In vivo structure/function and expression analysis of the CX3C chemokine fractalkine.

The CX3C chemokine family is composed of only one member, CX3CL1, also known as fractalkine, which in mice is the sole ligand of the G protein-coupled, 7-transmembrane receptor CX3CR1. Unlike classic small peptide chemokines, CX3CL1 is synthesized as a membrane-anchored protein that can promote integrin-independent adhesion. Subsequent cleavage by metalloproteases, either constitutive or induced, can generate shed CX3CL1 entities that potentially have chemoattractive activity. To study the CX3C interface in tissues of live animals, we generated transgenic mice (CX3CL1cherry:CX3CR1gfp), which express red and green fluorescent reporter genes under the respective control of the CX3CL1 and CX3CR1 promoters. Furthermore, we performed a structure/function analysis to differentiate the in vivo functions of membrane-tethered versus shed CX3CL1 moieties by comparing their respective ability to correct established defects in macrophage function and leukocyte survival in CX3CL1-deficient mice. Specifically, expression of CX3CL1(105Δ), an obligatory soluble CX3CL1 isoform, reconstituted the formation of transepithelial dendrites by intestinal macrophages but did not rescue circulating Ly6Clo CX3CR1hi blood monocytes in CX3CR1gfp/gfp mice. Instead, monocyte survival required the full-length membrane-anchored CX3CL1, suggesting differential activities of tethered and shed CX3CL1 entities.

C and CX3C chemokines: cell sources and physiopathological implications.

Within the fascinating world of chemokines, C and CX3C chemokines have long been regarded as two minor components, even though they present unique features and show less redundancy than the other chemokine families. Nevertheless, the body of data on their expression and role in various inflammatory disorders has grown in the past few years. The C chemokine family is represented by two chemokines, XCL1/lymphotactin-alpha and XCL2/lymphotactin-beta, whereas the CX3C chemokine family contains only one member, called CX3CL1/ fractalkine. In this review, we present an overview on the structure, expression and signaling properties of these chemokines and their respective receptors and examine how they contribute to inflammation and the regulation of leukocyte trafficking, as well as their potential role in the pathophysiology of human inflammatory diseases. Taken together, these data expand the biological importance of C and CX3C chemokines from that of simple immune modulators to a much broader biological role, even though their precise commitment within the framework of immune responses has still to be determined.

Fractalkine in vascular biology: from basic research to clinical disease.

Fractalkine (now also called CX3CL1) is a unique chemokine that functions not only as a chemoattractant but also as an adhesion molecule and is expressed on endothelial cells activated by proinflammatory cytokines, such as interferon-gamma and tumor necrosis factor-alpha. The fractalkine receptor, CX3CR1, is expressed on cytotoxic effector lymphocytes, including natural killer (NK) cells and cytotoxic T lymphocytes, which contain high levels of intracellular perforin and granzyme B, and on macrophages. Soluble fractalkine causes migration of NK cells, cytotoxic T lymphocytes, and macrophages, whereas the membrane-bound form captures and enhances the subsequent migration of these cells in response to secondary stimulation with other chemokines. Furthermore, stimulation through membrane-bound fractalkine activates NK cells, leading to increased cytotoxicity and interferon-gamma production. Recently, accumulating evidence has shown that fractalkine is involved in the pathogenesis of various clinical disease states or processes, such as atherosclerosis, glomerulonephritis, cardiac allograft rejection, and rheumatoid arthritis. In addition, polymorphisms in CX3CR1, which reduce its binding activity to fractalkine, have been reported to increase the risk of HIV disease and to reduce the risk of coronary artery disease. This review will examine new concepts underlying fractalkine-mediated leukocyte migration and tissue damage, focusing primarily on the pathophysiological roles of fractalkine in various clinical conditions, especially in atherosclerosis and vascular injury.

Expression and targeting of CX3CL1 (fractalkine) in renal tubular epithelial cells.

