been found to be expressed in human umbilical vein EC in vitro [28]. It is interesting to speculate that the extent of expression of macrophage derived angiogenic or angiostatic CXC chemokines during neovascularisation of atherosclerotic plaques may contribute to the overall regulation of this process. One can further envisage a highly controlled process of CXC chemokine production by macrophages and receptor expression by EC under normal conditions, which during the excessive inflammatory response characteristic of atherosclerosis, shifts in favour of an increase in angiogenic factors to cope with increased remodelling.

2.2. Monocyte/macrophages Monocyte migration into the vessel wall is one of earliest events in the pathogenesis of atherosclerosis. The earliest visible stage of the disease, the so called ‘fatty streak’ is characterised by the presence of macrophages and T-cells in the intima [87]. The CC chemokines MCP-1, MCP-4, MIP-1a, MIP-1b and RANTES, all known to be expressed in atherosclerotic plaques [54–56,61,62], are chemoattractants for monocytes [88]. MCP-1 has been postulated to be a major chemokine involved in monocyte infiltration during atherosclerosis. mRNA for its receptor, CCR2, is expressed by monocytes and found to be decreased during monocyte differentiation into macrophages [89]. Incubation of a monocyte cell line, THP-1s, with LDL, resulted in an increase in CCR2 mRNA and protein, resulting in an increased chemotactic response to MCP1 [90]. OxLDL reduced levels of CCR2 mRNA and this

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is probably due to its ability to stimulate monocyte to macrophage differentiation [90]. In addition, CCR2 mRNA expression and MCP-1 binding is inhibited by IFN g in monocytic cell lines [91]. These findings imply that CCR2s main function is the recruitment of monocytes from the bloodstream and that perhaps other chemokines/receptors are more important for movement of macrophages within lesions. Down-regulation of CCR2 receptors on monocytes once they have entered the lesion could serve to maintain the cells in a specific area of inflammation until other chemokine signals are transmitted. In support of this idea, CCR5 (receptor for RANTES, MIP-1a and MIP-1b) expression increases during monocyte differentiation [92] so one may envisage the ligands for this receptor being important for signalling to cells within the lesion. Interestingly, MIP-1a and b co-localise in atherosclerotic plaques [61] and are expressed by T-cells in macrophage/foam cell rich areas of the plaque, strongly suggesting cross-talk between T-cells and macrophages through chemokine signalling, resulting in movement of macrophages to the appropriate inflammatory zones. Macrophages are present throughout all stages of atherosclerosis and are considered to be the major inflammatory mediators during disease progression. Having entered the atherosclerotic lesion environment, macrophages function as scavenger cells internalising modified lipoprotein particles, becoming foam cells [87] and there is strong evidence to suggest that macrophages act as antigen-presenting cells to T-cells during the immune response which occurs during the disease [93]. In addition to responding to regulatory molecules produced by other cells in the vessel wall, macrophages themselves are a source of cytokines/ chemokines and growth factors [87]. Macrophages are certainly the richest source of chemokines in atherosclerotic lesions [54–56,62,63,67,68]. Chemokine production can be stimulated in monocytes through a vast number of mediators present in the atherosclerotic plaque, most notably inflammatory cytokines, produced by activated T-cells and macrophages [94] and modified LDL particles [40,43,44,46,95]. Thrombin-activated platelets can also induce the expression and secretion of MCP-1 and IL-8 by monocytes [96]. A new human CC chemokine has been described recently by several groups as MIP-5 [97], leukotactin-1 [98], HCC-2 [99] and MIP-1d [100]. This chemokine is part of a sub-group of the CC chemokines which contain two extra cysteines [101]. There are several murine chemokines of this class which are chemotactic for neutrophils, a property which is unusual amongst the CC chemokines [101]. Functional activity has been observed for MIP-5 on human neutrophils by one group [98], but not by others [97,99,100]. Of interest in the context of atherosclerosis and its typical inflammatory cell infiltrate is the reported chemotactic activity of

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MIP-5 on monocytes and T-cells [97–100]. In addition, MIP-5 is found to be expressed at low levels by unstimulated monocytic cell lines, but greatly enhanced by LPS or inflammatory cytokine stimulation [102].

2.3. T-cells The presence of T-cells in addition to monocyte/ macrophages in atherosclerotic lesions of both humans and animal models indicates that adaptive immunological events in conjunction with inflammatory ones are implicated in atherogenesis [51–53]. In addition to mounting a local immune response, T-cells along with macrophages occur in large numbers at the sites of plaque rupture [103]. T-cell secreted inflammatory cytokines are capable of inducing metalloproteinase expression by macrophages [93], strongly implicating this cell type in the development of unstable plaque. Several chemokines are chemoattractants for both T-cells and monocytes, such as MCP-1,-2,-3, RANTES, MIP-1a and b [104–107]. Recently, a number of CC chemokines have been described and shown to selectively attract lymphocytes — PARC [11,12], ELC [14], SLC [15], LARC [13] and TARC [16]. We examined advanced human plaques for the expression of PARC/DC-CK1, ELC, SLC and LARC and found both PARC and ELC, but not SLC or LARC, to be expressed [63]. Of particular interest in the context of atherosclerosis and the involvement of local immune responses, is the finding that PARC shows preferential activity for resting, antigen naive T-cells (CD45RA + ) [12]. This property is not shared by other T-cell chemoattractants such as IP-10, MIG, RANTES, IL-8, MIP-1a, MIP-1b or MCP-1 [12,27,105–107]. As illustrated in Fig. 2, we have found PARC mRNA to be highly expressed in human atherosclerotic plaques and to be exclusively expressed by macrophages [63]. The receptor for PARC has not yet been identified, but saturation studies have shown that human blood lymphocytes express a single class of high affinity receptors for PARC [11]. It is known that the T-cells in advanced human atherosclerotic plaques are of a polyclonal origin, due to their heterogenous T-cell antigen receptor gene rearrangement patterns [108]. Human atherosclerotic plaques contain both CD4+ and CD8 + T-cells of the CD45RO+ memory and the CD45RA+ antigen naive phenotypes, the majority being CD45RO+ [109]. It is at present unknown whether the activated T-cells present in the lesions, implicated in plaque rupture, are preferentially recruited as such or activated locally. Activated T-cells could be recruited from the blood compartment by chemokines such as MCP-1, RANTES, MIP-1a or IP-10. Alternatively, resting naive T-cells may be recruited into the plaque by chemokines such as PARC and activated as a consequence of antigen presentation within the plaque. Several antigens

