Mutating the CX3C Motif in the G Protein Should Make a Live Respiratory Syncytial Virus Vaccine Safer and More Effective.

Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Through a CX3C chemokine motif (182CWAIC186) in the G protein, RSV binds to the corresponding chemokine receptor, CX3CR1. Since RSV binding to CX3CR1 contributes to disease pathogenesis, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A186, within the CX3C motif, mutating it to CX4C (182CWAIAC187), which is known to block binding to CX3CR1, might decrease disease. We studied the effect of the CX4C mutation in two strains of RSV (A2 and r19F) in a mouse challenge model. We included RSV r19F because it induces mucus production and airway resistance, two manifestations of RSV infection in humans, in mice. Compared to wild-type (wt) virus, mice infected with CX4C had a 0.7 to 1.2 log10-fold lower virus titer in the lung at 5 days postinfection (p.i.) and had markedly reduced weight loss, pulmonary inflammatory cell infiltration, mucus production, and airway resistance after challenge. This decrease in disease was not dependent on decrease in virus replication but did correspond to a decrease in pulmonary Th2 and inflammatory cytokines. Mice infected with CX4C viruses also had higher antibody titers and a Th1-biased T cell memory response at 75 days p.i. These results suggest that the CX4C mutation in the G protein could improve the safety and efficacy of a live attenuated RSV vaccine.IMPORTANCE RSV binds to the corresponding chemokine receptor, CX3CR1, through a CX3C chemokine motif (182CWAIC186) in the G protein. RSV binding to CX3CR1 contributes to disease pathogenesis; therefore, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A186, within the CX3C motif, mutating it to CX4C (182CWAIAC187), known to block binding to CX3CR1, might decrease disease. The effect of this mutation and treatment with the F(ab')2 form of the anti-RSV G 131-2G monoclonal antibody (MAb) show that mutating the CX3C motif to CX4C blocks much of the disease and immune modulation associated with the G protein and should improve the safety and efficacy of a live attenuated RSV vaccine.

Respiratory syncytial virus G protein CX3C motif impairs human airway epithelial and immune cell responses.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infection in infants and young children and causes disease in the elderly and persons with compromised cardiac, pulmonary, or immune systems. Despite the high morbidity rates of RSV infection, no highly effective treatment or vaccine is yet available. The RSV G protein is an important contributor to the disease process. A conserved CX3C chemokine-like motif in G likely contributes to the pathogenesis of disease. Through this motif, G protein binds to CX3CR1 present on various immune cells and affects immune responses to RSV, as has been shown in the mouse model of RSV infection. However, very little is known of the role of RSV CX3C-CX3CR1 interactions in human disease. In this study, we use an in vitro model of human RSV infection comprised of human peripheral blood mononuclear cells (PBMCs) separated by a permeable membrane from human airway epithelial cells (A549) infected with RSV with either an intact CX3C motif (CX3C) or a mutated motif (CX4C). We show that the CX4C virus induces higher levels of type I/III interferon (IFN) in A549 cells, increased IFN-α and tumor necrosis factor alpha (TNF-α) production by human plasmacytoid dendritic cells (pDCs) and monocytes, and increased IFN-γ production in effector/memory T cell subpopulations. Treatment of CX3C virus-infected cells with the F(ab')2 form of an anti-G monoclonal antibody (MAb) that blocks binding to CX3CR1 gave results similar to those with the CX4C virus. Our data suggest that the RSV G protein CX3C motif impairs innate and adaptive human immune responses and may be important to vaccine and antiviral drug development.

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.

CX3CL1 deficiency ameliorates inflammation, apoptosis and accelerates osteogenic differentiation, mineralization in LPS-treated MC3T3-E1 cells via its receptor CX3CR1.

