Inhibition of CX3CL1 (fractalkine) improves experimental autoimmune myositis in SJL/J mice

Idiopathic inflammatory myopathy is a chronic inflammatory muscle disease characterized by mononuclear cell infiltration in the skeletal muscle. The infiltrated inflammatory cells express various cytokines and cytotoxic molecules. Chemokines are thought to contribute to the inflammatory cell migration into the muscle. We induced experimental autoimmune myositis (EAM) in SJL/J mice by immunization with rabbit myosin and CFA. In the affected muscles of EAM mice, CX3CL1 (fractalkine) was expressed on the infiltrated mononuclear cells and endothelial cells, and its corresponding receptor, CX3CR1, was expressed on the infiltrated CD4 and CD8 T cells and macrophages. Treatment of EAM mice with anti-CX3CL1 mAb significantly reduced the histopathological myositis score, the number of necrotic muscle fibers, and infiltration of CD4 and CD8 T cells and macrophages. Furthermore, treatment with anti-CX3CL1 mAb down-regulated the mRNA expression of TNF-alpha, IFN-gamma, and perforin in the muscles. Our results suggest that CX3CL1-CX3CR1 interaction plays an important role in inflammatory cell migration into the muscle tissue of EAM mice. The results also point to the potential therapeutic usefulness of CX3CL1 inhibition and/or blockade of CX3CL1-CX3CR1 interaction in idiopathic inflammatory myopathy.     

Fractalkine-mediated endothelial cell injury by NK cells

Endothelial cells (ECs) are primary targets of immunological attack, and their injury can lead to vasculopathy and organ dysfunction in vascular leak syndrome and in rejection of allografts or xenografts. A newly identified CX3C-chemokine, fractalkine, expressed on activated ECs plays an important role in leukocyte adhesion and migration. In this study we examined the functional roles of fractalkine on NK cell activity and NK cell-mediated endothelial cell injury. Freshly separated NK cells expressed the fractalkine receptor (CX3CR1) determined by FACS analysis and efficiently adhered to immobilized full-length fractalkine, but not to the truncated forms of the chemokine domain or mucin domain, suggesting that fractalkine functions as an adhesion molecule on the interaction between NK cells and ECs. Soluble fractalkine enhanced NK cell cytolytic activity against K562 target cells in a dose- and time-dependent manner. This enhancement correlated well with increased granular exocytosis from NK cells, which was completely inhibited by the G protein inhibitor, pertussis toxin. Transfection of fractalkine cDNA into ECV304 cells or HUVECs resulted in increased adhesion of NK cells and susceptibility to NK cell-mediated cytolysis compared with control transfection. Moreover, both enhanced adhesion and susceptibility of fractalkine-transfected cells were markedly suppressed by soluble fractalkine or anti-CX3CR1 Ab. Our results suggest that fractalkine plays an important role not only in the binding of NK cells to endothelial cells, but also in NK cell-mediated endothelium damage, which may result in vascular injury.     

Chemokine receptor CX3CR1 mediates skin wound healing by promoting macrophage and fibroblast accumulation and function

Wounds heal through a highly regulated, self-limited inflammatory response, however, precise inflammatory mediators have not been fully delineated. In this study, we report that in a mouse model of excisional skin wound healing the chemokine CX3CL1 and its receptor CX3CR1 were both highly induced at wound sites; CX3CL1 colocalized with macrophages and endothelial cells, whereas CX3CR1 colocalized mainly with macrophages and fibroblasts. Loss of CX3CR1 function delayed wound closure in both CX3CR1 knockout (KO) mice and in wild-type mice infused with anti-CX3CR1-neutralizing Ab. Conversely, transfer of bone marrow from donor wild-type mice, but not from donor CX3CR1 KO mice, restored wound healing to normal in CX3CR1 KO-recipient mice. Direct effects of CX3CR1 disruption at the wound site included marked reduction of macrophages and macrophage products, such as TGF-beta1 and vascular endothelial growth factor. Consistent with this, we observed reduced alpha-smooth muscle actin (a marker for myofibroblasts) and collagen deposition in skin from wounded CX3CR1 KO mice, as well as reduced neovascularization. Together, the data support a molecular model of skin wound repair in which CX3CR1 mediates direct recruitment of bone marrow-derived monocytes/macrophages which release profibrotic and angiogenic mediators.     

