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(Circulation. 2003;108:2498.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Smooth Muscle Cells in Human Atherosclerotic Plaques Express the Fractalkine Receptor CX₃CR1 and Undergo Chemotaxis to the CX₃C Chemokine Fractalkine (CX₃CL1)

Andrew D. Lucas, PhD^*; Christina Bursill, PhD^*; Tomasz J. Guzik, MD, PhD; Jerzy Sadowski, MD, PhD; Keith M. Channon, MD; David R. Greaves, PhD

From the Sir William Dunn School of Pathology, University of Oxford (A.D.L., C.B., D.R.G.), and Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford (C.B., T.J.G., K.M.C.), UK; and the Department of Internal Medicine, Jagiellonian University School of Medicine, J. Dietl Hospital, Kraków, Poland (T.J.G., J.S.).

Correspondence to David R. Greaves, Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK. E-mail david.greaves{at}path.ox.ac.uk

Received November 14, 2002; de novo received June 11, 2003; revision received August 14, 2003; accepted August 19, 2003.

	Abstract

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Background— Chemokines are important mediators of inflammatory cell recruitment that play a significant role in atherosclerosis. Fractalkine (CX₃CL1) is an unusual membrane-bound chemokine that mediates chemotaxis through the CX₃CR1 receptor. Recently, functional polymorphisms in the human CX3CR1 gene have been described that are associated with coronary artery disease.

Methods and Results— We investigated the expression of the CX₃C chemokine fractalkine and its receptor CX₃CR1 in human coronary artery plaques by immunocytometry. We show that a subset of mononuclear cells expresses high levels of fractalkine in human coronary atherosclerotic plaques and that smooth muscle cells within the neointima express the fractalkine receptor CX₃CR1. There is a positive correlation between the number of fractalkine-expressing cells and the number of CX₃CR1-positive cells in human atherosclerotic plaques (r=0.70, n=15 plaques). Furthermore, we demonstrate that cultured vascular smooth muscle cells express the CX₃CR1 receptor and undergo chemotaxis to fractalkine that can be inhibited by G protein inactivation by pertussis toxin.

Conclusions— These results suggest that in human atherosclerosis, fractalkine, rather than mediating inflammatory cell recruitment, can act as a mediator of smooth muscle cell migration.

Key Words: chemokines • atherosclerosis • macrophages • muscle, smooth • remodeling

	Introduction

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Local and systemic inflammation play critical roles in the development of atherosclerosis.^1–4 Monocytes that adhere to inflamed endothelium are recruited into the subendothelial space, where they differentiate into macrophages. Recruited macrophages endocytose modified forms of LDL via scavenger receptors to form foam cells, which are the predominant cell type present in fatty streak lesions.⁵ These lesions develop into fibrofatty plaques, which contain large numbers of macrophages and CD4+ T cells and show evidence of smooth muscle cell (SMC) migration and proliferation.^5–7

Chemokines are a superfamily of chemoattractant cytokines that play an essential role in leukocyte recruitment in acute and chronic inflammation.^5,8–10 A key role for the CC chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) has been demonstrated in murine models of atherogenesis. LDLR^-/- and apoE^-/- mice with deletion of either the MCP-1 or the CCR2 gene exhibit reduced atherosclerosis.^5,11,12 Recently, MCP-1 has been demonstrated to play an important role in other forms of vascular remodeling, including restenosis.^5,13,14 MCP-1 is expressed by a range of cell types in human atherosclerotic plaques, including macrophages and SMCs.¹⁵ Other CC chemokines that are expressed in human atherosclerotic plaques include eotaxin (CCL11), macrophage-derived chemokine (MDC/CCL22), and thymus- and activation-regulated chemokine (TARC/CCL17).^16,17 A potential role for CXC chemokines in the development of atherosclerotic lesions has also been demonstrated in gene knockout animals.^16,18,19