BACKGROUND: Osteoporosis is a devastating skeletal disease responsible for bone fragility and fracture. CX3C chemokine ligand 1 (CX3CL1) is an inflammatory chemokine which has been identified to possess increased expression in the serum of postmenopausal osteoporotic patients. This paper was to illuminate the impacts of CX3CL1 on inflammation, apoptosis and osteogenic differentiation, mineralization in LPS-treated osteoblasts and investigate the regulatory mechanism. METHODS: The viability of MC3T3-E1 cells exposed to elevating doses of LPS was detected by CCK-8 assay. CX3CL1 and C-X3-C motif chemokine receptor 1 (CX3CR1) expression were detected by RT-qPCR and western blot. CX3CR1 expression was examined again following CX3CL1 depletion. The binding of CX3CL1 with CX3CR1 was testified through Co-IP assay. In MC3T3-E1 cells co-transduced with CX3CL1 interference and CX3CR1 overexpression plasmids following LPS exposure, cell activity and inflammation were separately estimated via CCK-8 assay and RT-qPCR. Apoptosis was measured by TUNEL assay and western blot. Osteoblast differentiation was evaluated by ALP activity assay, RT-qPCR and western blot. Osteoblast mineralization was assessed by ARS staining, RT-qPCR and western blot. Results The experimental data presented that LPS attenuated the viability and enhanced CX3CL1 and CX3CR1 expression in MC3T3-E1 cells in a dose-dependent manner. CX3CR1 interacted with CX3CL1 and was positively modulated by CX3CL1. The suppressive role of CX3CL1 absence in LPS-evoked viability decrease, inflammation and apoptosis in MC3T3-E1 cells was reversed by CX3CR1 elevation. Besides, CX3CR1 reversed the promoted osteoblast differentiation and mineralization imposed by CX3CL1 interference. CONCLUSIONS: CX3CL1 knockdown eased inflammation, apoptosis and promoted osteogenic differentiation, mineralization in MC3T3-E1 cells upon LPS exposure through down-regulating CX3CR1.

Vaccination to induce antibodies blocking the CX3C-CX3CR1 interaction of respiratory syncytial virus G protein reduces pulmonary inflammation and virus replication in mice.

Respiratory syncytial virus (RSV) infection causes substantial morbidity and some deaths in the young and elderly worldwide. There is no safe and effective vaccine available, although it is possible to reduce the hospitalization rate for high-risk children by anti-RSV antibody prophylaxis. RSV has been shown to modify the immune response to infection, a feature linked in part to RSV G protein CX3C chemokine mimicry. This study determined if vaccination with G protein polypeptides or peptides spanning the central conserved region of the G protein could induce antibodies that blocked G protein CX3C-CX3CR1 interaction and disease pathogenesis mediated by RSV infection. The results show that mice vaccinated with G protein peptides or polypeptides containing the CX3C motif generate antibodies that inhibit G protein CX3C-CX3CR1 binding and chemotaxis, reduce lung virus titers, and prevent body weight loss and pulmonary inflammation. The results suggest that RSV vaccines that induce antibodies that block G protein CX3C-CX3CR1 interaction may offer a new, safe, and efficacious RSV vaccine strategy.

Respiratory Syncytial Virus (RSV) G Protein Vaccines With Central Conserved Domain Mutations Induce CX3C-CX3CR1 Blocking Antibodies.

Respiratory syncytial virus (RSV) infection can cause bronchiolitis, pneumonia, morbidity, and some mortality, primarily in infants and the elderly, for which no vaccine is available. The RSV attachment (G) protein contains a central conserved domain (CCD) with a CX3C motif implicated in the induction of protective antibodies, thus vaccine candidates containing the G protein are of interest. This study determined if mutations in the G protein CCD would mediate immunogenicity while inducing G protein CX3C-CX3CR1 blocking antibodies. BALB/c mice were vaccinated with structurally-guided, rationally designed G proteins with CCD mutations. The results show that these G protein immunogens induce a substantial anti-G protein antibody response, and using serum IgG from the vaccinated mice, these antibodies are capable of blocking the RSV G protein CX3C-CX3CR1 binding while not interfering with CX3CL1, fractalkine.

Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow.

