CX3CL1 up-regulation is associated with recruitment of CX3CR1+ mononuclear phagocytes and T lymphocytes in the lungs during cigarette smoke-induced emphysema

CX3CR1 is expressed on monocytes, dendritic cells, macrophages, subsets of T lymphocytes, and natural killer cells and functions in diverse capacities such as leukocyte adhesion, migration, and cell survival on ligand binding. Expression of the CX3CL1 gene, whose expression product is the sole ligand for CX3CR1, is up-regulated in human lungs with chronic cigarette smoke-induced obstructive lung disease. At present, it is unknown whether CX3CL1 up-regulation is associated with the recruitment and accumulation of immune cells that express CX3CR1. We show that mice chronically exposed to cigarette smoke up-regulate CX3CL1 gene expression, which is associated with an influx of CX3CR1⁺ cells in the lungs. The increase in CX3CR1⁺ cells is primarily comprised of macrophages and T lymphocytes and is associated with the development of emphysema. In alveolar macrophages, cigarette smoke exposure increased the expression of both CX3CR1 and CX3CL1 genes. The inducibility of CX3CR1 expression was not solely dependent on a chronic stimulus because lipopolysaccharide up-regulated CX3CR1 in RAW264.7 cells in vitro and in mononuclear phagocytes in vivo. Our findings suggest a mechanism by which macrophages amplify and promote CX3CR1⁺ cell accumulation within the lungs during both acute and chronic inflammatory stress. We suggest that one function of the CX3CR1-CX3CL1 pathway is to recruit and sustain divergent immune cell populations implicated in the pathogenesis of cigarette smoke-induced emphysema.     

Chemokines and common variable immunodeficiency; possible contribution of the fractalkine system (CX3CL1/CX3CR1) to chronic inflammation

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by defective immunoglobulin production and high frequency of bacterial infections, autoimmunity and manifestations of chronic inflammation. The chemokine Fractalkine (CX3CL1) and its receptor CX3CR1 is suggested to play an important role in the pathogenesis of several inflammatory disorders. We hypothesized that enhanced CX3CL1/CX3CR1 interaction could be involved in the chronic inflammation characterising subgroups of CVID. CVID patients were characterized by raised plasma levels of CX3CLl and enhanced expression of its corresponding receptor CX3CR1 on CD4(+) and CD8(+) T cells, including both CD45RA(+) and CD45RA(-) subsets. CX3CR1 expression was particularly enhanced in patients characterized by chronic inflammation in vivo. The high expression of the receptor in CVID patients was accompanied by enhanced chemotactic, adhesive, and other inflammatory cell responses to stimulation with CX3CL1. Our findings suggest that increased CX3CL1/CX3CR1 interaction could contribute to the inflammatory phenotype seen in subgroups of CVID patients.     

Chemoattraction,adhesion and activation of natural killer cells are involved in the antitumor immune response induced by fractalkine/CX3CL1

Fractalkine (FK, also called neurotactin or CX3CL1) is a CX3C chemokine that can chemoattract T lymphocytes, monocytes, dendritic cells (DC) and natural killer (NK) cells. One of our previous studies demonstrated that FK in soluble form can chemoattract T cells and DC and membrane-bound FK can adhere T cells and DC. Vaccination with 3LL lung carcinoma cells gene-modified with FK (3LL-FK) induces potent antitumor CTL response. The aim of the present study is to investigate whether NK cells participate in FK-induced antitumor immunity. We found that NK activity was increased in mice inoculated with 3LL-FK and in vivo depletion of NK cells resulted in the decreased tumor growth inhibition of 3LL-FK, indicating that NK cells play an important role in the antitumor immunity induced by FK. Further studies showed 3LL-FK could chemoattract, adhere NK cells and attract more NK cells to infiltrate into tumor tissue. Incubation of NK cells with 3LL-FK could increase the cytotoxicity of NK cells against YAC-1 cells and even against NK-resistant parental 3LL cells. IL-12 production increased more significantly in the 3LL-FK tumor nodules. Taken together with CTL response induced by 3LL-FK, our data demonstrate that FK, expressed by gene-modified tumor cells, can induce potent antitumor effect through different mechanisms, one of which involves chemoattraction of NK cells into tumor sites and activation of NK cells.     

