The fractalkine receptor CX3CR1 is a key mediator of atherogenesis

Myron I. Cybulsky, Robert A. Hegele

J Clin Invest. 2003;111(8):1118-1120. https://doi.org/10.1172/JCI18237.

Commentary

Recruitment of circulating to the arterial intima contributes to the formation of atherosclerotic lesions and may participate in their destabilization. Leukocyte emigration from blood into tissues is mediated by multiple adhesion molecules and , which orchestrate specific steps of emigration and regulate preferential recruitment of different leukocytes depending on their expression patterns of receptors. Over the last several years, a number of adhesion molecules, including VCAM-1, P-selectin and ICAM-1, the chemokines MCP-1 (also known as CCL2) and IL-8 (also known as CXCL8), and their respective receptors CCR2 and CXCR2, have been functionally implicated in atherosclerosis. Two studies — one recently published in the JCI (1), and the second reported in this issue (2) — expand this list to include the CX3CR1, the receptor for fractalkine (also known as CX3CL1). Fractalkine structure and functions Among more than 50 known chemokines, fractalkine is the sole member of the CX3C family, and has unique structural and functional attributes (3, 4). In contrast to many other chemokines, whose presentation on the cell surface requires interaction with proteoglycans, the N-terminal chemokine domain of fractalkine is anchored to the cell membrane through a contiguous extended mucin-like stalk, transmembrane and cytoplasmic domains (Figure 1). Fractalkine binding to its seven-transmembrane domain G –coupled receptor triggers signaling, but it also directly mediates cell adhesion (5). […]

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See the related article beginning on page 1241. triggers signaling, but it also directly mediates cell adhesion (5). Fractal- The fractalkine receptor CX3CR1 is a key kine binds CX3CR1 rapidly and firm- ly, which leads to tethering and arrest mediator of atherogenesis of leukocytes under conditions of physiological flow independent of Myron I. Cybulsky1 and Robert A. Hegele2 CX3CR1 signaling (5). TNF-α–con- verting enzyme (also known as 1Department of Laboratory Medicine and Pathobiology, University of Toronto, ADAM17) can cleave the mucin stalk Toronto General Research Institute, Toronto, Ontario, Canada of fractalkine and release soluble 2Robarts Research Institute, Blackburn Cardiovascular Genetics Laboratory, chemokine (6, 7). CX3CR1 has two London, Ontario, Canada common coding polymorphisms, J. Clin. Invest. 111:1118–1120 (2003). doi:10.1172/JCI200318237. namely V249I and T280M, that are in strong linkage disequilibrium (al- most always occurring on the same Recruitment of circulating monocytes and IL-8 (also known as CXCL8), and allele) and have been associated with to the arterial intima contributes to their respective receptors CCR2 and interindividual differences in suscep- the formation of atherosclerotic CXCR2, have been functionally impli- tibility to both HIV infection and ath- lesions and may participate in their cated in atherosclerosis. Two studies erosclerosis (8–10). If replicated, these destabilization. Leukocyte emigration — one recently published in the JCI findings may have clinical relevance. from blood into tissues is mediated by (1), and the second reported in this multiple adhesion molecules and issue (2) — expand this list to include The role of CX3CR1 in chemokines, which orchestrate specif- the chemokine receptor CX3CR1, the experimental atherogenesis ic steps of emigration and regulate receptor for fractalkine (also known The article by Lesnik et al. (1) demon- preferential recruitment of different as CX3CL1). strated that fractalkine expression was leukocytes depending on their expres- upregulated in atherosclerotic lesions sion patterns of chemokine receptors. Fractalkine structure and functions of apolipoprotein E–/– (apoe–/–) mice, pri- Over the last several years, a number of Among more than 50 known chemo- marily in intimal smooth muscle cells, adhesion molecules, including VCAM-1, kines, fractalkine is the sole member of which is consistent with the expression P-selectin and ICAM-1, the chemo- the CX3C family, and has unique pattern observed previously in human kines MCP-1 (also known as CCL2) structural and functional attributes (3, atherosclerosis. The function of CX3CR1 4). In contrast to many other chemo- in atherosclerosis was assessed by cross- –/– –/– Address correspondence to: Myron I. kines, whose presentation on the cell ing CX3CR1 mice into the apoe Cybulsky, Department of Laboratory surface requires interaction with pro- background and feeding these mice a Medicine and Pathobiology, University of teoglycans, the N-terminal chemokine Western-type diet for 5, 10, or 15 weeks. Toronto, Toronto General Research Institute, domain of fractalkine is anchored to Lesion formation throughout the UHN, 200 Elizabeth Street, Eaton-4, Toronto, Ontario, Canada M5G 2C4. the cell membrane through a con- aorta, including the aortic root, was sig- Phone: (416) 340-3578; Fax: (416) 340-3578; tiguous extended mucin-like stalk, nificantly reduced in the CX3CR1–/– E-mail: [email protected]. transmembrane and cytoplasmic groups. These elegant data provide con- Conflict of interest: The authors have declared that no conflict of interest exists. domains (Figure 1). Fractalkine bind- vincing evidence that CX3CR1 plays an Nonstandard abbreviations used: single ing to its seven-transmembrane important role in experimental athero- nucleotide polymorphism (SNP). domain G protein–coupled receptor genesis, are consistent with a recent