Leukocyte migration into sites of inflammation involves multiple molecular interactions between leukocytes and vascular endothelial cells, mediating sequential leukocyte capture, rolling, and firm adhesion. In this study, we tested the role of molecular interactions between fractalkine (FKN), a transmembrane mucin-chemokine hybrid molecule expressed on activated endothelium, and its receptor (CX3CR1) in leukocyte capture, firm adhesion, and activation under physiologic flow conditions. Immobilized FKN fusion proteins captured resting peripheral blood mononuclear cells at physiologic wall shear stresses and induced firm adhesion of resting monocytes, resting and interleukin (IL)-2-activated CD8(+) T lymphocytes and IL-2-activated NK cells. FKN also induced cell shape change in firmly adherent monocytes and IL-2-activated lymphocytes. CX3CR1-transfected K562 cells, but not control K562 cells, firmly adhered to FKN-expressing ECV-304 cells (ECV-FKN) and tumor necrosis factor alpha-activated human umbilical vein endothelial cells. This firm adhesion was not inhibited by pertussis toxin, EDTA/EGTA, or antiintegrin antibodies, indicating that the firm adhesion was integrin independent. In summary, FKN mediated the rapid capture, integrin-independent firm adhesion, and activation of circulating leukocytes under flow. Thus, FKN and CX3CR1 mediate a novel pathway for leukocyte trafficking.

Fractalkine preferentially mediates arrest and migration of CD16+ monocytes.

CD16+ monocytes represent 5-10% of peripheral blood monocytes in normal individuals and are dramatically expanded in several pathological conditions including sepsis, human immunodeficiency virus 1 infection, and cancer. CD16+ monocytes produce high levels of proinflammatory cytokines and may represent dendritic cell precursors in vivo. The mechanisms that mediate the recruitment of CD16+ monocytes into tissues remain unknown. Here we investigate molecular mechanisms of CD16+ monocyte trafficking and show that migration of CD16+ and CD16- monocytes is mediated by distinct combinations of adhesion molecules and chemokine receptors. In contrast to CD16- monocytes, CD16+ monocytes expressed high CX3CR1 and CXCR4 but low CCR2 and CD62L levels and underwent efficient transendothelial migration in response to fractalkine (FKN; FKN/CX3CL1) and stromal-derived factor 1 alpha (CXCL12) but not monocyte chemoattractant protein 1 (CCL2). CD16+ monocytes arrested on cell surface-expressed FKN under flow with higher frequency compared with CD16- monocytes. These results demonstrate that FKN preferentially mediates arrest and migration of CD16+ monocytes and suggest that recruitment of this proinflammatory monocyte subset to vessel walls via the CX3CR1-FKN pathway may contribute to vascular and tissue injury during pathological conditions.

Role of CX3C-chemokine CX3C-L/fractalkine expression in a model of slowly progressive renal failure.

BACKGROUND: The chemokine/chemokine receptor pair CX(3)C-L/CX(3)C-R is suspected to play a role in renal fibrogenesis. The aim of this study was to investigate their function in an animal model of slowly progressive chronic renal failure. METHODS: Functional data were analysed in folic acid nephropathy (FAN) at different time points (up to day 142 after induction). Immunostaining for CX(3)C-L, CD3, S100A4, collagen type I, fibronectin, alpha-smooth muscle actin, Tamm-horsfall protein, aquaporin 1 and 2 as well as quantitative real-time PCR (qRT-PCR) for CX(3)C-L, CX(3)C-R and fibroblast-specific protein 1 (FSP-1) were performed. Additionally, regulatory mechanisms and functional activity of CX(3)C-L in murine proximal and distal tubular epithelial cells as well as in fibroblasts were investigated. RESULTS: CX(3)C-L/GAPDH ratio was upregulated in FAN 3.4-fold at day 7 further increasing up to 7.1-fold at day 106. The expression of mRNA CX(3)C-L correlated well with CX(3)C-R (R(2) = 0.96), the number of infiltrating CD3+ cells (R(2) = 0.60) and the degree of tubulointerstitial fibrosis (R(2) = 0.56) and moderately with FSP-1 (R(2) = 0.33). Interleukin-1beta, tumour necrosis factor-alpha, transforming growth factor-beta as well as the reactive oxygen species (ROS) H(2)O(2) were identified by qRT-PCR as inductors of CX(3)C-L/fractalkine (FKN) in tubular epithelial cells. Functionally, CX(3)C-L/FKN chemoattracts peripheral blood mononuclear cells, activates several aspects of fibrogenesis and induces the mitogen-activated protein kinases in renal fibroblasts. CONCLUSIONS: In FAN, there is a good correlation between the expression of CX(3)C-L with markers of interstitial inflammation and fibrosis which may result from upregulation by pro-inflammatory and pro-fibrotic cytokines as well as by ROS in tubular epithelial cells. The FKN system may promote renal inflammation and renal fibrogenesis.