Fractalkine (CX₃CL1) is synthesized as a membrane-bound molecule with the chemokine motif attached to a long mucin stalk.²⁰ It is expressed by a range of cell types in vivo, including neurons,²¹ epithelial cells,²² and macrophages.¹⁷ The receptor for fractalkine, CX₃CR1, is a G protein-coupled receptor (GPCR) with 7 transmembrane (TM7)-spanning regions.²³ The CX₃CR1 receptor is able to mediate tight adhesion of cells to immobilized forms of fractalkine under flow conditions in vitro.^24,25 Fractalkine gene knockout mice have no obvious defects in leukocyte trafficking but are less susceptible to cerebral ischemia-reperfusion injury.^26,27 Further evidence for a possible role of fractalkine in cardiovascular disease has come from recent observations in human subjects suggesting that a single amino acid polymorphism at position 249 of the CX₃CR1 receptor is an independent risk factor for coronary artery disease^28,29 (reviewed by Alexander³⁰). However, the biological rationale for these observations remains unclear

Accordingly, we investigated the presence of fractalkine and its cognate receptor, CX₃CR1, in human coronary artery atherosclerotic plaques. We show that fractalkine expression is principally by mononuclear cells, whereas the fractalkine receptor CX₃CR1 is expressed by intimal SMCs. Furthermore, we demonstrate that human SMCs can undergo chemotaxis to fractalkine in vitro. These results lead us to propose fractalkine as a newly identified mediator of SMC migration in human coronary atherosclerosis.

	Methods

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Human Tissues
Sections of right and left coronary arteries were taken from hearts removed from transplant recipients (9 men, 1 woman; mean age, 51 years) with appropriate local ethical approval and written informed consent. For more details see Table 1.

View this table: [in this window] [in a new window]   TABLE 1. Coronary Artery Samples Used in This Study

Human Primary Cells and Cell Lines
Human coronary artery SMCs were prepared from a 21-year-old male donor (TCS CellWorks). Cells were typically passaged 2 times before use. For immunohistochemistry experiments, 1x10⁴ cells were seeded into glass chamber slides (Nunc) and grown until confluent. For Western blot analysis, cells (5x10⁵) were seeded into 6-well cluster dishes (Costar) and grown until confluent. Where indicated, cells were grown in the presence of tumor necrosis factor- (10 ng/mL) or interferon- (500 U/mL, PeproTech) for 24 hours before analysis. The human SMC line HCM-601EB (a kind gift of Professor Martin Bennett, Addenbrooke’s Hospital, Cambridge, UK) has been described previously.³¹ Transfected CHO-K1 and HEK 293 cells were grown in RPMI 1640 cell culture media supplemented with 10% FCS, glutamine (2 mmol/L), and penicillin/streptomycin (5 IU/mL, 50 µg/mL, respectively; InVitrogen Lifetech).

Chemotaxis Assays
Cell migration was assessed by use of transwell membranes (ChemoTX, 6-mm diameter, 8-µm pore size) as described previously.³² Cells were harvested, placed on transwell membranes, and allowed to migrate toward increasing concentrations of fractalkine or platelet-derived growth factor (PDGF)-AB. Migrated cells on the underside of membranes were fixed (paraformaldehyde-sucrose) and stained with Topro blue. Migration was quantified as total pixel count of blue cells under the confocal microscope (scans performed in triplicate, 3 scans/membrane). Signal transduction via G protein-coupled receptors was blocked by preincubating cells with pertussis toxin (PTX, 250 ng/mL, Sigma-Aldrich) for 2 hours before chemotaxis.

Western Blotting
The expression of CX₃CR1 and fractalkine by primary human coronary artery cells was evaluated by Western blotting, as described previously.²² Detergent lysates derived from human peripheral blood mononuclear cells (PBMCs) or CD14+ monocytes were used as a positive control.

