Chemoattractant-stimulated granule release from neutrophils, basophils and eosinophils is critical for the innate immune response against infectious bacteria. Interleukin 8 (IL-8) activation of the chemokine receptor CXCRI was found to stimulate rapid formation of beta-arrestin complexes with Hck or c-Fgr. Formation of beta-arrestin-Hck complexes led to Hck activation and trafficking of the complexes to granule-rich regions. Granulocytes expressing a dominant-negative beta-arrestin-mutant did not release granules or activate tyrosine kinases after IL-8 stimulation. Thus, beta-arrestins regulate chemokine-induced granule exocytosis, indicating a broader role for beta-arrestins in the regulation of cellular functions than was previously suspected.

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Background-Recent genetic data in mouse and humans suggest that the chemokine receptors CCR2 and CX3CR1 are involved in atherogenesis; however, detailed molecular and cellular mechanisms have not been fully delineated. Methods and Results-Here, we show that oxidized linoleic acid metabolites, which are components of oxidized LDL found in large amounts in atherosclerotic plaque, were able to specifically induce differentiation of human monocytes to macrophages with decreased expression of CCR2, confirming a previous report, and increased expression of CX3CR1. These macrophages acquired the ability to adhere to coronary artery smooth muscle cells. The adhesion was mediated directly and predominantly by CX3CR1. Reciprocal effects of these lipids on CCR2 and CX3CR1 expression were mediated by the nuclear receptor peroxisome proliferator-activated receptor (PPAR) ␥, and targeting the PPAR␥ gene with sRNAi dramatically reduced macrophage adhesion to coronary artery smooth muscle cells. Conclusions-These data suggest that in atherogenesis oxidized lipid-driven activation of macrophage PPAR␥ in the intima may result in a proadhesive chemokine receptor switch-CCR2 off, CX3CR1 on-causing cessation of CCR2-dependent migration and activation of CX3CR1-dependent retention mechanisms, which together promote macrophage accumulation in vessel wall. Our results may explain at the molecular and cell biology levels the genetic link between CX3CR1 and atherosclerosis. Moreover, they identify macrophage binding to coronary artery smooth muscle cells as the first primary cell setting in which CX3CR1 functions as the major adhesion system. (Circulation. 2006;114:807-819.)

Oxidative stress and inflammation are accepted as major factors in the pathogenesis of atherosclerosis, but how they interact to produce a plaque has not been delineated clearly. Recent data suggest that oxidized lipids may act in part by regulating production of chemokines and chemokine receptors, which in turn, may direct monocytes and other blood leukocytes to the vessel wall, where they may interact with endothelial cells and smooth muscle cells. The receptors may act at the level of recruitment, retention, and egress, not only through classic, chemotactic mechanisms but also through direct, intercellular adhesion. The results suggest a coordinated mechanism for inflammatory cell accumulation in plaque and identify novel targets, such as CCR2 and CX3CR1, for potential drug development in coronary artery disease.

Recent genetic evidence has implicated the adhesive chemokine CX3CL1 and its leukocyte receptor CX3CR1 in atherosclerosis. We previously proposed a mechanism involving foam cell anchorage to vascular smooth muscle cells because: 1) CX3CL1 and CX3CR1 are expressed by both cell types in mouse and human atherosclerotic lesions; 2) foam cells are reduced in lesions in cx3cr1 ؊/؊ apoE ؊/؊ mice; and 3) proatherogenic lipids (oxidized low density lipoprotein [oxLDL] and oxidized linoleic acid derivatives) induce adhesion of primary human macrophages to primary human coronary artery smooth muscle cells (CASMCs) in vitro in a macrophage CX3CR1-dependent manner. Here we analyze this concept further by testing whether atherogenic lipids regulate expression and function of CX3CL1 and CX3CR1 on CASMCs. We found that both oxLDL and oxidized linoleic acid derivatives indirectly up-regulated CASMC CX3CL1 at both the protein and mRNA levels through an autocrine feedback loop involving tumor necrosis factor ␣ production and NF-B signaling. Oxidized lipids also up-regulated CASMC CX3CR1 but through a different mechanism. Oxidized lipid stimulation also increased adhesion of macrophages to CASMCs when CASMCs were stimulated prior to assay, and a synergistic pro-adhesive effect was observed when both cell types were prestimulated. Selective inhibition with a CX3CL1-specific blocking antibody indicated that adhesion was strongly CASMC CX3CL1-dependent. These findings support the hypothesis that CX3CR1 and CX3CL1 mediate heterotypic anchorage of foam cells to CASMCs in the context of atherosclerosis and suggest that this chemokine/chemokine receptor pair may be considered as a pro-inflammatory target for therapeutic intervention in atherosclerotic cardiovascular disease.Atherosclerosis involves a complex interplay of inflammatory cells, vascular elements, and lipoproteins coordinated by adhesion molecules, cytokines, and chemokines (1, 2). Oxidation of low density lipoprotein (LDL) 3 and its accumulation in the subendothelial space are key initiating events that promote accumulation of leukocytes and other cell types that organize over time to form plaque (3). Leukocyte recruitment mechanisms are unclear; however, recent genetic data from mouse and man have implicated members of the chemokine family, a large group of leukocyte chemoattractants active at G proteincoupled receptors (4). Of these, the evidence for CX3CL1 (also known as fractalkine) and its receptor CX3CR1 is particularly strong (5-10). CX3CL1 is an atypical multimodular chemokine that exists both in membrane-tethered and shed forms. The immobilized form consists of a chemokine domain anchored to the plasma membrane through an extended mucin-like stalk, a transmembrane helix, and an intracellular domain (11). Transmembrane CX3CL1 is an adhesion molecule that mediates integrin-independent cell capture by binding to CX3CR1 on target cells (12). Following protease-mediated release of the chemokine domain (13, 14), CX3CL1 may also promote classical chemotactic responses of ...