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are thought to be associated with atherosclerosis, namely oxidised LDL and also heat shock proteins and bacterial and viral antigens [93,110] which could explain the polyclonal nature of the T-cells found in plaques. The most prevalent T-cell type found in lesions is the CD4+ helper cells that recognise antigen associated with class II MHC (major histocompatibility complex) molecules. All of the molecular components required for antigen presentation to CD4+ T-cells are present in the atherosclerotic plaque [93]. In order for naive T-cells to be completely activated and to mount an immune response after antigen presentation, signals from co-stimulatory molecules are required [111]. B7-1 and B7-2 (the major co-stimulatory molecules for T-cells) are expressed by macrophages in atheroscerotic plaques and their ligands by plaque T-cells [112] allowing T-cell activation to progress. The CD40 receptor and CD40L (ligand) are important co-stimulatory factors in antigen presentation and autoimmunity, as well as T-cell activation [113]. In addition to activated T-cells, both receptor and ligand have been detected on EC, SMC and macrophages in atherosclerotic plaques [114]. Due to the potential for over-responding, T-cell activation would need to be highly controlled and given the relatively small number of activated T-cells observed [79,112], it appears that this is the case. Taking this information together, plaque macrophages could initiate naive T-cell attraction and activation via PARC synthesis and antigen (e.g. oxidised LDL) presentation. This could spark a series of events that eventually could lead to a strong local inflammatory response, tissue destruction and plaque destabilisation. A study has shown that after activation, T-cells express different chemokine receptors as they develop into memory/effector cells [115]. Therefore, in the atherosclerotic plaque, once T-cells have been exposed to antigen and activated, different sets of chemokines/receptors probably take over in the recruitment of these cells to sites of inflammation. Atherosclerosis is associated with Th-1 type responses, as shown by the production of INF g by plaque T-cells [79]. Interestingly, CXCR3 is found to be preferentially expressed on Th-1 type cells as opposed to Th-2 type cells [115]. IP-10 and/or MIG could, therefore, be important in local T-cell responses during atherosclerosis. CCR5 is expressed on both Th-1 and Th-2 type cells but at higher levels on Th-1s. CCR5 is highly influenced by the activation state of the T-cell [115] which could highlight a role for RANTES, MIP 1a and MIP-1b in the full blown immune response. This may explain the high levels of RANTES expression detected in almost every cell type during transplant-associated accelerated atherosclerosis [64].

2.4. Vascular smooth muscle cells SMC proliferation and migration in vivo and in vitro are under the control of a host of growth factors and

cytokines produced by macrophages, T-cells, and endothelial cells [87,116]. However, in addition to being under the control of growth factors/cytokines, SMC themselves are a source of inflammatory mediators. As mentioned above, several chemokines have been found to be expressed by SMC in atherosclerotic lesions [54,57,63]. In vitro, chemokines can be up-regulated in VSMC treated with inflammatory stimuli. The CXC chemokine IP-10 can be induced in LPS or INF g stimulated SMC and this chemokine is also up-regulated after balloon injury of the rat carotid artery [117]. In contrast to the transient MCP-1 up-regulation observed following balloon injury [58], IP-10 expression is sustained for up to 2 weeks [117]. Our finding of high ELC expression by SMC in atherosclerotic plaques further indicates a direct inflammatory role of vascular SMC in atherosclerosis and indeed we found that in vitro, ELC mRNA could be up-regulated in human aortic SMC by a combination of INF g and TNF a [63]. Several other studies have shown increased levels of expression/secretion of chemokines in SMC by inflammatory cytokine treatment, e.g. human VSMC treated with IL-1 a or TNF a secrete increased amounts of IL-8, MCP-1 and RANTES [118], while human airway SMC can express RANTES mRNA and protein in response to treatment with TNF a alone, and to a greater extent with TNF a in combination with INF g [119]. Angiotensin II, a potent vasoconstrictor, can also directly up-regulate MCP-1 expression in rat SMC [120]. There are several reports of chemokine activity on VSMC proliferation and migration. IP-10 has been shown to be a more potent chemoattractant for SMC than IL-8, angiotensin II, basic fibroblast growth factor, endothelin, or transforming growth factor-b [117]. A study has shown that SMC constitutively express mRNA for the chemokine receptors CCR1 and CCR2, but not CCR3, CCR4, CCR5, nor CXCR1 or CXCR2 [30]. In addition, it was found that MIP-1a binding to VSMC caused an increase in intracellular Ca2 + levels, a signal frequently associated with proliferation and migration/ chemotaxis [30]. Receptors for the murine chemokine TCA3, have been demonstrated on rat SMC and this chemokine can stimulate SMC chemotaxis, proliferation, and increase adhesiveness to type III collagen [121]. Paradoxically, IL-8 has been shown to be both a mitogen and chemoattractant for VSMC [66]. It is unclear how this response may occur as VSMC are reported not to express either CXCR1 nor CXCR2 [30]. Regulation of surface chemokine receptor expression on VSMC has not been studied but some preliminary observations suggest that foetal bovine serum may be able to down-regulate chemokine receptor mRNA in VSMC [30]. Ambiguous results have also been reported for MCP-1 activity on VSMC — it has been shown to be both a positive and negative regulator of rat VSMC proliferation by two independent groups ([122,123], respectively). Therefore,

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the culture conditions under which these type of cell proliferation/migration experiments are carried out may profoundly influence the results obtained. In vivo studies using antibodies against chemokines in the balloon injury model should shed more light on the importance of these molecules on SMC activity. The current findings do suggest, however, that through production of chemokines in response to inflammatory stimuli, SMC themselves function as inflammatory mediators during atherosclerosis.