Immunohistochemistry
The following primary antibodies were used: rat anti-human CX₃CR1 (isotype IgG2b, clone 2A9-1, MBL); rabbit anti-human CX₃CR1 N-terminal peptide (ProSci Inc); goat anti-fractalkine chemokine domain (R&D Systems); mouse anti-hemagglutinin epitope (YPYDVPDYA) (isotype IgG1, clone 16B12, Covance); mouse anti-human CD14 (isotype IgG2a, clone TÜK4, Dako AS), mouse anti-human CD3 (isotype IgG1, clone UCHT1, Dako), and mouse anti-human CD68 (isotype IgG1, Clone EBM11, Dako). All antibodies were diluted in PBS containing 1% BSA and 0.2% Triton X-100, and sections were incubated with primary antibodies overnight at 4°C. In cases in which a red label was required for visualization, samples were incubated with anti-species-specific IgG conjugated to Alexa Fluor 568 (Molecular Probes) for 1 hour at 4°C. In cases in which a green label was required for visualization, samples were incubated with anti-species-specific IgG conjugated to biotin (Dako) for 1 hour at 4°C. Finally a streptavidin-fluorescein isothiocyanate (FITC) reagent (Amersham) was applied for 30 minutes at 4°C. Double immunofluorescent labeling involved sequential single-labeling procedures, with all incubations at 4°C. Immunohistochemical detection of single antigens was performed as described previously.^17,22

Validation of Anti-CX₃CR1 Antisera
To verify the specificity of a range of anti-CX₃CR1 reagents, we transfected HEK-293 cells with an expression vector encoding CX₃CR1 tagged at the C-terminus with the influenza hemagglutinin (HA) epitope tag. FACS analysis of transfected cells with mouse anti-hemagglutinin monoclonal antibody detected a strongly positive population of transfected cells, and a similar population was revealed by use of a rat anti-human CX₃CR1 monoclonal antibody. Two commercial rabbit polyclonal reagents varied in their ability to detect CX₃CR1 antigens in FACS and immunofluorescence, with the ProScience anti-N-terminal peptide reagent giving specific staining of CX₃CR1-transfected CHO cells (data not shown).

	Results

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Expression of Fractalkine and CX₃CR1 in Human Coronary Atherosclerosis
To address whether fractalkine could be detected in association with coronary artery disease, freshly isolated human coronary artery segments from heart transplant recipients were examined by immunohistochemistry. We examined 19 separate arteries sampled from 10 patients (Table 1). Most but not all arteries showed intimal areas characterized by an infiltrate of mononuclear cells, clearly counterstained with hematoxylin in the goat IgG isotype control experiment (Figure 1A). A proportion of the infiltrating mononuclear cells within the intima stained strongly positive for fractalkine (Figure 1B). Consistent with our previous studies on carotid and femoral arteries,²⁰ we did not detect fractalkine immunostaining of luminal endothelial cells in any of the sections studied. Indeed, occasional fractalkine-positive mononuclear cells were seen adhering to the unstained luminal endothelium that showed clear staining for von Willebrand factor in adjacent sections (data not shown).

View larger version (86K): [in this window] [in a new window]   Figure 1. Fractalkine expression in human coronary arteries. Adjacent frozen sections (8 to 10 µm) of coronary artery HCA007L were analyzed by immunohistochemistry. Sections were counterstained with hematoxylin and examined by light microscopy. A, Preimmune goat serum; B, goat anti-fractalkine polyclonal reagent. Similar results were obtained with 17 of 19 coronary artery samples.

Next, we performed double immunofluorescence labeling to identify the fractalkine-positive cells within coronary artery sections (red-staining cells in Figure 2, A–E, summarized in Table 2). Infiltrating CD3-positive T cells (stained green in Figure 2A) were present in most lesions examined (9 of 10 plaques), whereas CD14 monocytes (green in Figure 2C) were detected in 4 of 6 lesions examined and were only rarely double positive for fractalkine expression. These CD14 fractalkine double-positive cells were located just beneath the luminal endothelium, consistent with their being recently transmigrated monocytes (Figure 2C). A more substantial proportion of the fractalkine-positive cells double-stained for the CD68 antigen (stained yellow in Figure 2B; CD68 single-positive cells are green). In lesions containing large CD68-positive cells with the appearance of foam cells, there was a lack of double labeling with fractalkine (data not shown). Smooth muscle actin-positive cells were present within the intima (stained green in Figure 2D), and in 7 of 14 lesions examined, we were able to detect some SMCs that expressed fractalkine.