1118 The Journal of Clinical Investigation | April 2003 | Volume 111 | Number 8 Offspring Cohort, contrasts with most association studies principally because the authors included func- tional evaluation of the T280M/ V249I polymorphisms. Relative to T280, the atheroprotective M280/ I249 variant when expressed by trans- fected HEK 293 cells exhibited a lower on-rate of fractalkine binding. Trans- fected K562 cells or primary leuko- cytes from homozygous M280/I249 donors revealed markedly decreased adhesive function, signaling (calcium Figure 1 flux), and chemotaxis. These are Functions of fractalkine and its receptor CX3CR1 in recruitment during atherosclerosis. important and novel insights into the The recruitment of blood monocytes to the arterial intima is one of the earliest events in the for- significance of CX3CR1 coding poly- mation of an atherosclerotic lesion and persists even in advanced lesions. Monocyte emigration is morphisms. The association between a multistep process that includes tethering and rolling, arrest, stable adhesion, and diapedesis reduced risk of atherosclerotic cardio- (transendothelial migration). Shortly after initial tethers are established through binding of selectins or VCAM-1 on activated endothelium, monocytes encounter chemokines bound to vascular disease and CX3CR1-M280/ endothelial cell–surface proteoglycans. Chemokines initiate signaling via chemokine G protein–cou- I249 polymorphisms was based on a pled receptors, which then activates monocyte integrins and subsequently leads to reorganization dominant model, since the majority of the cytoskeleton. Activated integrins mediate arrest and stable adhesion, and contribute to dia- of individuals were heterozygotes pedesis by binding immunoglobulin gene superfamily adhesion molecules, including VCAM-1 and with a fully functional CX3CR1-T280 ICAM-1. Fractalkine, like other chemokines, activates multiple intracellular signaling pathways via allele. Considering that heterozygotes its receptor. However, it is unique, since it is a transmembrane protein that binds to CX3CR1 rap- have only a partial deficiency in idly and firmly, which may directly contribute to monocyte tethering and arrest. During atheroge- nesis, monocytes recruited to the arterial intima transform into macrophages, engulf lipids, and CX3CR1 function and that redun- exhibit morphological features of foam cells. dant pathways for monocyte recruit- ment may exist, assessment of inter- mediate phenotypes, such as monocyte study by Combadiere et al. (11), and inflammation, for example in devel- recruitment or abundance within establish CX3CR1 as a potential thera- opment. Intimal cells within athero- lesions, is required to bolster confi- peutic target. sclerotic lesions express fractalkine dence in the idea that the M280/I249 In the field of experimental athero- and VCAM-1 as well as other chemo- polymorphisms are truly responsible sclerosis, sophisticated transgenic kines and adhesion molecules, which for the significant association with and bone marrow transplantation may influence monocyte and/or reduced atherosclerotic disease. Other approaches have been used to modu- macrophage biology, including sur- possibilities should be explored. late the expression of candidate vival, migration, gene expression or There are several common single . However, the analysis, for the efflux. Unfortunately, little is known nucleotide polymorphisms (SNPs) most part, consists of simple deter- about the fate of individual mono- that lie within the CX3CR1 promoter mination of atherosclerotic lesion cytes and macrophages in atheroscle- (13) and it is necessary to determine area and/or volume and assessment rotic lesions and new approaches are if these affect CX3CR1 transcription of histological features. Similarly, required to elucidate this in trans- and are in linkage disequilibrium Lesnik et al. (1) did not provide direct genic mice. Similarly, new practical with the M280/I249 polymorphisms. evidence of reduced monocyte re- approaches are required to directly Furthermore, as is usually the case in cruitment. It is reasonable to assume assess monocyte recruitment to ath- genetic association studies, extra that deficiency of a chemokine, erosclerotic lesions. complexity arises from the variability chemokine receptor, or adhesion of these linkage relationships among molecule will result in decreased Effects of CX3CR1 polymorphisms different ethnic groups and popula- monocyte recruitment to the arterial on function and atherogenesis tion substrata, not to mention the intima, which will translate into The growing catalogue of genetic possibility that the observed associa- reduced lesion area and volume. Yet, association studies of atherosclerosis tions resulted from linkage disequi- one must keep in mind that there has tended to curb enthusiasm librium with unmeasured function- may be significant redundancy in regarding the role of any particular al variants in closely linked genes, molecular pathways contributing to gene or polymorphism in this com- such as CCR8 (13). Additional stud- leukocyte recruitment, that expres- plex phenotype (12). The paper by ies will be needed to replicate the sion of adhesion molecules and McDermott et al. (2), reporting asso- associations in other samples and chemokines is not restricted to the ciation between CX3CR1 coding poly- the trend in the future should be arterial endothelium, and that these morphisms and various atherosclero- towards analysis of all possible SNPs molecules have functions outside of sis end points in the Framingham at a .