The chemokine receptor CX3CR1 (CX3C chemokine receptor 1) is expressed in mouse blood on natural killer (NK) cells and on monocytes. Because interleukin-15 (IL-15) is an essential cytokine for NK cell development and maintenance, we hypothesized that it may induce CX3CR1 expression on this cell type. In contrast, we found that in primary mouse bone marrow-derived NK cells IL-15 specifically inhibited CX3CR1 protein and mRNA accumulation, whereas the related cytokine IL-2 did not inhibit but instead increased CX3CR1 expression. Consistent with this finding, intravenous injection of a single dose of recombinant IL-15 into C57BL/6 mice decreased steady-state CX3CR1 levels 24 hours after injection in freshly isolated peripheral blood mononuclear cells ( IntroductionDifferential expression of chemokine receptors coordinates specific leukocyte trafficking, which is important for development, distribution, and deployment of the immune system. 1 Lymphocyte subsets differentially express most of the 18 known human chemokine receptors and may be marked by them. For example, CXC chemokine receptor 3 (CXCR3) is preferentially expressed on T helper type 1 (Th1) effector T cells, and CC chemokine receptor 7 (CCR7) marks naive T cells that home to secondary lymphoid tissue. 2,3 In contrast to lymphocytes, neutrophils and eosinophils more uniformly express smaller subgroups of chemokine receptors. The interleukin-8 (IL-8) receptors CXCR1 and CXCR2 are strongly expressed on human neutrophils 4,5 and CCR3 is characteristic of human eosinophils. 6 CX3CR1 (CX3C chemokine receptor 1) is unusual among chemokine receptors because it is not expressed on mouse T cells but is expressed on 5% to 30% of mouse natural killer (NK) cells. 7 In humans, this receptor is expressed on cytotoxic CD8 ϩ and CD4 ϩ T cells, 8 as well as on most cells in the major subset of human NK cells, which have the immunophenotype CD56 dim CD16 ϩ . 9,10 In addition, CX3CR1 is expressed on mouse and human monocytes, 7,11 neurons, and microglia. 7,12,13 CX3CR1, like all chemokine receptors, is a 7-transmembrane domain G protein-coupled receptor (GPCR). 1 Fractalkine (CX3CL1) is its only known ligand, and CX3CR1 is fractalkine's only known mammalian receptor (the viral receptor US28 also binds CX3CL1). 14 CX3CR1 is classified among the inflammatory subgroup of chemokine receptors because it is selectively expressed on effector leukocytes. 8,11,15 In addition to its expression pattern, it is unusual compared with other chemokine receptors in 2 major ways. First, it is one of the few inflammatory chemokine receptors that has only one ligand. 16 Second, unlike most other chemokine receptors it functions as an adhesion molecule, 11 able to mediate integrin-and G protein-independent adhesion of receptor-bearing leukocytes to CX3CL1-expressing endothelial cells under both static and physiologic flow conditions. 17 The latter property is due in part to the unusual structure of CX3CL1, which in addition to having a typical chemokine domain has 3 other modules: a transmem...

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Atherosclerosis is a complex pathologic process in which chemokine-mediated leukocyte accumulation in arterial walls is thought to be an important mechanism of pathogenesis. An interesting exception to this paradigm is the chemokine CXCL16, also known as the scavenger receptor for phosphatidylserine and oxidized low density lipoprotein, which is highly expressed in mouse and human atherosclerotic lesions, yet appears to be atheroprotective. In this study, we address potential mechanisms responsible for this activity. Consistent with its presence in atherosclerotic plaque, we found that atherogenic lipids up-regulated CXCL16 in primary human monocyte-derived macrophages. However, the same lipids down-regulated the CXCL16-targeted protease ADAM10, resulting in preferential expression of CXCL16 as the transmembrane form, not the shed form. Although transmembrane CXCL16 is known to mediate cell-cell adhesion by binding its receptor CXCR6, and atherogenic lipids are known to stimulate macrophage adhesion to coronary artery smooth muscle cells, we found that heterotypic adhesion of these cell types occurred in a CXCL16-independent manner. Instead we found that in macrophages, CXCL16 promoted internalization of both oxidized low density lipoprotein and high density lipoprotein, as well as release of cholesterol. Moreover, CXCL16 deficiency in macrophages interfered with oxidized low density lipoprotein-induced up-regulation of atheroprotective genes: adenosine triphosphate-binding cassette transporter A1 and G1 as well as apolipoprotein E. Thus, our findings support the hypothesis that CXCL16 mediates atheroprotection through its scavenger role in macrophages and not by cell-cell adhesion.