Rapid progression to AIDS in HIV+ individuals with a structural variant of the chemokine receptor CX3CR1.

Human immunodeficiency virus (HIV) enters cells in vitro via CD4 and a coreceptor. Which of 15 known coreceptors are important in vivo is poorly defined but may be inferred from disease-modifying mutations, as for CCR5. Here two single nucleotide polymorphisms are described in Caucasians in CX3CR1, an HIV coreceptor and leukocyte chemotactic/adhesion receptor for the chemokine fractalkine. HIV-infected patients homozygous for CX3CR1-I249 M280, a variant haplotype affecting two amino acids (isoleucine-249 and methionine-280), progressed to AIDS more rapidly than those with other haplotypes. Functional CX3CR1 analysis showed that fractalkine binding is reduced among patients homozygous for this particular haplotype. Thus, CX3CR1-I249 M280 is a recessive genetic risk factor in HIV/AIDS.

Fractalkine (CX3CL1)- and interleukin-2-enriched neuroblastoma microenvironment induces eradication of metastases mediated by T cells and natural killer cells.

Fractalkine (FKN) is a unique CX3C chemokine (CX3CL1) known to induce both adhesion and migration of leukocytes mediated by a membrane-bound and a soluble form, respectively. Its function is mediated through CX3C receptor (CX3CR), which is expressed by T(H)1 immune cells including T cells and natural killer (NK) cells. FKN was shown to be expressed in >90% of 68 neuroblastoma samples as determined by cDNA microarray analysis. Here, we characterized the effect of FKN in the neuroblastoma microenvironment using a syngeneic model genetically engineered to secrete FKN. We show FKN-mediated migration, adhesion, and IFN-gamma secretion of immune effector cells, but limited antineuroblastoma activity, in vitro and in vivo. Therefore, we tested the hypothesis that a combined increase of FKN and interleukin-2 (IL-2) in the neuroblastoma microenvironment induces an effective antitumor immune response. For this purpose, IL-2 was targeted to ganglioside GD2, which is highly expressed on neuroblastoma tissue, using an anti-GD2 antibody IL-2 immunocytokine (ch14.18-IL-2). Only mice bearing FKN- and IL-2-enriched neuroblastoma tumors exhibited a reduction in primary tumor growth and a complete eradication of experimental liver metastases. The depletion of T cells and NK cells in vivo abrogated the effect, and these effector cells showed the highest cytolytic activity in vitro. Finally, only the FKN- and IL-2-enriched neuroblastoma microenvironment resulted in T-cell activation and the release of proinflammatory cytokines. In summary, we showed for the first time the immunologic mechanisms by which targeted IL-2 treatment of neuroblastoma with an FKN-rich microenvironment induces an effective antitumor response.

Chemokine fractalkine mediates leukocyte recruitment to inflammatory endothelial cells in flowing whole blood: a critical role for P-selectin expressed on activated platelets.