A role for fractalkine and its receptor (CX3CR1) in cardiac allograft rejection

The hallmark of acute allograft rejection is infiltration of the inflamed graft by circulating leukocytes. We studied the role of fractalkine (FKN) and its receptor, CX(3)CR1, in allograft rejection. FKN expression was negligible in nonrejecting cardiac isografts but was significantly enhanced in rejecting allografts. At early time points, FKN expression was particularly prominent on vascular tissues and endothelium. As rejection progressed, FKN expression was further increased, with prominent anti-FKN staining seen around vessels and on cardiac myocytes. To determine the capacity of FKN on endothelial cells to promote leukocyte adhesion, we performed adhesion assays with PBMC and monolayers of TNF-alpha-activated murine endothelial cells under low-shear conditions. Treatment with either anti-FKN or anti-CX(3)CR1-blocking Ab significantly inhibited PBMC binding, indicating that a large proportion of leukocyte binding to murine endothelium occurs via the FKN and CX(3)CR1 adhesion receptors. To determine the functional significance of FKN in rejection, we treated cardiac allograft recipients with daily injections of anti-CX(3)CR1 Ab. Treatment with the anti-CX(3)CR1 Ab significantly prolonged allograft survival from 7 +/- 1 to 49 +/- 30 days (p < 0.0008). These studies identify a critical role for FKN in the pathogenesis of acute rejection and suggest that FKN may be a useful therapeutic target in rejection.     

IL-15 alters expression and function of the chemokine receptor CX3CR1 in human NK cells

The chemokine receptor CX3CR1 is thought to regulate inflammation in part by modulating NK cell adhesion, migration, and killing in response to its ligand CX3CL1 (fractalkine). Recent reports indicate that IL-15, which is essential for development and survival of NK cells, may negatively regulate CX3CR1 expression, however, the effects of the cytokine on human NK cell CX3CR1 expression and function have not been fully delineated. Here, we demonstrate that short term culture in IL-15 decreases surface expression of CX3CR1 on cultured CD56+ cells from human blood resulting in diminished chemotaxis and calcium flux in response to CX3CL1. Cells cultured long term in IL-15 (more than five days) completely lost surface expression as well as mRNA and protein for CX3CR1. The effect was specific since mRNA for CCR5 was increased and mRNA for CXCR4 was unchanged in these cells by IL-15. Thus, exogenous IL-15 is a negative regulator of CX3CR1 expression and function in human CD56+ NK cells. The data imply that the use of IL-15 alone to expand NK cells ex vivo for immunotherapy may produce cells impaired in their ability to traffic to sites of inflammation.     

Serum level of soluble CX3CL1/fractalkine is elevated in patients with polymyositis and dermatomyositis, which is correlated with disease activity

Introduction

Polymyositis (PM) and dermatomyositis (DM) are chronic inflammatory muscle diseases, in which chemokines are thought to contribute to inflammatory cell migration into muscle. In this study, we retrospectively analyzed the expressions of CX3CL1/fractalkine and its corresponding receptor, CX3CR1, in muscle and lung with interstitial lung disease (ILD) of PM patients and DM patients, and determined the correlation between serum soluble CX3CL1 level and disease activity.

Methods

Expressions of CX3CL1 and CX3CR1 in muscle and lung tissue were analyzed by immunohistochemistry. Serum CX3CL1 concentrations were measured by ELISA. For evaluation of patients' disease activity, serum creatinine kinase, manual muscle testing, and the alveolar-arterial oxygen pressure difference were used independently.

Results

CX3CL1 was expressed on infiltrated mononuclear cells and endothelial cells in muscle affected by PM and DM and in lung with ILD, whereas CX3CR1 was expressed on some CD4⁺ T cells, a majority of CD8⁺ T cells, and most macrophages in muscle, and on infiltrated mononuclear cells in the lung. Serum soluble CX3CL1 was significantly higher in PM patients and DM patients than in healthy controls. The CX3CL1 level was correlated with serum creatinine kinase and manual muscle testing score. In patients with PM and DM with ILD, serum CX3CL1 was also correlated with alveolar-arterial oxygen pressure difference. Furthermore, CX3CL1 was significantly decreased after conventional treatment.

Conclusions

The interaction between CX3CL1 and CX3CR1 might contribute to the inflammatory cell infiltration into affected muscle and lung with ILD in PM patients and DM patients. Serum CX3CL1 level could be a surrogate marker of disease activity.     