View larger version (39K): [in this window] [in a new window]   Figure 2. Double immunofluorescence analysis of fractalkine- and CX3CR1-expressing cells in human atherosclerosis. A–E, Serial sections from a single representative infiltrated atherosclerotic plaque (HCA007L) double-immunofluorescently labeled with goat anti-human fractalkine (red). A, Anti-CD3 positive T cells (green); B, anti-CD68-positive macrophages (green); C, anti-CD14-positive monocytes (green); D, anti--actin-positive SMCs (green); and E, rat anti-CX3CR1-stained cells (green). F, Staining of same plaque with anti-CX3CR1 (green) and anti--actin (red) revealed that most CX3CR1-positive cells are also positive for SMC actin.

View this table: [in this window] [in a new window]   TABLE 2. Summary of Coronary Artery Sample Double-Staining Experiments

Thus, cells expressing fractalkine in human coronary artery plaques included CD68-positive macrophages, CD14-positive monocytes, some SMCs, and a significant population of mononuclear cells that were negative for these markers but not endothelial cells.

To determine the relationships between cells expressing fractalkine and those expressing the fractalkine receptor in human coronary artery plaques, we performed double immunofluorescent staining for the fractalkine receptor CX₃CR1. Strikingly, fractalkine receptor-positive cells with the characteristics of SMCs (green in Figure 2E) were present adjacent to fractalkine-positive cells (red in Figure 2E) within the intimal region in 16 of the 19 coronary lesions examined, and in 8 of 19 lesions examined, we detected some cells that were positive for both fractalkine and CX3CR1. Confirmation of the CX₃CR1-positive cells as SMCs was obtained by double staining with an anti-smooth muscle actin antibody (yellow cells in Figure 2F). When the numbers of fractalkine- and SMC actin-positive cells were determined, no relationship could be demonstrated (Figure 3A; r=0.1, P=NS). However, there was a strong positive correlation between the numbers of fractalkine-positive and fractalkine receptor-positive cells within lesions (Figure 3B; r=0.70, P<0.01).

View larger version (14K): [in this window] [in a new window]   Figure 3. Quantitative image analysis of fractalkine- and CX3CR1-positive cells in human atherosclerosis. Human coronary arteries were double-labeled for fractalkine and its receptor, CX3CR1. Representative views of human artery sections were electronically captured by CCD camera, and cells positive for either label were counted within a standardized area (1.5x104 µm2) that contained at least 1 fractalkine-positive cell in lesions double-labeled with CX3CR1 or smooth muscle actin (SMA). A, Numbers of fractalkine-positive and SMA-positive cells are shown as an xy plot, and correlation between co-incidence of both markers was calculated. B, Numbers of fractalkine-positive and CX3CR1-positive cells were plotted as an xy plot, and correlation between co-incidence of both markers was calculated. Distinct symbols represent data points from individual arteries.

Primary Human SMCs Express the Fractalkine Receptor CX₃CR1 and Undergo Chemotaxis to Fractalkine
To further investigate expression of the fractalkine receptor CX₃CR1 by SMCs, we cultured primary human coronary artery SMCs to a 60% to 70% confluence on glass chamber slides. Cells were fixed and stained for CX₃CR1 and smooth muscle actin expression. We detected strong, punctate cellular staining for the fractalkine receptor CX₃CR1 relative to rat IgG2b isotype control antibody (Figure 4A). This staining was both perinuclear and clearly distributed on the plasma membrane (Figure 4A, arrow). Western blotting of primary human PBMCs, CD14 positively selected monocytes, and primary human coronary artery SMC lysates for CX₃CR1 revealed a band of 45 kDa in all 3 cell preparations (Figure 4B). Human primary SMC lysates but not monocyte lysates were positive for smooth muscle actin expression (Figure 4C). These observations confirm that primary human SMCs express the fractalkine receptor CX₃CR1.