BACKGROUND: The membrane-bound chemokine fractalkine (CX3CL1) is expressed on various cell types such as activated endothelial cells and has been implicated in the inflammatory process of atherosclerosis. The aim of the present study was to dissect the role of fractalkine in leukocyte recruitment to inflamed endothelium under arterial shear forces. METHODS AND RESULTS: With the use of immunofluorescence and laminar flow assays, the present study shows that human umbilical vein endothelial cells stimulated with tumor necrosis factor-alpha and interferon-gamma abundantly express CX3CL1 and promote substantial leukocyte accumulation under arterial flow conditions. In the presence of high shear, firm adhesion of leukocytes to inflamed endothelial cells is reduced by approximately 40% by a function-blocking anti-fractalkine antibody or by an antibody directed against the fractalkine receptor (CX3CR1). With the use of intravital video-fluorescence microscopy we demonstrate that inhibition of fractalkine signaling attenuates leukocyte adhesion to the atherosclerotic carotid artery of apolipoprotein E-deficient mice, which suggests that the CX3CL1-CX3CR1 axis is critically involved in leukocyte adhesion to inflamed endothelial cells under high shear forces both in vitro and in vivo. Surprisingly, platelets were strictly required for fractalkine-induced leukocyte adhesion at high shear rates. Correspondingly, specific inhibition of platelet adhesion to inflamed endothelial cells also significantly reduced leukocyte accumulation. We show that both soluble and membrane-bound fractalkine induces platelet degranulation and subsequent surface expression of P-selectin, which thereby promotes direct platelet-leukocyte interaction. CONCLUSIONS: Fractalkine expressed by inflamed endothelial cells triggers P-selectin exposure on adherent platelets, which thereby initiates the local accumulation of leukocytes under arterial shear, an essential step in the development of atherosclerotic lesions.

The roles of chemokines in leukocyte recruitment and fibrosis in systemic sclerosis.

Systemic sclerosis (SSc, scleroderma) is an autoimmune disease characterized by excessive extracellular matrix deposition and vascular injury in the skin and other visceral organs. Although the pathogenesis remains unclear, interactions among leukocytes, endothelial cells, and fibroblasts are likely to be central to the pathogenesis of the disease. Chemokines mediate the leukocyte chemotaxis and migration through endothelia into the organ tissues, leading to the interaction between leukocytes and fibroblasts. While amounts of literatures reported chemokine abnormalities in SSc, which might explain the altered accumulation of effector leukocyte subsets in the affected tissues. Among various chemokines, monocyte chemoattractant protein-1 (MCP-1/CCL2) likely has the most critical role for tissue fibrosis in SSc. Although therapeutic effect for targeting MCP-1 has been demonstrated in mouse models of SSc or fibrotic disorders, it is unknown whether this strategy is effective in human clinical trials. Here recent data will be reviewed on the pathogenic role of chemokines and their receptors in SSc.

The CX3C chemokine fractalkine induces vascular dysfunction by generation of superoxide anions.

OBJECTIVE: The chemokine fractalkine activates platelets and induces leukocyte adhesion to the endothelium. Expression of fractalkine and its receptor, CX3CR1, is elevated in coronary artery disease. We assessed the effects of fractalkine on vascular function in isolated rat aorta. METHODS AND RESULTS: CX3CR1 expression was demonstrated in rat aortic endothelial and smooth muscle cells by immunohistochemistry, Western blot, and polymerase chain reaction (PCR). Fractalkine (up to 1 microg/mL) did not directly induce contractile or relaxant responses when applied to rat aortic rings in organ baths. Short-term incubation with fractalkine (1 microg/mL) for 5 minutes did not affect vascular reactivity. Pretreatment of isolated rat aortic rings with fractalkine for 2 hours impaired acetylcholine-induced nitric oxide (NO)-mediated relaxation after preconstriction with phenylephrine in a concentration-dependent manner. The concentration response to the NO donor DEA-NONOate was significantly shifted to the right. The radical scavenger tiron normalized the attenuated acetylcholine-induced relaxation after fractalkine incubation. Aortic superoxide formation was enhanced by fractalkine, which was inhibited by diphenyleneiodonium but not by inhibitors of xanthine oxidase or NO synthase. CONCLUSIONS: In addition to its role as a chemokine and adhesion molecule, fractalkine induces vascular dysfunction by stimulating vascular reactive oxygen species resulting in reduced NO bioavailability.

Intervention of MAdCAM-1 or fractalkine alleviates graft-versus-host reaction associated intestinal injury while preserving graft-versus-tumor effects.