3. Evidence from gene knockout mice for the involvement of MCP-1and CCR2 in atherosclerosis Direct evidence for a role of MCP-1 and its receptor in the pathogenesis of atheroscerosis has come from studies where mice lacking the CCR2 receptor [124] were crossed with ApoE knockout mice [125]. CCR2 ( − / − ) mice show significant defects in leukocyte adhesion [126], monocyte/macrophage recruitment, [124,126,127] and a reduction in INF g production when exposed to both antigenic and nonantigenic stimuli [124]. When these mice were crossed with the ApoE ( − / − ) mice and fed a Western type diet, they displayed a marked decrease in lesion formation [125,128]. There was not only a decrease in lesion size, but also a decrease in the number of macrophages in these lesions, supporting the hypothesis that MCP-1 is a major chemokine involved in monocyte recruitment during atherosclerosis and that the process of monocyte recruitment is a major determinant of lesion size and complexity. An independent study has shown that mice lacking the ligand (MCP-1 (−/ −) mice), when crossed with LDL receptor (LDLR) ( −/ − ) mice, show decreased lesion size and a significant reduction of macrophages in lesions [129]. Another important finding in both of these studies is the fact that lesion lipid accumulation was reduced without an effect on total plasma lipids. A recent study has described that overexpression of MCP-1 in ApoE (−/ −) mice results in accelerated atherosclerosis through an increase in lesion macrophage number and lipid accumulation [130]. Inhibition of chemokines/receptors in atherosclerosis could provide a route for treatment of atherosclerosis in normolipidemic, as well as hyperlipidemic subjects, for the latter probably in combination with lipid lowering drugs. Taken together, these studies provide compelling evidence for a direct role of MCP-1 and CCR2 in monocyte recruitment during atherosclerosis. The fact that there are still macrophages in the lesions, albeit fewer, do point to other chemokines/cytokines having a role in the disease, and indeed it remains to be seen in the long term whether lesions in mice lacking MCP-1 or its receptor would remain less severe. Nevertheless, these studies show that interfering with a single

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chemokine/receptor can have profound effects on disease progression in the ApoE and LDLR (− /−) mice without an effect on plasma lipids.

4. Conclusion There is no doubt that the description of the chemokine superfamily has filled a gap in our understanding of basic immunological mechanisms. In terms of atherosclerosis, proof of concept for short term inhibition of lesion progression has been demonstrated in animal models. The next question is, whether in order to effect the pathogenesis of the disease longterm, targeting of multiple chemokines/receptors will be necessary. As the role of chemokines in the pathophysiology of atherosclerosis and other inflammatory conditions is better understood, we will undoubtedly hear much more about the therapeutic benefits of manipulating this fascinating family of receptor/ligands.

References [1] Baggiolini M. Chemokines and leukocyte traffic. Nature 1998;392:565 – 8. [2] Wang JM, Su S, Gong W, Oppenheim JJ. Chemokines, receptors, and their role in cardiovascular pathology. Int J Clin Lab Res 1998;28:83 – 90. [3] Luster AD. Chemokines — chemotactic cytokines that mediate inflammation. New Engl J Med 1998;338:436 – 45. [4] Robinson EA, Yoshimura T, Leonard EJ, Tanaka S, Griffen PR, Shabanowitz J, Hunt DF, Appella E. Complete amino acid sequence of a human monocyte chemoattractant, a putative mediator of cellular immune reactions. Proc Natl Acad Sci USA 1989;86:1850 – 4. [5] Baggiolini M, Walz A, Kunkel SL. Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that attracts neutrophils. J Clin Invest 1989;84:1045 – 9. [6] Kennedy J, Kelner G, Kleyensteuber S, Schall TJ, Weiss MC, Yssel H, Schneider PV, Cocks BG, Bacon KB, Zlotnik A. Molecular cloning and functional characterization of human lymphotactin. J Immunol 1995;155:203 – 9. [7] Fernando-Bazan J, Bacon KB, Hadiman G, Wang W, Soo K, Rossi D, Greaves DR, Zlotnik A, Schall TJ. A new class of membrane-bound chemokine with a CX3C motif. Nature 1997;385:640 – 4. [8] Baggiolini M, Dewald B, Moser B. Human chemokines: an update. Annu Rev Immunol 1997;15:675 – 705. [9] Strieter RM, Polverini PJ, Kunkel SL, Arenberg DA, Burdick MD, Kasper J, Dzuiba J, Van Damme J, Walz A, Marriot D, Chan S-Y, Roczniak S, Shanafelt AB. The functional role of the ELR motif in CXC chemokine-mediated angiogenesis. J Biol Chem 1995;270:27348– 57. [10] Graves DT, Jiang Y. Chemokines, a family of chemotactic cytokines. Crit Rev Oral Biol Med 1995;6:109 – 18. [11] Hieshima K, Imai T, Baba M, Shoudai K, Ishizuka K, Nakagawa T, Tsuruta J, Takeya M, Sakaki Y, Takatsuki K, Miura R, Opdenakker G, Van Damme J, Yoshie O, Nomiyama H. A novel human CC chemokine PARC that is most homologous to macrophage-inflammatory protein-15aLD78a and chemotactic for T lymphocytes, but not for monocytes. J Immunol 1997;159:1140– 9.

