Production and neuroprotective functions of fractalkine in the central nervous system

The CX3C-chemokine, fractalkine is reportedly to be expressed in the central nervous system, and up-regulated in certain pathological conditions, such as HIV encephalopathy and multiple sclerosis. In the present study, we examined the production of fractalkine and the expression of its receptor, CX3CR1 in murine glial and neuronal cell in vitro, and investigated its neuroprotective functions. Both fractalkine and CX3CR1 were expressed constitutively in neurons, microglia, and astrocytes. Neither the production of fractalkine nor its receptor expression was up-regulated by lipopolysaccharide (LPS), as measured by mRNA expression and protein synthesis. Fractalkine dose-dependently suppressed the production of nitric oxide (NO), interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha with activated microglia. It also significantly suppressed neuronal cell death induced by microglia activated with LPS and interferon-gamma, in a dose-dependent manner. These results suggest the possible functions of fractalkine as an intrinsic inhibitor against neurotoxicity by activated microglia.     

Expression of fractalkine (CX3CL1) and its receptor, CX3CR1, during acute and chronic inflammation in the rodent CNS

In this study, we investigate the expression of fractalkine (CX3CL1) and the fractalkine receptor (CX3CR1) in the naive rat and mouse central nervous system (CNS). We determine if the expression of this chemokine and its receptor are altered during chronic or acute inflammation in the CNS. In addition, we determine if CX3CL1, which has been reported to be chemoattractant to leukocytes in vitro, is capable of acting as a chemoattractant in the CNS in vivo. Immunohistochemistry was performed using primary antibodies recognizing soluble and membrane-bound CX3CL1 and the N-terminus of the CX3CR1. We found that neurons in the naive rodent brain are immunoreactive for CX3CL1 and CX3CR1, both showing a perinuclear staining pattern. Resident microglia associated with the parenchyma and macrophages in the meninges and choroid plexus constituitively express CX3CR1. In a prion model of chronic neurodegeneration and inflammation, CX3CL1 immunoreactivity is upregulated in astrocytes and CX3CR1 expression is elevated on microglia. In surviving neurons, expression of CX3CL1 appears unaltered relative to normal neurons. There is a decrease in neuronal CX3CR1 expression. Acute inflammatory responses in the CNS, induced by stereotaxic injections of lipopolysaccharide or kainic acid, results in activation of microglia and astrocytes but no detectable changes in the glial expression of CX3CL1 or CX3CR1. The expression of CX3CL1 and CX3CR1 by glial cells during inflammation in the CNS may be influenced by the surrounding cytokine milieu, which has been shown to differ in acute and chronic neuroinflammation.     

Fractalkine (CX3CL1) and fractalkine receptor (CX3CR1) distribution in spinal cord and dorsal root ganglia under basal and neuropathic pain conditions

Fractalkine is a unique chemokine reported to be constitutively expressed by neurons. Its only receptor, CX3CR1, is expressed by microglia. Little is known about the expression of fractalkine and CX3CR1 in spinal cord. Given that peripheral nerve inflammation and/or injury gives rise to neuropathic pain, and neuropathic pain may be partially mediated by spinal cord glial activation and consequent glial proinflammatory cytokine release, there must be a signal released by affected neurons that triggers the activation of glia. We sought to determine whether there is anatomical evidence implicating spinal fractalkine as such a neuron-to-glia signal. We mapped the regional and cellular localization of fractalkine and CX3CR1 in the rat spinal cord and dorsal root ganglion, under basal conditions and following induction of neuropathic pain, employing both an inflammatory (sciatic inflammatory neuropathy; SIN) as well as a traumatic (chronic constriction injury; CCI) model. Fractalkine immunoreactivity and mRNA were observed in neurons, but not glia, in the rat spinal cord and dorsal root ganglia, and levels did not change following either CCI or SIN. By contrast, CX3CR1 was expressed by microglia in the basal state, and the microglial cellular concentration was up-regulated in a regionally specific manner in response to neuropathy. CX3CR1-expressing cells were identified as microglia by their cellular morphology and positive OX-42 and CD4 immunostaining. The cellular distribution of fractalkine and CX3CR1 in the spinal circuit associated with nociceptive transmission supports a potential role in the mechanisms that contribute to the exaggerated pain state in these models of neuropathy.     