The Journal of Clinical Investigation | April 2003 | Volume 111 | Number 8 1119 Conclusion a role for fractalkine in atherogenesis. J. Clin. Invest. (CX3CL1). J. Biol. Chem. 276:37993–38001. 111:333–340. doi:10.1172/JCI200315555. 8. Faure, S., et al. 2000. Rapid progression to AIDS So where do we stand today? We now 2.McDermott, D.H., et al. 2003. Chemokine in HIV+ individuals with a structural variant of appreciate that CX3CR1 and presum- receptor mutant CX3CR1-M280 has impaired the chemokine receptor CX3CR1. Science. ably fractalkine are important players adhesive function and correlates with protec- 287:2274–2277. tion from cardiovascular disease in man. 9. McDermott, D.H., et al. 2001. Association in atherogenesis, and that CX3CR1 J. Clin. Invest. 111:1241–1250. doi:10.1172/ between polymorphism in the chemokine recep- contains polymorphisms that modu- JCI200316790. tor CX3CR1 and coronary vascular endothelial 3. Bazan, J.F., et al. 1997. A new class of membrane- dysfunction and atherosclerosis. Circ. Res. late its adhesive and signaling func- bound chemokine with a CX3C motif. Nature. 89:401–407. tions and are associated with reduced 385:640–644. 10. Moatti, D., et al. 2001. Polymorphism in risk of atherosclerotic cardiovascular 4. Pan, Y., et al. 1997. Neurotactin, a membrane- the fractalkine receptor CX3CR1 as a genetic anchored chemokine upregulated in brain risk factor for coronary artery disease. Blood. disease. But to truly understand the inflammation. Nature. 387:611–617. 97:1925–1928. mechanism of CX3CR1 and other 5. Imai, T., et al. 1997. Identification and molecular 11. Combadiere, C., et al. 2003. Decreased athero- characterization of fractalkine receptor CX3CR1, sclerotic lesion formation in CX3CR1/apolipo- genes in a highly complex and chronic which mediates both leukocyte migration and protein E double knockout mice. Circulation. disease process such as atherosclerosis, adhesion. Cell. 91:521–530. 107:1009–1016. new practical intermediate markers are 6. Tsou, C.L., Haskell, C.A., and Charo, I.F. 2001. 12. Hegele, R.A. 2002. SNP judgments and freedom Tumor necrosis factor-alpha-converting enzyme of association. Arterioscler. Thromb. Vasc. Biol. required in both experimental models mediates the inducible cleavage of fractalkine. 22:1058–1061. and clinical studies. J. Biol. Chem. 276:44622–44626. 13. DeVries, M.E., et al. 2003. Genomic organization 7. Garton, K.J., et al. 2001. Tumor necrosis factor- and evolution of the CX3CR1/CCR8 chemokine 1. Lesnik, P., Haskell, C.A., and Charo, I.F. 2003. alpha-converting enzyme (ADAM17) mediates receptor locus. J. Biol. Chem. 278:11985–11994. –/– Decreased atherosclerosis in CX3CR1 mice reveals the cleavage and shedding of fractalkine doi:10.1074/jcb.M211422200.