Eotaxin-3/CC chemokine ligand 26 is a functional ligand for CX3CR1

Eotaxin-3/CCL26 is a functional ligand for CCR3 and abundantly produced by IL-4-/IL-13-stimulated vascular endothelial cells. CCL26 also functions as a natural antagonist for CCR1, CCR2, and CCR5. In this study, we report that CCL26 is yet a functional ligand for CX3CR1, the receptor for fractalkine/CX3CL1, which is expressed by CD16(+) NK cells, cytotoxic effector CD8(+) T cells, and CD14(low)CD16(high) monocytes. Albeit at relatively high concentrations, CCL26 induced calcium flux and chemotaxis in mouse L1.2 cells expressing human CX3CR1 but not mouse CX3CR1 and competed with CX3CL1 for binding to CX3CR1. In chemotaxis assays using human PBMCs, CCL26 attracted not only eosinophils but also CD16(+) NK cells, CD45RA(+)CD27(-)CD8(+) T cells, and CD14(low)CD16(high) monocytes. Intraperitoneal injection of CCL26 into mice rapidly recruited mouse eosinophils and intravenously transferred human CD16(+) NK cells into the peritoneal cavity. IL-4-stimulated HUVECs produced CCL26 and efficiently induced adhesion of cells expressing CX3CR1. Real-time PCR showed that skin lesions of psoriasis consistently contained CX3CL1 mRNA but not CCL26 mRNA, whereas those of atopic dermatitis contained CCL26 mRNA in all samples but CX3CL1 mRNA in only about half of the samples. Nevertheless, the skin lesions from both diseases consistently contained CX3CR1 mRNA at high levels. Thus, CCL26 may be partly responsible for the recruitment of cells expressing CX3CR1 in atopic dermatitis particularly when the expression of CX3CL1 is low. Collectively, CCL26 is another agonist for CX3CR1 and may play a dual role in allergic diseases by attracting eosinophils via CCR3 and killer lymphocytes and resident monocytes via CX3CR1.     

The chemokines, CX3CL1, CCL14, and CCL4, promote human trophoblast migration at the feto-maternal interface

Human embryo implantation is a complex process involving blastocyst attachment to the endometrial epithelium and subsequent trophoblast invasion of the decidua. Chemokines, critical regulators of leukocyte migration, are abundant in endometrial epithelial and decidual cells at this time. We hypothesized that endometrial chemokines stimulate trophoblast invasion. Chemokine receptors CX3CR1 and CCR1 were immunolocalized in human first-trimester implantation sites, specifically to endovascular extravillous trophoblasts, but not to the invading interstitial EVTs (iEVTs), with weak staining also on syncytium. CCR3 was localized to invading iEVTs and to microvilli on the syncytial surface. Expression of CX3CL1 (fractalkine), CCL7 (MCP-3), and their receptors (CX3CR1, CCR1, CCR2, CCR3, and CCR5) mRNA was examined in cellular components of the maternal-embryonic interface by RT-PCR. Both chemokines were abundant in entire endometrium and placenta, endometrial cells (primary cultures and HES, a human endometrial epithelial cell line) and trophoblast cell lines (JEG-3, ACIM-88, and ACIM-32). Chemokine receptor mRNA was expressed by placenta and trophoblast cell lines: CCR1 by all trophoblast cell types, whereas CCR2, CCR3, and CX3CR1 were more variable. CX3CR1, CCR1, CCR2, and CCR5 were also expressed by endometrial cells. Migration assays used the trophoblast cell line most closely resembling extravillous cytotrophoblast (AC1M-88). Trophoblast migration occurred in response to CX3CL1, CCL14, and CCL4, but not CCL7. Endometrial cell-conditioned media also stimulated trophoblast migration; this was attenuated by neutralizing antibodies to CX3CL1 and CCL4. Thus, chemokines are expressed by maternal and embryonic cells during implantation, whereas corresponding receptors are on trophoblast cells. Promotion of trophoblast migration by chemokines and endometrial cell conditioned medium indicates an important involvement of chemokines in maternal-fetal communication.     