222

T.J. Reape, P.H.E. Groot / Atherosclerosis 147 (1999) 213–225

[12] Adema GJ, Hartgers F, Verstraten R, deVries E, Marland G, Menon S, Foster J, Xu G, Nooyen P, McClanahan T, Bacon KB, Figdor CG. A dendritic-cell-derived C–C chemokine that preferentially attracts naive T-cells. Nature 1997;387:713 – 7. [13] Hieshima K, Imai T, Opdenakker G, Van Damme J, Kusuda J, Tei H, Sakaki Y, Takagi S, Nishimura M, Kakizaki M, Nomiyama H, Yoshie O. Molecular cloning of a novel human CC chemokine liver and activation-regulated chemokine (LARC) expressed in liver. J Biol Chem 1997;272:5846– 53. [14] Yoshida R, Imai T, Hieshima K, Kusuda J, Baba M, Kitaura M, Nishimura M, Kakizaki M, Nomiyama H, Yoshie O. Molecular cloning of a novel human CC chemokine EBI1-ligand chemokine that is a specific functional ligand for EBI1, CCR7. J Biol Chem 1997;272:13803–9. [15] Nagira M, Imai T, Hieshima K, Kusuda J, Ridanpaa M, Takagi S, Nishimura M, Kakizaki M, Nomiyama H, Yoshie O. Molecular cloning of a novel CC chemokine secondary lymphoid-tissue chemokine (SLC) that is a potent chemoattractant for lymphocytes and mapped to chromosome 9p13. J Biol Chem 1997;272:19518– 24. [16] Imai T, Yoshida T, Baba M, Nishimura M, Kakizaki M, Yoshie O. Molecular cloning of a novel T-cell-directed CC chemokine expressed in thymus by signal sequence trap using Epstein – Barr virus vector. J Biol Chem 1996;271:21514–21. [17] Nomiyama H, Imai T, Kusuda J, Miura R, Callen DF, Yoshie O. Assignment of the human CC chemokine gene TARC (SCYA17) to chromosome 16q13. Genomics 1997;40:211 – 3. [18] Shirozu M, Nakano T, Inazawa J, Tashiro K, Tada H, Shinohara T, Honjo T. Structure and chromosomal localization of the human stromal cell derived factor 1. Genomics 1995;28:495 – 500. [19] Horuk R. Molecular properties of the chemokine receptor family. Trends Pharmacol Sci 1994;151:159–65. [20] Murphy PM. Chemokine receptors: structure, function and role in microbial pathogenesis. Cyt Growth Fact Rev 1996;7:47 – 64. [21] Bonini JA, Martin SK, Dralyuk F, Roe MW, Philipson LH, Steiner DF. Cloning, expression, and chromosomal mapping of a novel human CC-chemokine receptor (CCR10) that displays high-affinity binding for MCP-1 and MCP-3. DNA Cell Biol 1997;16:1249 – 56. [22] Nibbs RJB, Wylie SM, Yang J, Landau NR, Graham GJ. Cloning and characterization of a novel promiscuous human b-chemokine receptor D6. J Biol Chem 1997;272:32078– 83. [23] Wells TNC, Power CA, Proudfoot AEI. Definition, function and pathophysiological significance of chemokine receptors. TIPS 1998;19:376 – 80. [24] Imai T, Hieshima K, Haskwell C, Baba M, Nagira M, Nishimura M, Kakizaki M, Takagi S, Nomiyama H, Schall TJ, Yoshie O. Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion, Cell 1997:521–530. [25] Yoshida T, Imai T, Kakizaki M, Nishimura M, Takagi S, Yoshie O. Identification of single C motif-1/lymphotactin receptor XCR1. J Biol Chem 1998;273:16551–4. [26] Farber JM. Mig and IP-10: CXC chemokines that target lymphocytes. J Leuk Biol 1997;61:246–57. [27] Xu L, Kelvin DJ, Ye GQ, Taub DD, Ben-Baruch A, Oppenheim JJ, Wang JM. Modulation of IL-8 receptor expression on purified human T lymphocytes is associated with changed chemotactic responses to IL-8. J Leuk Biol 1995;57:335 – 42. [28] Gupta SK, Lysko PG, Pillarisetti K, Ohlstein E, Stadel JM. Chemokine receptors in human endothelial cells — functional expression of CXCR4 and it’s transcriptional regulation by inflammatory cytokines. J Biol Chem 1998;273:4282–7. [29] Volin MV, Joseph L, Shockley MS, Davies PF. Chemokine receptor CXCR4 expression in endothelium. Biochem Biophys Res Commun 1998;242:46–53.