The roles of fractalkine/CX3CR1 system in neuronal death following pilocarpine-induced status epilepticus

Although fractalkine is one of chemokines involved in mediation of neuronal/microglial interaction, it is not known whether fractalkine/CX3CR1-mediated pathogenesis occurs in the rat brain following epileptogenic insults. In order to elucidate the roles of the fractalkine/CX3CR1 system in microglial activation and neurodegeneration induced by status epilepticus (SE), we investigated changes in fractalkine/CX3CR1 system within the rat hippocampus following SE. In non-SE induced animals, fractalkine and CX3CR1 immunoreactivity was detected in neurons and microglia, respectively. Following SE, fractalkine immunoreactivity was transiently increased in neurons and astrocytes. CX3CR1 immunoreactivity was also transiently detected in neurons (particularly in CA1 pyramidal cells). Intracerebroventricular infusions of recombinant rat fractalkine aggravated SE-induced neuronal damage, while fractalkine IgG or CX3CR1 IgG infusion alleviated it, compared to saline-infused animals. These findings suggest that fractalkine/CX3CR1 system may play an important role in SE-induced neuronal damages via neuron-microglial interactions.     

TGF-beta1 upregulates CX3CR1 expression and inhibits fractalkine-stimulated signaling in rat microglia

Following peripheral nerve transection, CX3CR1 and TGF-beta1 are increased in a time-dependent manner within the injured facial motor nucleus. To explore the relationship between TGF-beta1 and CX3CR1 in the CNS, the effects of TGF-beta1 on CX3CR1 mRNA, protein and fractalkine-dependent stimulation of signal transduction cascades in primary cultures of rat microglia were examined. TGF-beta1 increased steady state levels of CX3CR1 mRNA, 125I-fractalkine binding sites and blunted fractalkine-stimulated ERK1/2 phosphorylation. The half-life of CX3CR1 mRNA was unaltered by TGF-beta1 and two potential Smad binding elements (SBEs) were identified in the rat CX3CR1 promoter. TGF-beta1 may shift fractalkine-dependent signaling away from activation of ERK1/2 towards other pathways and/or may provide a mechanism for microglia to more strongly adhere to neurons.     

Expression of fractalkine and its receptor,CX3CR1, in response to ischaemia-reperfusion brain injury in the rat

Fractalkine is a neuronally expressed chemokine that acts through its G-protein-coupled receptor CX3CR1, localized on microglial and immune cells. Fractalkine might be involved in neuroinflammatory processes secondary to neuronal damage, which normally occur in a time frame of days after ischaemia. We evaluated by in situ hybridization and immunohistochemistry the expression of fractalkine and CX3CR1 in the rat brain, after a transient occlusion of the middle cerebral artery. We found that at 12 h after ischaemia neuronal fractalkine expression was transiently increased in scattered necrotic neurons of the cortex and lost from the ischaemic striatum. At 24 and 48 h after ischaemia, fractalkine immunoreactivity was strongly increased in morphologically intact cortical neurons of the ischaemic penumbra where also the stress-inducible HSP-72 was strongly up-regulated. The intensity of fractalkine immunoreactivity of neurons in the penumbra returned to basal levels at 7 days after ischaemia. Fractalkine synthesis was also induced in endothelial cells of the infarcted area, at 48 h and 7 days after ischaemia. CX3CR1 expression was detected in the activated microglial cells of the ischaemic tissue 24 and 48 h after ischaemia, and became strongly up-regulated in macrophages/phagocytic microglia inside the infarcted tissue 7 days after ischaemia. These data suggest that fractalkine may participate in the activation and chemoattraction of microglia into the infarcted tissue, and contribute to the control of leucocyte trafficking from blood vessels into the injured area.     