View larger version (49K): [in this window] [in a new window]   Figure 4. Human SMCs express fractalkine receptor CX3CR1. Human primary coronary artery SMCs grown in normal growth media in glass chamber slides were fixed with 4% paraformaldehyde and labeled with following reagents. A, Inset, Rat IgG2a isotype control followed by anti-rat IgG-FITC and mouse anti-human smooth muscle actin followed by anti-mouse IgG-Cy3. Main panel, rat anti-human CX3CR1 followed by anti-rat IgG-FITC and mouse anti-human SMA followed by anti-mouse IgG-Cy3. Arrow highlights punctate CX3CR1-FITC labeling on surface of a cell extension. B, Western blot analysis of human PBMCs, purified CD14+ human monocytes, and primary coronary artery SMCs probing for CX3CR1 expression. Cell lysates were separated by 8% polyacrylamide-SDS gel electrophoresis, transferred to nitrocellulose membranes, and probed for CX3CR1 expression with a polyclonal rabbit anti-CX3CR1 NT reagent (ProScience), which detects a specific band of 45 kDa. C, Monocyte and SMC lysates were probed for smooth muscle actin expression.

To investigate the responsiveness of human SMCs to recombinant human fractalkine, we used the human SMC line HCM-601EB and primary SMCs in chemotaxis assays in modified Boyden chambers. HCM-601EB cells migrated in response to both recombinant fractalkine (Figure 5A) and PDGF-AB (Figure 5B), showing a characteristic "bell-shaped" curve typical of chemokine migration assays. The maximal migration occurred to 25 nmol/L fractalkine and 4 nmol/L PDGF-AB. As a further test of specificity, we preincubated HCM-601EB cells with PTX (250 ng/mL), an inhibitor of G protein-coupled receptor signaling. As expected, the migratory response to fractalkine was blocked by PTX (Figure 5C), because the fractalkine receptor is a G_i-coupled receptor. In contrast, the response to PDGF, mediated by the tyrosine kinase PDGF receptor, was not affected (Fig 5D). Importantly, we demonstrated that primary human coronary artery SMCs undergo chemotaxis to fractalkine (Figure 5E) and that this chemotaxis is sensitive to PTX inhibition (Figure 5F).

View larger version (23K): [in this window] [in a new window]   Figure 5. Fractalkine mediates SMC chemotaxis. In transwell membranes, human HCM-601EB SMCs were exposed to a range of increasing doses of (A) recombinant human fractalkine (0 to 80 nmol/L) or (B) recombinant PDGF-AB (0 to 24 nmol/L). Migrated cells were fixed and stained on underside of transwell membranes. Migration was quantified as total pixel count of blue fluorescent nuclei imaged by confocal microscopy (triplicate experiments, 3 scans/membrane). C, HCM-601EB cells were allowed to migrate toward buffer alone (blank), fractalkine (Frac) (25 nmol/L) with or without preincubation with PTX (250 ng/mL). By ANOVA, P<0.05 between Blank vs Fractalkine and Fractalkine vs PTX. D, HCM-601EB cells were allowed to migrate toward buffer alone (blank) or PDGF (4 nmol/L) with or without preincubation with PTX (250 ng/mL). P<0.05 between blank vs PDGF and blank vs PDGF+PTX. E, Human coronary artery primary SMCs were allowed to migrate toward increasing concentrations of recombinant human fractalkine (0 to 80 nmol/L). F, Human primary SMCs were allowed to migrate toward buffer alone (blank) or recombinant human fractalkine (40 nmol/L) with or without preincubation with PTX (250 ng/mL). P<0.001 between blank vs fractalkine and fractalkine±PTX.