[30] Hayes IM, Jordon NJ, Towers S, Smith G, Paterson JR, Earnshaw JJ, Roach AG, Westwick J, Williams RJ. Human vascular smooth muscle cells express receptors for CC chemokines. Arterioscler Thromb Vasc Biol 1998;18:397 – 403. [31] Schall TJ, Bacon KB. Chemokines, leukocyte trafficking, and inflammation. Curr Opin Immunol 1994;6:865 – 73. [32] McFadden G, Kelvin D. New strategies for chemokine inhibition and modulation: you take the high road and I’ll take the low road. Biochem Pharmacol 1997;12:1271 – 80. [33] Ross R. Atherosclerosis — An inflammatory disease. New Engl J Med 1999;340:115 – 26. [34] Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 1994;76:301– 14. [35] Proost P, Wuyts A, Van Damme J. The role of chemokines in inflammation. Int J Clin Lab Res 1996;26:211 – 23. [36] Ross R. Atherosclerosis: a defence mechanism gone awry. Am J Pathol 1993;143:987 – 1002. [37] Pettersson K, Bjo¨rk H, Bondjers G. Endothelial integrity and injury in atherogenesis. Transplant Proc 1993;25:2054–6. [38] Jeng JR, Chang CH, Shieh SM, Chiu HC. Oxidised low-density lipoprotein enhances monocyte-endothelial cell binding against shear-stress-induced detachment. Biochem Biophys Acta 1993;1178:221– 7. [39] Erl W, Weber PC, Weber C. Monocytic cell adhesion to endothelial cells stimulated by oxidised low density lipoprotein is mediated by distinct endothelial ligands. Atherosclerosis 1998;136:297 – 303. [40] Cushing SD, Berliner JA, Valente AJ, Territo MC, Navab M, Parhami F, Gerrity R, Schwartz CJ, Fogelman AM. Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells. Proc Natl Acad Sci USA 1990;87:5134 – 8. [41] Steinbrecher UP, Parthasarathy S, Leake DS, Witzum JL, Steinberg D. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids. Proc Natl Acad Sci USA 1984;81:3883 – 7. [42] Hurt-Camejo E, Camejo G. Potential involvement of type II in atherosclerosis. Atherosclerosis phospholipase A2 1997;132:1 – 8. [43] Takahara N, Kashiwagi A, Maegawa H, Shigeta Y. Lysophosphatidylcholine stimulates the expression and production of MCP-1 by human vascular endothelial cells. Metabolism 1996;45:559 – 64. [44] Klouche M, Gottschling S, Gerl V, Hell W, Husmann M, Dorweiler B, Messner M, Bhakdi S. Atherogenic properties of enzymatically degraded LDL. Selective induction of MCP-1 and cytoxic effects on human macrophages. Arterioscler Thromb Vasc Biol 1998;18:1376 – 85. [45] Torzewski M, Klouche M, Hock J, Messner M, Dorweiler B, Torzewski J, Gabbart HE, Bhakdi S. Immunohisochemical demonstration of enzymatically modified human LDL and its colocalization with the terminal complement complex in the early atherosclerotic lesion. Arterioscler Thromb Vasc Biol 1998;18:369 – 78. [46] Klouche M, May AE, Hemmes M, Messner M, Kanse SM, Preissner KT, Bhakdi S. Enzymatically modified, nonoxidised LDL induces selective adhesion and transmigration of monocytes and T lymphocytes through human endothelial cell monolayers. Arterioscler Thromb Vasc Biol 1999;19:784 – 93. [47] Kol A, Libby P. The mechanisms by which infectious agents may contribute to atherosclerosis and its clinical manifestations. Trends Cardiovasc Med 1998;8:191 – 9. [48] Nagel T, Resnick N, Atkinson WJ, Dewey CF Jr, Gimbrone MA Jr. Shear stress selectively upregulates intercellular adhesion

T.J. Reape, P.H.E. Groot / Atherosclerosis 147 (1999) 213–225 molecule – 1 expression in cultured human vascular cells. J Clin Invest 1994;94:885 – 91. [49] Shyy YJ, Hsieh HJ, Usami S, Chien S. Fluid shear stress induces a biphasic response of human monocyte chemotactic protein 1 gene expression in vascular endothelium. Proc Natl Acad Sci USA 1994;91:4678 –82. [50] Campbell JJ, Hedrick J, Zlotnik A, Siani MA, Thompson DA, Butcher EC. Chemokines and the arrest of lymphocytes rolling under flow conditions. Science 1998;279:381–3. [51] Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK. Regional accumulations of T-cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis 1986;6:131 – 8. [52] Yokota T, Hansson GK. Immunological mechanisms in atherosclerosis. Int J Med 1995;238:479–89. [53] Watanabe T, Haraoka S, Shimokama T. Inflammatory and immunological nature of atherosclerosis. Int J Cardiol 1996;54:S51 – 60. [54] Nelken NA, Coughlin SR, Gordon D, Wilcox JN. Monocyte chemoattractant protein-1 in human atheromatous plaques. J Clin Invest 1991;88:1121–7. [55] Yla¨-Herttuala S, Lipton BA, Rosenfeld ME, Sa¨rkioja T, Yoshimura T, Leonard EJ, Witztum JL, Steinberg D. Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Proc Natl Acad Sci USA 1991;88:5252 –6. [56] Takeya M, Yoshimura T, Leonard EJ, Takahashi K. Detection of monocyte chemoattractant protein-1 in human atherosclerotic lesions by an anti-monocyte chemoattractant protein-1 monoclonal antibody. Hum Pathol 1993;24:539–43. [57] Yu X, Dluz S, Graves DT, Zhang L, Antoniades HN, Hollander W, Prusty S, Valente AJ, Schwartz CJ, Sonenshein GE. Elevated expression of monocyte chemoattractant protein 1 by vascular smooth muscle cells in hypercholesterolemic primates. Proc Natl Acad Sci USA 1992;89:6953–7. [58] Taubman MB, Rollins BJ, Poon M, Marmur J, Green RS, Berk BC, Nadal-Ginard. JE mRNA accumulates rapidly in aortic injury and in platelet-derived growth factor-stimulated vascular smooth muscle cells. Circ Res 1992;70:314–25. [59] Stark VK, Hoch JR, Warner TF, Hullett DA. Monocyte chemotactic protein-1 expression is associated with the development of vein graft intimal hyperplasia. Arterioscler Thromb Vasc Biol 1997;17:1614 – 21. [60] Wysocki SJ, Zheng MH, Smith A, Lamawansa MD, Iacopetta BJ, Robertson TA, Papadimitriou JM, House AK, Norman PE. Monocyte chemoattractant protein-1 gene expression in injured pig artery coincides with early appearance of infiltrating monocyte/macrophages. J Cell Biochem 1996;62:303–13. [61] Wilcox JN, Nelken NA, Coughlin SR, Gordon D, Schall TJ. Local expression of inflammatory cytokines in human atherosclerotic plaques. J Atheroscler Thromb 1994;1(Suppl 1):S3 – S10. [62] Berkhout TA, Sarau HM, Moores K, White JR, Elshourbagy N, Appelbaum E, Reape TJ, Brawner M, Makwana J, Foley JJ, Schmidt DB, Imburgia C, McNulty D, Matthews J, O’Donnell K, O’Shannessy D, Scott M, Groot PHE, Macphee C. Cloning, in vitro expression and functional characterization of a novel human CC chemokine of the MCP family (MCP-4), which binds and signals through the CC chemokine receptor 2B. J Biol Chem 1997;272:16404– 13. [63] Reape TJ, Rayner K, Manning CD, Gee AN, Barnette MS, Burnand KG, Groot PHE. Expression and cellular localisation of the CC chemokines PARC and ELC in human atheroscerotic plaques. Am J Pathol 1999;154:365–74. [64] Pattison JM, Nelson PJ, Huie P, Sibley RK, Krensky AM. RANTES chemokine expression in transplant-associated accelerated atherosclerosis. J Heart Lung Transplant 1996;15:1194 – 9.