CX3CL1 induces cell migration and invasion through ICAM-1 expression in oral squamous cell carcinoma cells

Human oral squamous cell carcinoma (OSCC) has been associated with a relatively low survival rate over the years and is characterized by a poor prognosis. C-X3-C motif chemokine ligand 1 (CX3CL1) has been involved in advanced migratory cells. Overexpressed CX3CL1 promotes several cellular responses related to cancer metastasis, including cell movement, migration and invasion in tumour cells. However, CX3CL1 controls the migration ability, and its molecular mechanism in OSCC remains unknown. The present study confirmed that CX3CL1 increased cell movement, migration and invasion. The CX3CL1-induced cell motility is upregulated through intercellular adhesion molecule-1 (ICAM-1) expression in OSCC cells. These effects were significantly suppressed when OSCC cells were pre-treated with CX3CR1 monoclonal antibody (mAb) and small-interfering RNA (siRNA). The CX3CL1-CX3CR1 axis activates promoted PLCβ/PKCα/c-Src phosphorylation. Furthermore, CX3CL1 enhanced activator protein-1 (AP-1) activity. The CX3CR1 mAb and PLCβ, PKCα, c-Src inhibitors reduced CX3CL1-induced c-Jun phosphorylation, c-Jun translocation into the nucleus and c-Jun binding to the ICAM-1 promoter. The present results reveal that CX3CL1 induces the migration of OSCC cells by promoting ICAM-1 expression through the CX3CR1 and the PLCβ/PKCα/c-Src signal pathway, suggesting that CX3CL1-CX3CR1-mediated signalling is correlated with tumour motility and appealed to be a precursor for prognosis in human OSCC.     

Involvement of CX3CL1/CX3CR1 Signaling in Spinal Long Term Potentiation

The long-term potentiation (LTP) of spinal C-fiber-evoked field potentials is considered as a fundamental mechanism of central sensitization in the spinal cord. Accumulating evidence has showed the contribution of spinal microglia to spinal LTP and pathological pain. As a key signaling of neurons-microglia interactions, the involvement of CX3CL1/CX3CR1 signaling in pathological pain has also been investigated extensively. The present study examined whether CX3CL1/CX3CR1 signaling plays a role in spinal LTP. The results showed that 10-trains tetanic stimulation (100 Hz, 2s) of the sciatic nerve (TSS) produced a significant LTP of C-fiber-evoked field potentials lasting for over 3 h in the rat spinal dorsal horn. Blockade of CX3CL1/CX3CR1 signaling with an anti-CX3CR1 neutralizing antibody (CX3CR1 AB) markedly suppressed TSS-induced LTP. Exogenous CX3CL1 significantly potentiated 3-trains TSS-induced LTP in rats. Consistently, spinal LTP of C-fiber-evoked field potentials was also induced by TSS (100 Hz, 1s, 4 trains) in all C57BL/6 wild type (WT) mice. However, in CX3CR1^-/- mice, TSS failed to induce LTP and behavioral hypersensitivity, confirming an essential role of CX3CR1 in spinal LTP induction. Furthermore, blockade of IL-18 or IL-23, the potential downstream factors of CX3CL1/CX3CR1 signaling, with IL-18 BP or anti-IL-23 neutralizing antibody (IL-23 AB), obviously suppressed spinal LTP in rats. These results suggest that CX3CL1/CX3CR1 signaling is involved in LTP of C-fiber-evoked field potentials in the rodent spinal dorsal horn.     

Dual functions of fractalkine/CX3C ligand 1 in trafficking of perforin+/granzyme B+ cytotoxic effector lymphocytes that are defined by CX3CR1 expression

Fractalkine/CX3C ligand 1 and its receptor CX3CR1 are known to mediate both cell adhesion and cell migration. Here we show that CX3CR1 defines peripheral blood cytotoxic effector lymphocytes commonly armed with intracellular perforin and granzyme B, which include NK cells, gammadelta T cells, and terminally differentiated CD8(+) T cells. In addition, soluble fractalkine preferentially induced migration of cytotoxic effector lymphocytes. Furthermore, interaction of cytotoxic effector lymphocytes with membrane-bound fractalkine promoted subsequent migration to the secondary chemokines, such as macrophage inflammatory protein-1beta/CC ligand 4 or IL-8/CXC ligand 8. Thus, fractalkine expressed on inflamed endothelium may function as a vascular regulator for cytotoxic effector lymphocytes, regardless of their lineage and mode of target cell recognition, through its ability to capture them from blood flow and to promote their emigration in response to other chemokines.     

Atherogenic lipids induce adhesion of human coronary artery smooth muscle cells to macrophages by up-regulating chemokine CX3CL1 on smooth muscle cells in a TNFalpha-NFkappaB-dependent manner

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 alpha production and NF-kappaB 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.     