CX3CL1/fractalkine is a novel regulator of normal and malignant human B cell function

CX(3)CL1, or fractalkine, the unique member of the CX(3)C chemokine family, exists as a transmembrane glycoprotein, as well as in soluble form, each mediating different biological activities, and is constitutively expressed in many hematopoietic and nonhematopoietic tissues. CX(3)CR1, the CX(3)CL1 exclusive receptor, is a classical GPCR, expressed on NK cells, CD14(+) monocytes, and some subpopulation of T cells, B cells, and mast cells. A recent paper by our group has demonstrated for the first time that highly purified human B cells from tonsil and peripheral blood expressed CX(3)CR1 at mRNA and protein levels. In particular, tonsil na?ve, GC, and memory B cells expressed CX(3)CR1, but only GC centrocytes were attracted by soluble CX(3)CL1, which with its receptor, are also involved in the pathogenesis of several inflammatory disorders, as well as of cancer. Previous studies have shown that CX(3)CR1 is up-regulated in different types of B cell lymphoma, as well as in B-CLL. Recently, we have demonstrated that the CX(3)CL1/CX(3)CR1 axis is involved in the interaction of B-CLL cells with their microenvironment. Taken together, our data delineate a novel role for the CX(3)CL1/CX(3)CR1 complex in the biology of normal B cells and B-CLL cells. These topics are the subject of this review article.     

Altered expression of CX3CL1 in patients with epilepsy and in a rat model

Chemokine C-X3-C motif ligand 1 (CX3CL1, alias fractalkine), is highly expressed in the central nervous system and participates in inflammatory responses. Recent studies indicated that inflammatory processes within the brain constitute a common and crucial mechanism in the pathophysiological characteristics of epilepsy. This study investigated the expression pattern of CX3CL1 in epilepsy and its relationship with neuronal loss. Double immunolabeling, IHC, and immunoblotting results showed that CX3CL1 expression was up-regulated in the temporal neocortex of patients with temporal lobe epilepsy. In a rat model of epilepsy, CX3CL1 up-regulation began 6 hours after epilepsy, with relatively high expression for 60 days. In addition, ELISA revealed that the concentrations of CX3CL1 in cerebrospinal fluid and serum were higher in epileptic patients than in patients with neurosis but lower than in patients with inflammatory neurological diseases. Moreover, H&E staining demonstrated significant neuronal loss in the brains of epileptic patients and in the rat model. Finally, the expression of tumor necrosis factor-related apoptosis-inducing ligand was significantly increased in both patients and the animal model, suggesting that tumor necrosis factor-related apoptosis-inducing ligand may play a role in CX3CL1-induced cell death. Thus, our results indicate that CX3CL1 may serve as a possible biomarker of brain inflammation in epileptic patients.     

Hypertrophy of the ligament flavum in degenerative lumbar stenosis associated with the increased expression of fractalkine (CX3CL1)/CX3CR1 chemokine

Abstract

Fractalkine (CX3CL1) and its receptor (CX3CR1) comprise a chemokine system involved in leukocyte recruitment and adhesion in chronic inflammatory disease, but its role in spinal diseases is unknown. The purpose of this study is to investigate the role of CX3CL1/CX3CR1 chemokine on hypertrophy of the ligamentum flavum (LF) in degenerative lumbar stenosis (DLS) compared with that of non-degenerative spinal condition (NDS) of the lumbar spine and correlation between expression of CX3CL1/CX3CR1 chemokine and thickness of LF. The mRNA concentrations of CX3CL1/CX3CR1 chemokine were analyzed in the surgically obtained LF specimens from DLS (n?=?10) and NDS (n?=?11) by real-time PCR. The localization of CX3CL1/CX3CR1 chemokine within the LF was determined using immunohistochemical study. Plasma levels of soluble FKN (sFKN) were measured by enzyme-linked immunosorbent assay, respectively. The thickness of the LF was measured with axial T1-weighted MRI. The cells that express CX3CL1/CX3CR1 chemokine ratio in the LF observed in DLS group were substantially higher than in NDS group. In ELISA, the plasma levels of sFKN was significantly increased in DLS compared with patients in the other groups (p?=?0.006). There was greater CX3CL1/CX3CR1 expression in DLS as quantified by RT-PCR (p?=?0.004, 0.010). Thickness of LF in patients was significantly correlated with serum CX3CL1 level (R^2?=?0.824, p?=?0.003) and with mRNA expression of CX3CL1/CX3CR1 (R^2?=?0.671, p?=?0.000) (R^2?=?0.514, p?=?0.001). This study identified for the first time that increases in CX3CL1 and CX3CR1-expressing cells are significantly related to LF hypertrophy.     