Coincidence of the beneficial graft-vs.-tumor (GVT) effects and the detrimental graft-vs.-host disease (GVHD) remains the major obstacle against the widespread use of allogeneic bone marrow transplantation (BMT) as tumor immunotherapy. We here demonstrate that intervention of MAdCAM-1 (mucosal vascular addressin cell adhesion molecule-1) or fractalkine/CX3CL1 after the expansion of allo-reactive donor CD8 T cells selectively inhibits the recruitment of effector donor CD8 T cells to the intestine and alleviates the graft-vs.-host reaction (GVHR) associated intestinal injury without impairing GVT effects. In a nonirradiated acute GVHD model, donor CD8 T cells up-regulate the expression of intestinal homing receptor alpha4beta7 and chemokine receptors CXCR6 and CX3CR1, as they differentiate into effector cells and subsequently infiltrate into the intestine. Administration of anti-MAdCAM-1 antibody or anti-fractalkine antibody, even after the expansion of alloreactive donor CD8 T cells, selectively reduced the intestine-infiltrating donor CD8 T cells and the intestinal crypt cell apoptosis without affecting the induction of donor derived anti-host CTL or the infiltration of donor CD8 T cells in the hepatic tumor. Moreover, in a clinically relevant GVHD model with myeloablative conditioning, these antibodies significantly improved the survival and loss of weight without impairing the beneficial GVT effects. Thus, interruption of alpha4beta7-MAdCAM-1 or CX3CR1-fractalkine interactions in the late phase of GVHD would be a novel therapeutic approach for the separation of GVT effects from GVHR-associated intestinal injury.

An oxidized lipid-peroxisome proliferator-activated receptor gamma-chemokine pathway in the regulation of macrophage-vascular smooth muscle cell adhesion.

Recent genetic studies have implicated pro-inflammatory chemokines and chemokine receptors in atherogenesis. Studies at the molecular and cellular levels have suggested specific atherogenic mechanisms for two chemokine-chemokine receptor pairs, CCL2-CCR2 and CX3CL1-CX3CR1, involving differential receptor regulation by the transcription factor peroxisome proliferator-activated receptor gamma. This pathway is triggered by oxidized proatherogenic lipids, such as oxidized low-density lipoprotein and linoleic acid derivatives, which promote differentiation of CCR2(hi)CX3CR1(lo) human monocytes to CCR2(lo)CX3CR1(hi) macrophages that adhere to coronary artery smooth muscle cells in a CX3CR1- and peroxisome proliferator-activated receptor gamma-dependent manner. Switching CX3CR1 on and CCR2 off in vivo may result in cessation of CCR2-dependent migration and activation of CX3CR1-dependent retention that together may promote foam cell accumulation in the vessel wall.

Differential CXC and CX3C Chemokine Expression Profiles in Aqueous Humor of Patients With Specific Endogenous Uveitic Entities.

Purpose: To determine the levels of the neutrophil chemoattractants CXCL1, CXCL2, CXCL5, CXCL6, and CXCL8, the T helper 1 chemoattractants CXCL9, CXCL10 and CXCL11, the lymphoid chemokines CXCL12 and CXCL13 and the soluble form of the transmembrane chemokines CXCL16 and CX3CL1, in aqueous humor samples from patients with specific uveitic entities. Methods: Aqueous humor samples from patients with active uveitis associated with Behçet's disease (n = 13), sarcoidosis (n = 8), HLA-B27-related inflammation (n = 12), Vogt-Koyanagi-Harada (VKH) disease (n = 12), and healthy controls (n = 9) were assayed with the use of a multiplex assay. Results: All chemoattractant levels were significantly higher in all patients than in the controls. The levels of all neutrophil chemoattractants and CXCL10, CXCL16, and CX3CL1 were significantly higher in nongranulomatous uveitis (Behçet's disease and HLA-B27-associated uveitis) than in granulomatous uveitis (sarcoidosis and VKH disease), whereas the levels of the B cell chemoattractant CXCL13 were significantly higher in granulomatous uveitis than in nongranulomatous uveitis. CXCL13 levels were highest in the patients with VKH disease. CXCL9, CXCL11, and CXCL12 levels did not differ significantly. Conclusions: Inflammation in nongranulomatous uveitis appears to be driven by neutrophils and T helper 1 lymphocytes, whereas B lymphocytes may contribute to the inflammatory process in granulomatous uveitis, particularly in VKH disease.