See the related article beginning on page 1221. In laboratory animals, estrogen defi- ciency leads to increased osteoclasto- RANK ligand and the regulation genesis and bone loss, and inhibition of osteoclastogenesis is the major of skeletal remodeling means by which estrogen prevents the loss of bone. This is accomplished by Norman H. Bell diminishing production of IL-1, IL-6, and TNF-α — cytokines that enhance Department of Medicine, Medical University of South Carolina, Charleston, production of M-CSF and RANKL (1, South Carolina, USA 3–5) — and downregulating NF-κB and J. Clin. Invest. 111:1120–1122 (2003). doi:10.1172/JCI200318358. RANKL-induced activation of JNK1 and osteoclastogenic activator pro- tein–1 transcription factors c-Fos and It has long been known that estrogen lifespan, and function. These include c-Jun (7). Estrogen upregulates OPG deficiency in animal models and post- parathyroid hormone (PTH), calcitriol, (8) and TGF-β (9), and TGF-β increas- menopausal women is associated with PTH-related protein, prostaglandin E2, es OPG expression by osteoblasts and increased osteoclastic bone resorption thyroxine, and IL-11 (3–5). The forma- stromal cells (10) and inhibits bone and bone loss (1–5). In the past decade, tion of active osteoclasts requires resorption by increasing apoptosis of several important discoveries of some M-CSF (1, 3–6) and involves cell-to-cell osteoclasts (11) (Figure 1). of the key factors involved in osteoclast contact between precursors of the mo- formation, survival, function, and reg- nocyte/macrophage lineage and osteo- RANKL and postmenopausal ulation by estrogen have been made. blasts, marrow stromal cells, and T and bone loss A number of hormones and cytokines B cells. These cells express the receptor Whether the OPG/RANKL/RANK sys- modulate osteoclastogenesis by enhanc- activator of NF-κB ligand (RANKL), a tem is involved in bone loss caused by ing osteoclast differentiation, activation, member of the TNF ligand family, estrogen deficiency in humans was not which is essential for this process. known until now. In this issue of the JCI Address correspondence to: Norman H. Bell, RANKL attaches to RANK, a receptor (12), Eghbali-Fatourechi and colleagues Department of Medicine, Medical University on the cell surface of osteoclasts and employ an elegant set of experiments to of South Carolina, 114 Doughty Street, osteoclast precursors, to stimulate pro- examine the possible role of RANKL in Charleston, South Carolina 29425, USA. liferation and differentiation of cells to postmenopausal bone loss. The authors Phone: (843) 876-5162; Fax: (843) 876-5163; E-mail: [email protected]. form the osteoclast phenotype and inhib- obtained bone marrow mononuclear Conflict of interest: The author has declared it apoptosis. (OPG), a cells and used surface markers and flow that no conflict of interest exists. soluble decoy receptor produced by cytometry to isolate and identify pre- Nonstandard abbreviations used: osteoblasts, marrow stromal cells, and osteoblastic marrow stromal cells, T parathyroid hormone (PTH); receptor other cells, profoundly modifies the lymphocytes, and B lymphocytes in activator of NF-κB (RANK); RANK ligand (RANKL); osteoprotegerin (OPG); selective effects of RANKL by inhibiting RANKL/ groups of premenopausal women, estrogen receptor modulator (SERM). RANK interaction (1, 3–5). untreated postmenopausal women, and

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