Extracellular adenosine signaling induces CX3CL1 expression in the brain to promote experimental autoimmune encephalomyelitis

ABSTRACT: BACKGROUND: Multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) are debilitating neuroinflammatory diseases mediated by lymphocyte entry into the central nervous system (CNS). While it is not known what triggers lymphocyte entry into the CNS during neuroinflammation, blockade of lymphocyte migration has been shown to be effective in controlling neuroinflammatory diseases. Since we have previously shown that extracellular adenosine is a key mediator of lymphocyte migration into the CNS during EAE progression, we wanted to determine which factors are regulated by adenosine to modulate EAE development. Methods: We performed a genetic analysis of wild type and CD73-/- (that are unable to produce extracellular adenosine and are protected from EAE development) to identify factors that are both important for EAE development and controlled by extracellular adenosine signaling. Results: We show that extracellular adenosine triggered lymphocyte migration into the CNS by inducing the expression of the specialized chemokine/adhesion molecule CX3CL1 at the choroid plexus. In wild type mice, CX3CL1 is upregulated in the brain on Day 10 post EAE induction, which corresponds with initial CNS lymphocyte infiltration and the acute stage of EAE. Conversely, mice that cannot synthesize extracellular adenosine (CD73-/- mice) do not upregulate CX3CL1 in the brain following EAE induction and are protected from EAE development and its associated lymphocyte infiltration. Additionally, blockade of the A2A adenosine receptor following EAE induction prevents disease development and the induction of brain CX3CL1 expression. The CX3CL1 induced during EAE is found on the choroid plexus, which is the barrier between the blood and cerebral spinal fluid in the brain and is a prime entry point into the CNS for immune cells. Furthermore, CX3CL1 expression can be induced in the brains of mice and in choroid plexus cell line following A2A adenosine receptor agonist administration. Most importantly, we show that CX3CL1 blockade protects against EAE development and inhibits lymphocyte entry into the CNS. Conclusions: We conclude that extracellular adenosine is an endogenous modulator of neuroinflammation during EAE that induces CX3CL1 at the choroid plexus to trigger lymphocyte entry into the brain.     

Macrophage migration inhibitory factor of Sciaenops ocellatus regulates immune cell trafficking and is involved in pathogen-induced immune response

Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine involved in immunoregulation and inflammation. In this study, we examined the expression and biological function of a MIF, SoMIF, from red drum Sciaenops ocellatus. SoMIF is composed of 115 residues and shares 85–99% overall sequence identities with the MIF of a number of teleost. SoMIF expression was detected in a wide range of tissues and upregulated by bacterial and viral infection in a time-dependent manner. In head kidney (HK) leukocytes, pathogen infection induced SoMIF expression, and the expressed SoMIF was secreted into the extracellular milieu. Recombinant SoMIF (rSoMIF) purified from Escherichia coli inhibited the migration of both HK monocytes and lymphocytes, and this inhibitory effect was abolished by the presence of anti-rSoMIF antibodies. When rSoMIF was administered into red drum, it stimulated the production of reactive oxygen species in HK monocytes both in the presence and absence of pathogen infection. In vivo infection study showed that compared to untreated fish, fish pre-treated with rSoMIF before bacterial infection exhibited significantly lower bacterial loads in blood, kidney, spleen, and liver. Taken together, these results indicate that SoMIF is a secreted protein that regulates immune cell trafficking and is involved in pathogen-induced immune response.     