Chemokine fractalkine/CX3CL1 negatively modulates active glutamatergic synapses in rat hippocampal neurons.

We examined the effects of the chemokine fractalkine (CX3CL1) on EPSCs evoked by electrical stimulation of Schaffer collaterals in patch-clamped CA1 pyramidal neurons from rat hippocampal slices. Acute application of CX3CL1 caused a sustained reduction of EPSC amplitude, with partial recovery after washout. CX3CL1-induced EPSC depression is postsynaptic in nature, because paired-pulse ratio was maintained, amplitude distribution of spontaneous excitatory postsynaptic currents shifted to lower values, and whole-cell current responses to AMPA were reversibly inhibited. EPSC depression by CX3CL1 is mediated by CX3CL1 receptor (CX3CR1), because CX3CL1 was unable to influence EPSC amplitude in CA1 pyramidal neurons from CX3CR1 knock-out mice. CX3CL1-induced depression of both EPSC and AMPA current was not observed in the absence of afferent fiber stimulation or AMPA receptor activation, respectively, indicating the requirement of sustained receptor activity for its development. Findings obtained from hippocampal slices, cultured hippocampal neurons, and transfected human embryonic kidney cells indicate that a Ca2+-, cAMP-, and phosphatase-dependent process is likely to modulate CX3CL1 effects because of the following: (1) CX3CL1-induced depression was antagonized by intracellular BAPTA, 8Br-cAMP, phosphatase inhibitors, and pertussis toxin (PTX); (2) CX3CL1 inhibited forskolin-induced cAMP formation sensitive to PTX; and (3) CX3CL1 inhibited forskolin-induced Ser845 GluR1 phosphorylation, which was sensitive to PTX and dependent on Ca2+ and phosphatase activity. Together, these findings indicate that CX3CL1 negatively modulates AMPA receptor function at active glutamatergic synapses through cell-signaling pathways by influencing the balance between kinase and phosphatase activity.

Chemokines: what chemokine is that?

The discovery of a new and unusual member of the chemokine family illustrates the importance of chemoattractant diversity in the regulation of leukocyte movement through the body. The chemokines are now divisible into four clearly defined subgroups on the basis of structural and functional properties.

Chemokine receptor mutant CX3CR1-M280 has impaired adhesive function and correlates with protection from cardiovascular disease in humans.

The chemokine receptor CX3CR1 is a proinflammatory leukocyte receptor specific for the chemokine fractalkine (FKN or CX3CL1). In two retrospective studies, CX3CR1 has been implicated in the pathogenesis of atherosclerotic cardiovascular disease (CVD) based on statistical association of a common receptor variant named CX3CR1-M280 with lower prevalence of atherosclerosis, coronary endothelial dysfunction, and acute coronary syndromes. However, the general significance of CX3CR1-M280 and its putative mechanism of action have not previously been defined. Here we show that FKN-dependent cell-cell adhesion under conditions of physiologic shear is severely reduced in cells expressing CX3CR1-M280. This was associated with marked reduction in the kinetics of FKN binding as well as reduced FKN-induced chemotaxis of primary leukocytes from donors homozygous for CX3CR1-M280. We also show that CX3CR1-M280 is independently associated with a lower risk of CVD (adjusted odds ratio, 0.60, P = 0.008) in the Offspring Cohort of the Framingham Heart Study, a long-term prospective study of the risks and natural history of this disease. These data provide mechanism-based and consistent epidemiologic evidence that CX3CR1 may be involved in the pathogenesis of CVD in humans, possibly by supporting leukocyte entry into the coronary artery wall. Moreover, they suggest that CX3CR1-M280 is a genetic risk factor for CVD.