223

[65] Koch AE, Polverini PJ, Kunkel SL, Harlow LA, DiPietro LA, Elner VM, Elner SG, Strieter RM. Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science 1992;258:1798– 801. [66] Yue TL, Wang X, Sung CP, Olson B, McKenna PJ, Gu JL, Feuerstein GZ. Interleukin-8: a mitogen and chemoattractant for vascular smooth muscle cells. Circ Res 1994;75:1 – 7. [67] Apostolopoulos J, Davenport P, Tipping PG. Interleukin-8 production by macrophages from atheromatous plaques. Arterioscl Thromb Vasc Biol 1996;16:1007 – 12. [68] Wang N, Tabas I, Winchester R, Ravalli S, Rabbani LE, Tall A. Interleukin 8 is induced by cholesterol loading of macrophages and expressed by macrophage foam cells in human atheroma. J Biol Chem 1996;271:8837 – 42. [69] Boisvert WA, Santiago R, Curtiss LK, Terkeltaub RA. A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice. J Clin Invest 1998;101:353 – 63. [70] Gerszten RE, Garcia-Zepeda EA, Lim YC, Yoshida M, Ding HA, GimbroneJr MA, Luster AD, Luscinskas FW, Rosenzweig A. MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature 1999;398:718 – 23. [71] Koch AE, Kunkel SL, Pearce WH, Shah MR, Parikh D, Evanoff HL, Haines GK, Burdick MD, Strieter RM. Enhanced production of the chemotactic cytokines interleukin-8 and monocyte chemoattractant protein-1 in human abdominal aortic aneurysms. Am J Pathol 1993;142:1423 – 31. [72] Uguccioni M, Loetscher P, Forssmann U, Dewald B, Li H, Lima SH, Li Y, Kreider B, Garotta G, Thelen M, Baggiolini M. Monocyte chemotactic protein 4 (MCP-4), a novel structural and functional analogue of MCP-3 and eotaxin. J Exp Med 1996;183:2379– 84. [73] Garcia-Zepeda EA, Combadiere C, Rothenberg ME, Sarafi MN, Lavigne F, Hamid Q, Murphy PM, Luster AD. Human monocyte chemoattractant protein (MCP)-4 is a novel CC chemokine with activities on monocytes, eosinophils, and basophils induced in allergic and nonallergic inflammation that signals through the CC chemokine receptors (CCR)-2 and -3. J Immunol 1996;157:5613– 26. [74] Yoshida R, Nagira M, Imai T, Baba M, Takagi S, Tabira Y, Akagi J, Nomiyama H, Yoshie O. EBI1-ligand chemokine (ELC) attracts a broad spectrum of lymphocytes-activated Tcells strongly up-regulate CCR7 and efficiently migrate toward ELC. Int Immunol 1998;10:901 – 10. [75] Butcher EC. Leukocyte – endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 1991;67:1033–6. [76] Foxman EF, Campbell JJ, Butcher EC. Multistep navigation and the combinatorial control of leukocyte chemotaxis. J Cell Biol 1997;139:1349– 60. [77] MarfaingKola A, Devergne O, Gorgone G, Portier A, Schall TJ, Galanaud P, Emilie D. Regulation of the production of the RANTES chemokine by endothelial cells. Synergistic induction by INF g plus TNF a and inhibition by IL-4 and IL-3. J Immunol 1995;154:1870– 8. [78] Brown Z, Gerritsen ME, Carley WW, Strieter RM, Kunkel SL, Westwick J. Chemokine gene expression and secretion by cytokine-activated human microvascular endothelial cells. Differential regulation of monocyte chemoattractant protein-1 and interleukin-8 in response to interferon-g. Am J Pathol 1994;145:913 – 21. [79] Hansson GK, Holm J, Jonasson L. Detection of activated T lymphocytes in the human atherosclerotic plaque. Am J Pathol 1989;135:169 – 75. [80] Rus HG, Niculescu F, Vlaicu R. Tumour necrosis factor-alpha in human arterial wall with atherosclerosis. Atherosclerosis 1991;89:247 – 54.