Thrombin-induced expression of endothelial CX3CL1 potentiates monocyte CCL2 production and transendothelial migration

CX3CL1 (fractalkine, neurotactin) is the sole CX3C chemokine. It induces monocyte locomotion in its cleaved form, but in its membrane-anchored form, it also acts as an adhesion molecule. The expression of CX3CL1 is up-regulated in endothelial cells by proinflammatory cytokines such as IL-1 or TNF-alpha. Here, we studied the effect of the serine protease thrombin on endothelial CX3CL1 induction and its putative relevance for monocyte function. In HUVEC, thrombin triggered a time- and concentration-dependent expression of CX3CL1 at the mRNA and the protein level as shown by RT-PCR, Western immunoblotting, and flow cytometric analysis. Thrombin induced CX3CL1 by activating protease-activated receptor 1 (PAR1) as demonstrated by the use of PAR1-activating peptide and the PAR1-specific antagonist SCH 79797. The thrombin-induced CX3CL1 expression was NF-kappaB-dependent, as shown by EMSA, ELISA, and by inhibition of the NF-kappaB signaling pathway by the IkappaB kinase inhibitor acety-11-keto-beta-boswellic acid or by transient overexpression of a transdominant-negative form of IkappaBalpha. Upon cocultivation of human monocytes with HUVEC, the thrombin-dependent induction of membrane-anchored CX3CL1 in HUVEC triggered monocyte adhesion and an enhanced release of the MCP-1/CCL2 by monocytes and potentiated the monocyte transendothelial migration. Accordingly, the recombinant extracellular domain of CX3CL1 induced CCL2 release by monocytes. Thus, the thrombin-induced monocyte/endothelial cell cross-talk mediated by increased CX3CL1 expression potentiates the CCL2 chemokine generation that might contribute to the recruitment of monocytes into inflamed areas.     

结肠癌患者CX3CL1的表达情况与术后预后的相关性分析

目的探讨结肠癌组织中CX3CL1的表达情况、临床意义及其与术后预后分析的相关性。方法应用免疫组织化学法检测104例结肠癌病人的石蜡切片中CX3CL1的表达情况,用χ2检验来分析CX3CL1的临床意义,用Kaplan-Meier方法及建立Cox回归模型来分析CX3CL1表达情况与结肠癌术后预后的相关性。结果 CX3CL1高表达组占所有结肠癌病人的61.54%。CX3CL1的表达与年龄、性别、肿瘤大小、肿瘤位置、Dukes分期、组织分化程度无明显相关（P〉0.05）,但与淋巴结转移相关（P〈0.05）。CX3CL1高表达组（n=64）比低表达组（n=40）有更长的生存期（P=0.0096）。Cox回归模型分析表明,CX3CL1表达可作为判断结肠癌患者术后生存和预后的独立指标（RR=2.4;P=0.0135）。结论 CX3CL1可作为结肠癌的独立预后因素,结肠癌中CX3CL1高表达提示更好的预后。     

Antitumor immune response by CX3CL1 fractalkine gene transfer depends on both NK and T cells

The CX3C chemokine fractalkine (CX3CL1) exists as both a membrane-bound form promoting firm cell-cell adhesion and a soluble form chemoattracting leukocytes expressing its receptor CX3CR1. When adenoviral vector expressing mouse fractalkine (AdFKN) was transduced to the tumor cells, fractalkine was expressed as both membrane-bound form on the tumor cells and soluble form in the supernatant in vitro. Intratumoral injection of AdFKN (1 x 10(9)PFU/tumor) into C26 and B16F10 tumors resulted in marked reduction of tumor growth compared to control (C26: 86.5%, p<0.001; B16F10: 85.5%, p<0.001). Histological examination of tumor tissues revealed abundant infiltration of NK cells, dendritic cells, and CD8(+) T lymphocytes 3 and/or 6 days after treatment with AdFKN. Splenocytes from mice treated by AdFKN developed tumor-specific cytotoxic T cells, and thereby protected from rechallenging with parental tumor cells. Antitumor effects by AdFKN were completely abrogated in both NK cell-depleted mice and CD8(-/-) mice, and partially blocked in CD4(-/-) mice. These data indicated that fractalkine mediates antitumor effects by both NK cell-dependent and T cell-dependent mechanisms. This study suggests that fractalkine can be a suitable candidate for immunogene therapy of cancer because fractalkine induces both innate and adaptive immunity.     