Fibroblast Growth Factor Receptor 1 Activation in Mammary Tumor Cells Promotes Macrophage Recruitment in a CX3CL1-Dependent Manner

Tumor formation is an extensive process requiring complex interactions that involve both tumor cell-intrinsic pathways and soluble mediators within the microenvironment. Tumor cells exploit the intrinsic functions of many soluble molecules, including chemokines and their receptors, to regulate pro-tumorigenic phenotypes that are required for growth and progression of the primary tumor. Previous studies have shown that activation of inducible FGFR1 (iFGFR1) in mammary epithelial cells resulted in increased proliferation, migration, and invasion in vitro and tumor formation in vivo. These studies also demonstrated that iFGFR1 activation stimulated recruitment of macrophages to the epithelium where macrophages contributed to iFGFR1-mediated epithelial cell proliferation and angiogenesis. The studies presented here further utilize this model to identify the mechanisms that regulate FGFR1-induced macrophage recruitment. Results from this study elucidate a novel role for the inflammatory chemokine CX3CL1 in FGFR1-induced macrophage migration. Specifically, we illustrate that activation of both the inducible FGFR1 construct in mouse mammary epithelial cells and endogenous FGFR in the triple negative breast cancer cell line, HS578T, leads to expression of the chemokine CX3CL1. Furthermore, we demonstrate that FGFR-induced CX3CL1 is sufficient to recruit CX3CR1-expressing macrophages in vitro. Finally, blocking CX3CR1 in vivo leads to decreased iFGFR1-induced macrophage recruitment, which correlates with decreased angiogenesis. While CX3CL1 is a known target of FGF signaling in the wound healing environment, these studies demonstrate that FGFR activation also leads to induction of CX3CL1 in a tumor setting. Furthermore, these results define a novel role for CX3CL1 in promoting macrophage recruitment during mammary tumor formation, suggesting that the CX3CL1/CX3CR1 axis may represent a potential therapeutic approach for targeting breast cancers associated with high levels of tumor-associated macrophages.     

Regulated shedding of transmembrane chemokines by the disintegrin and metalloproteinase 10 facilitates detachment of adherent leukocytes

CX3CL1 (fractalkine) and CXCL16 are unique members of the chemokine family because they occur not only as soluble, but also as membrane-bound molecules. Expressed as type I transmembrane proteins, the ectodomain of both chemokines can be proteolytically cleaved from the cell surface, a process known as shedding. Our previous studies showed that the disintegrin and metalloproteinase 10 (ADAM10) mediates the largest proportion of constitutive CX3CL1 and CXCL16 shedding, but is not involved in the phorbolester-induced release of the soluble chemokines (inducible shedding). In this study, we introduce the calcium-ionophore ionomycin as a novel, very rapid, and efficient inducer of CX3CL1 and CXCL16 shedding. By transfection in COS-7 cells and ADAM10-deficient murine embryonic fibroblasts combined with the use of selective metalloproteinase inhibitors, we demonstrate that the inducible generation of soluble forms of these chemokines is dependent on ADAM10 activity. Analysis of the C-terminal cleavage fragments remaining in the cell membrane reveals multiple cleavage sites used by ADAM10, one of which is preferentially used upon stimulation with ionomycin. In adhesion studies with CX3CL1-expressing ECV-304 cells and cytokine-stimulated endothelial cells, we demonstrate that induced CX3CL1 shedding leads to the release of bound monocytic cell lines and PBMC from their cellular substrate. These data provide evidence for an inducible release mechanism via ADAM10 potentially important for leukocyte diapedesis.     

Decreased expression of the fractalkine receptor CX3CR1 on circulating monocytes as new feature of sepsis-induced immunosuppression

Although it is known that septic shock rapidly induces immune dysfunctions, which contribute to the impaired clearance of microorganisms observed in patients, the mechanisms for this phenomenon remain incompletely understood. We recently observed, in a microarray study, an altered circulating leukocyte CX3CR1 mRNA expression associated with patients' mortality. As monocytes play a central role in septic shock pathophysiology and express high levels of CX3CR1, we therefore further investigated the alteration of CX3CR1 expression and of its ligand fractalkine (CX3CL1) on those cells in this clinical condition. We observed that CX3CR1 expression (both mRNA and protein) was severely down-regulated in monocytes and consequently associated with a lack of functionality upon fractalkine challenge. Importantly, nonsurvivors presented with significantly sustained lower expression in comparison with survivors. This down-regulation was reproduced by incubation of cells from healthy individuals with LPS, whole bacteria (Escherichia coli and Staphylococcus aureus), and, to a lower extent, with corticosteroids-in accordance with the concept of LPS-induced monocyte deactivation. In addition, CX3CL1 serum concentrations were elevated in patients supporting the hypothesis of increased cleavage of the membrane-anchored form expressed by endothelial cells. As CX3CR1/CX3CL1 interaction preferentially mediates arrest and migration of proinflammatory cells, the present observations may contribute to patients' inability to kill invading microorganisms. This could represent an important new feature of sepsis-induced immunosuppression.     