The chemokine CX3CL1 plays a key role in glomerulonephritis and can act as both chemoattractant and adhesion molecule. CX3CL1 also is upregulated in tubulointerstitial injury, but little is known about the subcellular distribution and function of CX3CL1 in renal tubular epithelial cells (RTEC). Unexpectedly, it was found that CX3CL1 is expressed predominantly on the apical surface of tubular epithelium in human renal transplant biopsy specimens with acute rejection or acute tubular necrosis. For studying the targeting of CX3CL1 in polarized RTEC, MDCK cells that expressed untagged or green fluorescent protein-tagged CX3CL1 were generated. The chemokine was present on the apical membrane and in subapical vesicles. Apical targeting of CX3CL1 was not due to signals that were conferred by its intracellular domain, to associations with lipid rafts, or to O-glycosylation but, rather, depended on N-linked glycosylation of the protein. With the use of fluorescence recovery after photobleaching, it was found that CX3CL1 is immobile in the apical membrane. However, CX3CL1 partitioned with the triton-soluble rather than -insoluble cellular fraction, indicating that it is not associated directly with the actin cytoskeleton or with lipid rafts. Accordingly, disruption of rafts through cholesterol depletion did not render CX3CL1 mobile. For exploration of potential functions of apical CX3CL1, binding of CX3CR1-expressing leukocytes to polarized RTEC was examined. Leukocyte adhesion to the luminal surface was enhanced significantly when CX3CL1 was present. These data demonstrate that CX3CL1 is expressed preferentially on the apical membrane of RTEC and suggest a novel function for the chemokine in recruitment and retention of leukocytes in tubulointerstitial inflammation.

Fractalkine targeting with a receptor-mimicking peptide-amphiphile.

In this study we have designed the NTFR peptide-amphiphile that mimics a fragment of the N-terminus of the fractalkine receptor (CX(3)CR1) and specifically targets fractalkine, a novel adhesion molecule expressed on the surface of inflamed endothelial cells. Bioartificial membranes were constructed from mixtures of NTFR peptide-amphiphiles and DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) phospholipids, and the affinity and specificity of fractalkine for the synthetic NTFR was investigated with an atomic force microscope (AFM). Fractalkine was immobilized onto the AFM tips, and forces were collected between fractalkine and the bioartificial membranes. The adhesive interactions were studied at the collective level, when each adhesion event corresponded to the rupture of multiple biomolecular bonds. Retraction force profiles for the fractalkine-NTFR system exhibited single or multiple peaks and a small percentage of the force curves demonstrated stretching of the fractalkine-NTFR complex. Strong adhesion was measured when both DPPC and NTFR were present, compared to pure NTFR surfaces. This may be due to the fact that the DPPC molecule is shorter, and thus it can provide more space for the peptide headgroup to bend and expose its sequence at the interface. Specificity was demonstrated by comparing the NTFR-fractalkine adhesion to the forces between the alpha(5)beta(1) integrin (an adhesion receptor expressed on the surface of endothelial cells) and other surfaces such as GRGDSP (the specific ligand for alpha(5)beta(1)), GRGESP (an inactive sequence), and NTFR.

Elucidating the structural mechanisms for biological activity of the chemokine family.

Chemokines are a family of proteins involved in inflammatory and immune response. They share a common fold, made up of a three-stranded beta-sheet, and an overlaying alpha-helix. Chemokines are mainly categorized into two subfamilies distinguished by the presence or absence of a residue between two conserved cysteines in the N-terminus. Although dimers and higher-order quaternary structures are common in chemokines, they are known to function as monomers. Yet, there is quite a bit of controversy on how the actual function takes place. The mechanisms of binding and activation in the chemokine family are investigated using the gaussian network model of proteins, a low-resolution model that monitors the collective motions in proteins. It is particularly suitable for elucidating the global dynamic characteristics of large proteins or the common properties of a group of related proteins such as the chemokine family presently investigated. A sample of 16 proteins that belong to the CC, CXC, or CX(3)C subfamilies are inspected. Local packing density and packing order of residues are used to determine the type and range of motions on a global scale, such as those occurring between various loop regions. The 30s-loop, although not directly involved in the binding interface like the N-terminus and the N-loop, is identified as having a prominent role in both binding/activation and dimerization. Two mechanisms are distinguished based on the communication among the three flexible regions. In these two-step mechanisms, the 30s-loop assists either the N-loop or the N-terminus during binding and activation. The findings are verified by molecular mechanics and molecular dynamics simulations carried out on the detailed structure of representative proteins from each mechanism type. A basis for the construction of hybrids of chemokines to bind and/or activate various chemokine receptors is presented. Proteins 2001;43:150-160.

The G glycoprotein of respiratory syncytial virus depresses respiratory rates through the CX3C motif and substance P.

Respiratory syncytial virus (RSV) infection in the neonate can alter respiratory rates, i.e., lead to episodes of apnea. We show that RSV G glycoprotein reduces respiratory rates associated with the induction of substance P (SP) and G glycoprotein-CX3CR1 interaction, an effect that is inhibited by treatment with anti-G glycoprotein, anti-SP, or anti-CX3CR1 monoclonal antibodies. These data suggest new approaches for treating some aspects of RSV disease.