224

T.J. Reape, P.H.E. Groot / Atherosclerosis 147 (1999) 213–225

[81] Moore BB, Arenberg DA, Strieter RM. The role of CXC chemokines in the regulation of angiogenesis in association with lung cancer. Trends Cardivasc Med 1998;8:51–8. [82] O’Brien ER, Garvin MR, Dev R, Stewart DK, Hinohara T, Simpson JB, Schwartz SM. Angiogenesis in human coronary atherosclerotic plaques. Am J Pathol 1994;145:883–94. [83] Thompson MM, Jones L, Nasim A, Sayers RD, Bell PRF. Angiogenesis in abdominal aortic aneurysms. Eur J Vasc Endovasc Surg 1996;11:464–9. [84] Fan TPD, Jaggar R, Bicknell R. Controlling the vasculature: angiogenesis, anti-angiogenesis and vascular targeting of gene therapy. Trends Pharmacol Sci 1995;16:57–66. [85] Sunderko¨tter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C. Macrophages and angiogenesis. J Leuk Biol 1994;55:410 – 22. [86] Moore BB, Arenberg DA, Strieter RM. The role of CXC chemokines in the regulation of angiogenesis in association with lung cancer. Trends Cardiovasc Med 1998;8:51–8. [87] Ross R. The pathogenesis of atherosclerosis: a perspective of the 1990s. Nature 1993;362:801–8. [88] Uguccioni M, D’Apuzzo M, Loetscher M, Dewald B, Baggiolini M. Actions of the chemotactic cytokines MCP-1, MCP-2, MCP-3, RANTES, MIP-1a and MIP-1b on human monocytes. Eur J Immunol 1995;25:64–8. [89] Wong L-M, Myers SJ, Tsou C-L, Gosling J, Arai H, Charo IF. Organization and differential expression of the human monocyte chemoattractant protein 1 receptor gene. Evidence for the role of the carboxyl-terminal tail in receptor trafficking. J Biol Chem 1997;272:1038–45. [90] HoonHan K, Tangirala RK, Green SR, Quehenberger O. Chemokine receptor CCR2 expression and monocyte chemoattractant protein-1-mediated chemotaxis in human monocytes: a regulatory role for plasma LDL. Arterioscler Thromb Vasc Biol 1998;18:1983 –91. [91] Tangirla RK, Murao K, Quehenberger O. Regulation of expression of the human monocyte chemotactic protein-1 receptor (HCCR2) by cytokines. J Biol Chem 1997;272:8050– 6. [92] Tuttle DL, Harrison JK, Anders C, Sleasman JW, Goodenow MM. Expression of CCR5 increases during monocyte differentiation and directly mediates macrophage susceptibility to infection by human immunodeficiency virus type 1. J Virol 1998;72:4962 – 9. [93] Hansson GK. Cell-mediated immunity in atherosclerosis. Curr Opin Lipidol 1997;8:301–11. [94] Mantovani A. The interplay between primary and secondary cytokines: cytokines involved in the regulation of monocyte recruitment. Drugs 1997;54(Suppl 1):15–23. [95] Terkeltaub R, Banka CL, Solan J, Santoro S, Brand K, Curtiss LK. Oxidised LDL induces monocytic cell expression of interleukin-8, a chemokine with T lymphocyte chemotactic activity. Arterioscler Thromb 1994;14:47–53. [96] Weyrich AS, Elstad MR, McEver RP, McIntyre TM, Moore KL, Morrissey JH, Prescott SM, Zimmerman GA. Activated platelets signal chemokine synthesis by human monocytes. J Clin Invest 1996;97:1525–34. [97] Coulin F, Power CA, Alouani S, Peitsh MC, Schroeder JM, Moshizuki M, Clark-Lewis I, Wells TNC. Characterisation of macrophage inflammatory protein-5/human CC cytokine-2, a member of the macrophage-inflammatory-protein family of chemokines. Eur J Biochem 1997;248:507–15. [98] Youn B-S, Zhang SM, Lee EK, Park DH, Broxmeyer HE, Murphy PM, Locati M, Pease JE, Kim KK, Antol K, Kwon BS. Molecular cloning of leukotactin: a novel human bchemokine, a chemoattractant for neutrophils, monocytes, and lymphocytes, and a potent agonist at CC chemokine receptors 1 and 3. J Immunol 1997;159:5201–5. [99] Pardigol A, Forssmann U, Zucht HD, Loetscher P, SchulzKnappe P, Baggiolini M, Forssmann WG, Ma¨gert HJ. HCC-2,

[100]

[101] [102]

[103]

[104]

[105]

[106]

[107]

[108]

[109]

[110]

[111] [112]

[113]

[114]

[115]

[116] [117]

[118]