Hydrogen sulfide inhibits the development of atherosclerosis with suppressing CX3CR1 and CX3CL1 expression

Hydrogen sulfide, as a novel gaseous mediator, has been suggested to play a key role in atherogenesis. However, the precise mechanisms by which H(2)S affects atherosclerosis remain unclear. Therefore, the present study aimed to investigate the potential role of H(2)S in atherosclerosis and the underlying mechanism with respect to chemokines (CCL2, CCL5 and CX3CL1) and chemokine receptors (CCR2, CCR5, and CX3CR1) in macrophages. Mouse macrophage cell line RAW 264.7 or mouse peritoneal macrophages were pre-incubated with saline or NaHS (50 μM, 100 μM, 200 μM), an H(2)S donor, and then stimulated with interferon-γ (IFN-γ) or lipopolysaccharide (LPS). It was found that NaHS dose-dependently inhibited IFN-γ or LPS-induced CX3CR1 and CX3CL1 expression, as well as CX3CR1-mediated chemotaxis in macrophages. Overexpression of cystathionine γ-lyase (CSE), an enzyme that catalyzes H(2)S biosynthesis resulted in a significant reduction in CX3CR1 and CX3CL1 expression as well as CX3CR1-mediated chemotaxis in stimulated macrophages. The inhibitory effect of H(2)S on CX3CR1 and CX3CL1 expression was mediated by modulation of proliferators-activated receptor-γ (PPAR-γ) and NF-κB pathway. Furthermore, male apoE(-/-) mice were fed a high-fat diet and then randomly given NaHS (1 mg/kg, i.p., daily) or DL-propargylglycine (PAG, 10 mg/kg, i.p., daily). NaHS significantly inhibited aortic CX3CR1 and CX3CL1 expression and impeded aortic plaque development. NaHS had a better anti-atherogenic benefit when it was applied at the early stage of atherosclerosis. However, inhibition of H(2)S formation by PAG increased aortic CX3CR1 and CX3CL1 expression and exacerbated the extent of atherosclerosis. In addition, H(2)S had minimal effect on the expression of CCL2, CCL5, CCR2 and CCR5 in vitro and in vivo. In conclusion, these data indicate that H(2)S hampers the progression of atherosclerosis in fat-fed apoE(-/-) mice and downregulates CX3CR1 and CX3CL1 expression on macrophages and in lesion plaques.     

Two novel fully functional isoforms of CX3CR1 are potent HIV coreceptors

We identified two novel isoforms of the human chemokine receptor CX3CR1, produced by alternative splicing and with N-terminal regions extended by 7 and 32 aa. Expression of the messengers coding these isoforms, compared with that of previously described V28 messengers, is lower in monocytes and NK cells, but higher in CD4(+) T lymphocytes. CX3CR1 and its extended isoforms were expressed in HEK-293 cells and compared for expression, ligand binding, and cellular responses. In steady state experiments, all three CX3CR1 isoforms bound CX3CL1 with similar affinity. In kinetic binding studies, however, k(on) and k(off) were significantly greater for the extended CX3CR1 isoforms, thereby suggesting that the N-terminal extensions may alter the functions induced by CX3CL1. In signaling studies, all three CX3CR1 isoforms mediated agonist-dependent calcium mobilization, but the EC(50) was lower for the extended than for the standard isoforms. In addition, chemotactic responses for these extended isoforms shifted left, also indicating a more sensitive response. Finally, the longer variants appeared to be more potent HIV coreceptors when tested in fusion and infection assays. In conclusion, we identified and characterized functionally two novel isoforms of CX3CR1 that respond more sensitively to CX3CL1 and HIV viral envelopes. These data reveal new complexity in CX3CR1 cell activation and confirm the critical role of the N-terminal domain of the chemokine receptors in ligand recognition and cellular response.     

Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine)

CX3CL1 (fractalkine), the only member of the delta subclass of chemokines, is a known chemotactic factor for monocytes/macrophages as well as NK cells and T lymphocytes. In several pathologies, excessive production of CX3CL1 at specific sites leads primarily to monocyte/macrophage recruitment, which causes tissue and vascular damage. Despite their clinical relevance, the mechanisms underlying monocyte/macrophage chemotaxis to CX3CL1 remain poorly documented. The present report addresses this issue and identifies cell signaling crucial for this process. Using the murine monocyte/macrophage RAW cell line, we show that CX3CL1 treatment elicits a rapid and transient increase in F-actin and the formation of F-actin-enriched cell protrusions. CX3CL1 also triggers tyrosine phosphorylation of proteins localized in those protrusions. The protein tyrosine kinase Syk is activated upon CX3CL1 treatment, and reduction of Syk expression using RNA-mediated interference results in a specific and massive impairment of RAW cell migration to CX3CL1. Similar results are obtained using the Syk inhibitor, piceatannol. Cells with reduced Syk expression also exhibit a major defect in CX3CL1-induced cytoskeletal remodeling. These data suggest that in monocytes/macrophages, Syk is essential for proper reorganization of the actin cytoskeleton in response to CX3CL1 and is therefore required for cell chemotaxis to CX3CL1.     

The CC chemokine MCP-1 stimulates surface expression of CX3CR1 and enhances the adhesion of monocytes to fractalkine/CX3CL1 via p38 MAPK

The membrane-anchored form of CX3CL1 has been proposed as a novel adhesion protein for leukocytes. This functional property of CX3CL1 is mediated through CX3CR1, a chemokine receptor expressed predominantly on circulating white blood cells. Thus far, it is still uncertain at what stage of the trafficking process CX3CR1 becomes importantly involved and how the CX3CR1-dependent adhesion of leukocytes is regulated during inflammation. The objective of this study was to examine the functional effects of chemokine stimulation on CX3CR1-mediated adhesion of human monocytes. Consistent with previous reports, our data indicate that the activity of CX3CR1 on resting monocytes is sufficient to mediate cell adhesion to CX3CL1. However, the basal, nonstimulated adhesion activity is low, and we hypothesized that like the integrins, CX3CR1 may require a preceding activation step to trigger firm leukocyte adhesion. Compatible with this hypothesis, stimulation of monocytes with MCP-1 significantly increased their adhesion to immobilized CX3CL1, under both static and physiological flow conditions. The increase of the adhesion activity was mediated through CCR2-dependent signaling and obligatory activation of the p38 MAPK pathway. Stimulation with MCP-1 also induced a rapid increase of CX3CR1 protein on the cell surface. Inhibition of the p38 MAPK pathway prevented this increase of CX3CR1 surface expression and blunted the effect of MCP-1 on cell adhesion, indicating a causal link between receptor surface density and adhesion activity. Together, our data suggest that a chemokine signal is required for firm CX3CR1-dependent adhesion and demonstrate that CCR2 is an important regulator of CX3CL1-dependent leukocyte adhesion.     

CX3CR1 is required for airway inflammation by promoting T helper cell survival and maintenance in inflamed lung

Allergic asthma is a T helper type 2 (T(H)2)-dominated disease of the lung. In people with asthma, a fraction of CD4(+) T cells express the CX3CL1 receptor, CX3CR1, and CX3CL1 expression is increased in airway smooth muscle, lung endothelium and epithelium upon allergen challenge. Here we found that untreated CX3CR1-deficient mice or wild-type (WT) mice treated with CX3CR1-blocking reagents show reduced lung disease upon allergen sensitization and challenge. Transfer of WT CD4(+) T cells into CX3CR1-deficient mice restored the cardinal features of asthma, and CX3CR1-blocking reagents prevented airway inflammation in CX3CR1-deficient recipients injected with WT T(H)2 cells. We found that CX3CR1 signaling promoted T(H)2 survival in the inflamed lungs, and injection of B cell leukemia/lymphoma-2 protein (BCl-2)-transduced CX3CR1-deficient T(H)2 cells into CX3CR1-deficient mice restored asthma. CX3CR1-induced survival was also observed for T(H)1 cells upon airway inflammation but not under homeostatic conditions or upon peripheral inflammation. Therefore, CX3CR1 and CX3CL1 may represent attractive therapeutic targets in asthma.