Anti-G protein antibody responses to respiratory syncytial virus infection or vaccination are associated with inhibition of G protein CX3C-CX3CR1 binding and leukocyte chemotaxis.

Respiratory syncytial virus (RSV) is an important cause of severe lower respiratory tract illness in infants and the elderly. Presently, no safe and efficacious RSV vaccine exists; however, advances in our understanding of immunity and the pathogenesis of disease associated with RSV infection may lead to new vaccine strategies. RSV G protein contains a CX3C chemokine motif that interacts with the CX3CR1 chemokine receptor and modifies the activities of fractalkine. In the present study, we show that anti-RSV G protein antibody responses after recent RSV infection or vaccination are associated with inhibition of RSV G protein CX3C-CX3CR1 interaction and RSV G protein-mediated leukocyte chemotaxis.

Mutational analysis of the fractalkine chemokine domain. Basic amino acid residues differentially contribute to CX3CR1 binding, signaling, and cell adhesion.

Fractalkine (FKN/CX3CL1) is a unique member of the chemokine gene family and contains a chemokine domain (CD), a mucin-like stalk, a single transmembrane region, and a short intracellular C terminus. This structural distinction affords FKN the property of mediating capture and firm adhesion of FKN receptor (CX3CR1)-expressing cells under physiological flow conditions. Shed forms of FKN also exist, and these promote chemotaxis of CX3CR1-expressing leukocytes. The goal of the present study was to identify specific residues within the FKN-CD critical for FKN-CX3CR1 interactions. Two residues were identified in the FKN-CD, namely Lys-7 and Arg-47, that are important determinants in mediating an FKN-CX3CR1 interaction. FKN-K7A and FKN-R47A mutants exhibited 30-60-fold decreases in affinity for CX3CR1 and failed to arrest efficiently CX3CR1-expressing cells under physiological flow conditions. However, these mutants had differential effects on chemotaxis of CX3CR1-expressing cells. The FKN-K7A mutant acted as an equipotent partial agonist, whereas the FKN-R47A mutant had marked decreased potency and efficacy in measures of chemotactic activity. These data identify specific structural features of the FKN-CD that are important in interactions with CX3CR1 including steady state binding, signaling, and firm adhesion of CX3CR1-expressing cells.

Fractalkine and vascular injury.

The vascular endothelium plays a central role in the recruitment and migration of circulating effector cells into sites of inflammation and immune responses. The unique CX(3)C-chemokine, fractalkine, is expressed on activated endothelial cells, and its receptor, CX(3)CR1, is expressed on natural killer cells, monocytes and some CD8+ T cells, all of which possess cytolytic function. Accumulating evidence that fractalkine is expressed on endothelial cells during glomerulonephritis and cardiac allograft rejection, as well as on cardiac endothelial cells activated by pro-inflammatory cytokines, might provide insight into the pathogenesis of vascular injury. Here, we propose a model in which fractalkine mediates vascular injury through the accumulation and activation of killer cells.

Molecular determinants of receptor binding and signaling by the CX3C chemokine fractalkine.

Fractalkine/CX3CL1 is a membrane-tethered chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's chemokine domain was undertaken. The results reveal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48) and one aromatic (Phe-50) that are critical for binding and/or signaling. The mutant R48A could bind but not induce chemotaxis, demonstrating that Arg-48 is a signaling trigger. This result also shows that signaling residues are not confined to chemokine N termini, as generally thought. F50A showed no detectable binding, underscoring its importance to the stability of the complex. K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors.

Tumor necrosis factor-alpha stimulates fractalkine production by mesangial cells and regulates monocyte transmigration: down-regulation by cAMP.