a human chemokine: gene structure, expression pattern, and biological activity. Proc Natl Acad Sci USA 1998;95:6308–13. Wang W, Bacon KB, Oldham ER, Schall TJ. Molecular cloning and functional characterization of human MIP-1d, a new C–C chemokine related to mouse CCF-18 and C10. J Clin Immunol 1998;18:214 – 22. Zlotnik A, Morales J, Hedrick JA. Recent advances in chemokines and chemokine receptors. Crit Rev Immunol 1999;19:1–47. Zhang S, Youn BS, Gao JL, Murphy PM, Kwon BS. Differential effects of leukotactin-1 and macrophage inflammatory protein-1a on neutrophils mediated by CCR1. J Immunol 1999;162:4938– 42. van der Wal AC, Becker AE, vanderLoos CM, Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterised by an inflammatory process irrespective of the dominant plaque morphology. Circulation 1994;89:36 – 44. Taub DD, Proost P, Murphy WJ, Anver M, Longo DL, VanDamme J, Oppenheim JJ. Monocyte chemotactic protein-1 (MCP-1), -2, and -3 are chemotactic for human T lymphocytes. J Clin Invest 1995;95:1370 – 6. Woldemar Carr M, Roth SJ, Luther E, Rose SS, Springer TA. Monocyte chemoattractant protein 1 acts as a T lymphocyte chemoattractant. Proc Natl Acad Sci USA 1994;91:3652–6. Schall TJ, Bacon K, Toy KJ, Goeddel DV. Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES. Nature 1990;347:669 – 71. Taub DD, Conlon K, Lloyd AR, Oppenheim JJ, Kelvin DJ. Preferential migration of activated CD4+ and CD8+ T-cells in response to MIP-1a and MIP-1b. Science 1993;260:355–8. Stemme S, Rymo L, Hansson GK. Polyclonal origin of T lymphocytes in human atherosclerotic plaques. Lab Invest 1991;65:654 – 60. Stemme S, Holm J, Hansson GK. T lymphocytes in human atherosclerotic plaques are memory cells expressing CD45RO and the integrin VLA-1. Arterioscler Thromb 1992;12:206–11. Stemme S, Faber B, Holm J, Wiklund O, Witztum JL, Hansson GK. T lymphocytes from human atherosclerotic plaques recognize oxidized low density lipoprotein. Proc Natl Acad Sci USA 1995;92:3893 – 7. Janeway CA Jr, Bottomly K. Signals and signs for lymphocyte responses. Cell 1994;76:275 – 85. de Boer OJ, Hirsch F, vanderWal AC, vanderLoos CM, Das PK, Becker AE. Costimulatory molecules in human atherosclerotic plaques: an indication of antigen specific T lymphocyte activation. Atherosclerosis 1997;133:227 – 34. Mach F, Scho¨nbeck U, Libby P. CD40 signaling in vascular cells: a key role in atherosclerosis? Atherosclerosis 1998;137(Suppl):S89 – 95. Mach F, Scho¨nbeck U, Sukhova GK, Bourcier T, Bonnefoy JY, Pober JS, Libby P. Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for CD40 – CD40 ligand signaling in atherosclerosis. Proc Natl Acad Sci USA 1997;94:1931–6. Sallusto F, Lenig D, Mackay CR, Lanzavecchia A. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J Exp Med 1998;187:875–83. Raines EW, Ross R. Smooth muscle cells and the pathogenesis of the lesions of atherosclerosis. Br Heart J 1993;69:S30 –7. Wang X, Yue TL, Ohlstein EH, Sung CP, Feuerstein GZ. Interferon-inducible protein-10 involves vascular smooth muscle cell migration, proliferation, and inflammatory response. J Biol Chem 1996;271:24286– 93. Jordon NJ, Watson ML, Williams RJ, Roach AG, Yoshimura T, Westwick J. Chemokine production by human vascular smooth muscle cells: modulation by IL-13. Br J Pharmacol 1997;122:749 – 57.

T.J. Reape, P.H.E. Groot / Atherosclerosis 147 (1999) 213–225 [119] John M, Hirst SJ, Jose PJ, Robichaud A, Berkman N, Witt C, Twort CHC, Barnes PJ, FanChung K. Human airway smooth muscle cells express and release RANTES in response to T helper 1 cytokines: regulation by T helper 2 cytokines and corticosteroids. J Immunol 1997;158:1841–7. [120] Chen X-L, Tummala PE, Olbrych MT, Alexander RW, Medford RM. Angiotensin II induces monocyte chemoattractant protein-1 gene expression in rat vascular smooth muscle cells. Circ Res 1998;83:952–9. [121] Luo Y, D’Amore PA, Dorf ME. b-Chemokine TCA3 binds to and activates rat vascular smooth muscle cells. J Immunol 1996;157:2143– 8. [122] Porreca E, DiFebbo C, Reale M, Castellani ML, Baccante G, Barbacane R, Conti P, Cuccurullo F, Poggi A. Monocyte chemotactic protein 1 (MCP-1) is a mitogen for cultured rat vascular smooth muscle cells. J Vasc Res 1997;34:58–65. [123] Ikeda U, Okada K, Ishikawa S-E, Saito T, Kasahara T, Shimada K. Monocyte chemoattractant protein 1 inhibits growth of rat vascular smooth muscle cells. Am J Physiol (Heart Circ Physiol 37) 1995;268:H1021–6. [124] Boring L, Gosling J, Chensue SW, Kunkel SL, Farese RV Jr, Broxmeyer HE, Charo IF. Impaired monocyte migration and reduced type 1 (Th1) cytokine responses in C – C

.

[125]

[126]

[127]

[128]

[129]

[130]

225

chemokine receptor 2 knockout mice. J Clin Invest 1997;100:2552– 61. Boring L, Gosling J, Cleary M, Charo IF. Decreased lesion formation in CCR2 −/ − mice reveals a role for chemokines in the initiation of atherosclerosis. Nature 1998;394:894 –7. Kuziel WA, Morgan SJ, Dawson TC, Griffen S, Smithies O, Ley K, Maeda N. Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2. Proc Natl Acad Sci USA 1997;94:12053– 8. Kurihara T, Warr G, Loy J, Bravo R. Defects in macrophage recruitment and host defense in mice lacking the CCR2 chemokine receptor. J Exp Med 1997;186:1757– 62. Dawson TC, Kuziel WA, Osahar TA, Maeda N. Absence of CC chemokine receptor-2 reduces atherosclerosis in apolipoprotein E-deficient mice. Atherosclerosis 1999;143:205 –11. Gu L, Okada Y, Clinton SK, Gerard C, Sukhova GK, Libby P, Rollins BJ. Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice. Mol Cell 1998;2:275 – 81. Aiello RJ, Bourassa PAK, Lindsey S, Weng W, Natoli E, Rollins BJ, Milos PM. Monocyte chemoattractant protein-1 accelerates atherosclerosis in apolipoprotein E-deficient mice, Arterioscler. Thromb. Vasc. Biol. 1999:1518 – 1525.