	Discussion

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We have undertaken a detailed analysis of the expression pattern of the CX₃C chemokine fractalkine and its receptor, CX₃CR1, in human coronary atherosclerosis. Fractalkine is expressed at a high level by a subset of inflammatory cells but not by endothelial cells. The number of fractalkine-expressing cells in the intima is directly correlated with the presence of CX₃CR1⁺ cells, most of which are SMCs. Furthermore, we have demonstrated that primary human SMCs express the CX₃CR1 receptor and can undergo specific CX₃CR1-mediated chemotaxis to fractalkine in vitro. Our results suggest that fractalkine, rather than acting as a proinflammatory cytokine in human atherosclerosis, may play an important role in vascular remodeling through the recruitment of SMCs into the atherosclerotic plaque.

In vitro SMCs³³ and cultured endothelial cells²⁰ treated with a range of inflammatory mediators express fractalkine mRNA and protein. However, we saw no evidence of endothelial expression of fractalkine in human coronary atherosclerosis (Figure 1). Rather, infiltrating mononuclear cells present in the intima of human coronary artery atherosclerotic plaques express high levels of fractalkine. These cells are primarily CD14- and CD68-negative and may represent recruited monocytes that are in the process of differentiation. We used validated anti-CX₃CR1 antibodies to identify fractalkine receptor-expressing cells in human atherosclerosis. Quantitative image analysis shows a positive correlation between the presence of CX₃CR1⁺ cells in the neointima and fractalkine-expressing mononuclear cells (Figure 3B). The majority of these CX₃CR1⁺ cells were shown by double immunofluorescence to be SMCs. Taken together with the chemotaxis data presented in Figure 5, our observations strongly suggest that rather than playing a role in continuing inflammatory cell recruitment, fractalkine expression within human atherosclerotic plaques may be acting to recruit SMCs. By recruiting cells capable of synthesizing substantial amounts of extracellular matrix, fractalkine expression may favor the development of stable atherosclerotic lesions rather than macrophage-rich vulnerable plaques. It will be interesting to determine whether developing atherosclerotic plaques that express high levels of fractalkine within the neointima progress to stable plaques with high numbers of SMCs and fibrous caps. In a recent article, Chandrasekar et al³⁴ reported CX₃CR1-dependent proliferation of primary rat aortic SMCs in response to fractalkine. However, an overenthusiastic recruitment of SMCs into a developing plaque followed by fractalkine-driven SMC proliferation could result in SMC-dominated intimal hyperplasia.

Recently, coding region polymorphisms, V249I and T280M, have been identified in the human CX3CR1 gene and appear to be associated with reduced risk of developing coronary artery disease.^28,29,35 PBMCs prepared from human donors with different CX3CR1 genotypes exhibit differences in the number of fractalkine-binding sites per cell, and cells transfected with variant receptor show impaired ligand-dependent cell adhesion.³⁵ However, the biological rationale for an association between genetic variation in CX₃CR1 and coronary artery disease has remained unclear. Our study now provides novel data on CX₃CR1 expression in human coronary atherosclerosis. Our observations suggest that a potential mechanistic link between CX3CR1 gene polymorphisms in human populations and the development of coronary artery disease may be exerted not only at the level of monocyte recruitment but also at the level of atherosclerotic plaque stability through modulation of SMC recruitment. The only other identified high-affinity receptor for fractalkine is encoded by the US28 open reading frame of human cytomegalovirus, and evidence has been presented for a link between cytomegalovirus infection and SMC proliferation in atherosclerosis.^36,37

More than 40 different human chemokines have been described, and the majority of CC chemokines have been shown to be potent mediators of either monocyte or T-cell chemotaxis.¹⁰ Indeed, CX3CR1^-/- apoE double-knockout mice exhibit reduced macrophage recruitment into atherosclerotic lesions.^38,39 However, our identification of fractal-kine as a chemoattractant for SMCs suggests that this unusual CX₃C chemokine merits further investigation in human atherosclerosis and other forms of vascular remodeling, including restenosis and arteriosclerosis.

	Acknowledgments

 
This work was supported by a Wellcome Trust Cardiovascular Research Initiative Fellowship to Dr Lucas, a Wellcome Trust International fellowship to Dr Guzik, and a British Heart Foundation project grant to Drs Channon and Greaves.

	Footnotes

 
*Drs Lucas and Bursill contributed equally to this work.

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