BACKGROUND: Fractalkine is a CX3C chemokine for mononuclear cells that has been implicated in the recruitment and accumulation of monocytes seen in glomerular diseases. We investigated the mechanisms by which tumor necrosis factor (TNF)-alpha stimulates mesangial cell (MC) fractalkine expression, and the effects of MC-derived fractalkine on monocyte transmigration. METHODS: Cultured rat MCs were incubated with TNF-alpha, with or without pretreatment with pharmacologic inhibitors of protein kinases or transcriptional factors downstream to TNF-alpha. Fractalkine mRNA and protein were analyzed by Northern and Western blotting. Translocation of nuclear factor (NF)-kappaB was evaluated by immunocytochemical staining. Monocyte transmigration was determined by in vitro chemotaxis assay. RESULTS: TNF-alpha stimulated MC fractalkine mRNA as well as cell-bound and soluble protein expression in a dose- and time-dependent manner. The soluble fractalkine was shed from the cell-bound form via metalloproteinase-dependent cleavage, and mediated in part TNF-alpha-induced monocyte transmigration in vitro. The incubation of MCs with calphostin C [a selective inhibitor of protein kinase C (PKC)] or PD98059 [a selective inhibitor of p42/44 mitogen-activated protein kinase (MAPK) kinase] attenuated TNF-alpha-stimulated fractalkine mRNA and protein expression. Coincubation of MCs with calphostin C and PD98059 resulted in a synergistic inhibition of TNF-alpha-stimulated fractalkine mRNA and protein expression. Incubation of MCs with phorbol myristate acetate (PMA) for four hours resulted in an increase in fractalkine mRNA expression that could be suppressed by calphostin C or depletion of PKC by pretreatment with PMA for 24 hours. Further, activation of PKC-depleted MCs with TNF-alpha stimulated fractalkine mRNA expression that could be blocked by calphostin C. PD 98059, but not calphostin C, inhibited TNF-alpha-activated phospho-p42/44 MAPK and phospho-c-Jun levels, whereas only calphostin C inhibited TNF-alpha-activated phosphorylation of PKCzeta/iota. The incubation of MCs with MG132, a NF-kappaB inhibitor, abolished TNF-alpha-induced degradation of inhibitory protein of NF-kappaB (I-kappaB)alpha, nuclear translocation of NF-kappaB, and fractalkine expression, without affecting phospho-c-Jun levels. In contrast, curcumin, an activating protein (AP)-1 inhibitor, attenuated TNF-alpha-stimulated phospho-c-Jun levels and fractalkine expression without discernible effects on TNF-alpha-induced degradation of I-kappaBalpha or NF-kappaB nuclear translocation. Neither PD 98059 nor calphostin C affected TNF-alpha-induced degradation of I-kappaBalpha or NF-kappaB nuclear translocation. Additional experiments examining the role of cAMP on MC fractalkine expression showed that the incubation of MCs with TNF-alpha and either db-cAMP or forskolin attenuated TNF-alpha-stimulated fractalkine mRNA and protein expression, preceded by attenuation of TNF-alpha-activated phosphorylation of p42/44 MAPK, and c-Jun, but not phosphorylation of PKCzeta/iota or nuclear translocation of NF-kappaB. CONCLUSION: The present data indicate that TNF-alpha activation of PKCzeta/iota, p42/44 MAPK, c-Jun/AP-1, and p65/NF-kappaB are involved in TNF-alpha-stimulated MC fractalkine expression, with the soluble fractalkine mediating in part the TNF-alpha-induced monocyte transmigration in vitro. Uncoupling of p42/44 MAPK or c-Jun/AP-1 signals may contribute to cAMP inhibition of MC fractalkine expression activated by TNF-alpha.

Expression cloning of the STRL33/BONZO/TYMSTRligand reveals elements of CC, CXC, and CX3C chemokines.

STRL33/BONZO/TYMSTR is an orphan chemokine and HIV/SIV coreceptor receptor that is expressed on activated T lymphocytes. We describe an expression cloning strategy whereby we isolated a novel chemokine, which we name CXCL16. CXCL16 is an alpha (CXC) chemokine but also has characteristics of CC chemokines and a structure similar to fractalkine (neurotactin) in having a transmembrane region and a chemokine domain suspended by a mucin-like stalk. A recombinant version of CXCL16 fails to mediate chemotaxis to all known chemokine receptor transfectants tested but does mediate robust chemotaxis, high affinity binding, and calcium mobilization to Bonzo receptor transfectants, indicating that this is a unique receptor ligand interaction. In vitro polarized T cell subsets including Th1, Th2, and Tr1 cells express functional Bonzo, suggesting expression of this receptor in chronic inflammation, which we further verified by demonstration of CXCL16-mediated migration of tonsil-derived CD4(+) T lymphocytes. CXCL16 is expressed on the surface of APCs including subsets of CD19(+) B cells and CD14(+) monocyte/macrophages, and functional CXCL16 is also shed from macrophages. The combination of unique structural features of both Bonzo and CXCL16 suggest that this interaction may represent a new class of ligands for this receptor family. Additionally, this chemokine might play a unique dual role of attracting activated lymphocyte subsets during inflammation as well as facilitating immune responses via cell-cell contact.