The transmembrane form of the CX3CL1 chemokine fractalkine is expressed predominantly by epithelial cells in vivo

Fractalkine (CX3CL1) is synthesized as a type I transmembrane protein. Its unique CX(3)C chemokine domain is attached to a 241-amino acid mucin stalk, a 19-amino acid transmembrane domain, and a 37-amino acid intracellular domain of unknown function. A soluble form of fractalkine can be generated by proteolytic cleavage at the base of the mucin stalk. Novel monoclonal and polyclonal antibodies that specifically recognize only the amino- or carboxyl-terminal ends of the human fractalkine molecule have revealed that epithelial cells are the predominant cell type expressing transmembrane forms of fractalkine in human skin, the tonsil, and the large intestine. Using these specific anti-fractalkine reagents we do not detect high-level expression of fractalkine on endothelial cells in normal or inflamed colon samples obtained from patients with Crohn's disease or ulcerative colitis. In contrast to previous reports we do not detect fractalkine expression by Langerhans cells or immature dendritic cells in mucosal-associated lymphoid tissues in vivo. We show that the reagent used in previous studies, an anti-fractalkine N-terminal peptide antisera, cross-reacts with human CD84. Finally we discuss potential roles for fractalkine in constitutive leukocyte trafficking based on its observed pattern of expression in epithelia.     

CX3CL1/fractalkine在肺动脉高压过程中的表达变化及葛根素的干预作用

目的: 观察趋化因子CX₃CL1/fractalkine(FKN)在野百合碱诱导的肺动脉高压大鼠模型中的变化并探讨葛根素的干预效应。方法: 复制大鼠肺动脉高压模型,随机分为溶剂对照组(C组)、野百合碱造模组(M组)和葛根素干预组(M+P组)。测量并比较各组肺动脉平均压(mPAP)、右心室平均压(mRVP)、颈动脉平均压(mCAP)、右心室游离壁(RV)和左心室加室间隔(LV＋S)重量比。观察肺细小动脉显微结构变化,测定肺细小动脉管壁面积/管总面积(WA/TA)、肺细小动脉中膜厚度(PAMT),测定血浆可溶性fractalkine(sFKN)浓度,肺细小动脉FKN蛋白,肺组织FKN mRNA的表达。结果: M组mPAP、mRVP、RV/(LV+S)、WA/TA和PAMT显著高于C组(P<0.01),M+P组RV/(LV+S)、WA/TA和PAMT较M组显著降低(P<0.01),M组血浆sFKN、肺组织FKN mRNA和肺动脉壁FKN含量显著大于C组(P<0.01),M+P组显著降低(P<0.05),sFKN与PAMT呈正相关(r＝0.719,P<0.01),与RV/(LV+S)呈正相关(r＝0.685,P<0.01)。结论: 葛根素具有抑制FKN表达、肺动脉高压发展和肺血管结构重建的作用。     

CX3CL1 in allergic diseases: not just a chemotactic molecule

Allergic asthma and atopic dermatitis (AD) are two allergic diseases that are primarily driven by the activation of T helper (Th)2 cells. Th2 cells produce cytokines that directly contribute to the symptoms of these diseases. The recruitment and maintenance of Th2 cells into the target tissues are two key events in the pathogenesis of allergic asthma and AD. While migration is mediated by both chemokines and lipid mediators such as leukotrienes and prostaglandins, very little is known about the molecules involved in lymphocyte survival and maintenance in inflamed tissues. However, chemokines could also play a role in this phenomenon. An example of this could be illustrated by CX3CL1, also known as fractalkine. CX3CL1 is a chemokine that is upregulated in some inflammatory diseases including allergic pathologies and that was recently demonstrated to provide a survival signal upon binding to its unique receptor CX3CR1.