Role of the CX3CR1/p38 MAPK pathway in spinal microglia for the development of neuropathic pain following nerve injury-induced cleavage of fractalkine

Accumulating evidence suggests that microglial cells in the spinal cord play an important role in the development of neuropathic pain. However, it remains largely unknown how glia interact with neurons in the spinal cord after peripheral nerve injury. Recent studies suggest that the chemokine fractalkine may mediate neural/microglial interaction via its sole receptor CX3CR1. We have examined how fractalkine activates microglia in a neuropathic pain condition produced by spinal nerve ligation (SNL). SNL induced an upregulation of CX3CR1 in spinal microglia that began on day 1, peaked on day 3, and maintained on day 10. Intrathecal injection of a neutralizing antibody against CX3CR1 suppressed not only mechanical allodynia but also the activation of p38 MAPK in spinal microglia following SNL. Conversely, intrathecal infusion of fractalkine produced a marked p38 activation and mechanical allodynia. SNL also induced a dramatic reduction of the membrane-bound fractalkine in the dorsal root ganglion, suggesting a cleavage and release of this chemokine after nerve injury. Finally, application of fractalkine to spinal slices did not produce acute facilitation of excitatory synaptic transmission in lamina II dorsal horn neurons, arguing against a direct action of fractalkine on spinal neurons. Collectively, our data suggest that (a) fractalkine cleavage (release) after nerve injury may play an important role in neural-glial interaction, and (b) microglial CX3CR1/p38 MAPK pathway is critical for the development of neuropathic pain.     

Evidence that exogenous and endogenous fractalkine can induce spinal nociceptive facilitation in rats

Recent evidence suggests that spinal cord glia can contribute to enhanced nociceptive responses. However, the signals that cause glial activation are unknown. Fractalkine (CX3C ligand-1; CX3CL1) is a unique chemokine expressed on the extracellular surface of spinal neurons and spinal sensory afferents. In the dorsal spinal cord, fractalkine receptors are primarily expressed by microglia. As fractalkine can be released from neurons upon strong activation, it has previously been suggested to be a neuron-to-glial signal that induces glial activation. The present series of experiments provide an initial investigation of the spinal pain modulatory effects of fractalkine. Intrathecal fractalkine produced dose-dependent mechanical allodynia and thermal hyperalgesia. In addition, a single injection of fractalkine receptor antagonist (neutralizing antibody against rat CX3C receptor-1; CX3CR1) delayed the development of mechanical allodynia and/or thermal hyperalgesia in two neuropathic pain models: chronic constriction injury (CCI) and sciatic inflammatory neuropathy. Intriguingly, anti-CX3CR1 reduced nociceptive responses when administered 5-7 days after CCI, suggesting that prolonged release of fractalkine may contribute to the maintenance of neuropathic pain. Taken together, these initial investigations of spinal fractalkine effects suggest that exogenous and endogenous fractalkine are involved in spinal sensitization, including that induced by peripheral neuropathy.     

Fractalkine modulates TNF-alpha secretion and neurotoxicity induced by microglial activation

Among the chemokine family, fractalkine shows unusual properties: it exists as a membrane-bound and soluble protein, and both fractalkine and its receptor CX(3)CR1 are expressed predominantly in the central nervous system. In rat cell culture models, the chemokine fractalkine was expressed in neurons and microglia, but not in astrocytes and its receptor exclusively localized to microglial cells, where its expression was downregulated by treatment with the bacterial endotoxin (LPS). In microglial cultures, LPS (10 ng/ml) induced a marked increase in the release of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). The effects of LPS on TNF-alpha secretion were partially blocked (30%) by fractalkine and the effects of fractalkine were reversed by a polyclonal anti-fractalkine antibody. When microglial-associated fractalkine was neutralized by anti-fractalkine antibody, the LPS response was increased by 80%, suggesting tonic activation of microglial fractalkine receptors by endogenous fractalkine. The effects of the antibody were antagonized by the addition of fractalkine. LPS-activated microglia were neurotoxic when added to neuronal hippocampal culture, producing 20% neuronal death, as measured by NeuN-positive cell counting. An anti-fractalkine antibody produced neurotoxic effects of similar magnitude in this co-culture system and also markedly potentiated the neurotoxic effects of LPS-activated microglia (40% neuronal death). These results suggest that endogenous fractalkine might act tonically as an anti-inflammatory chemokine in cerebral tissue through its ability to control and suppress certain aspects of microglial activation. These data may have relevance to degenerative conditions such as multiple sclerosis, in which cerebral inflammatory processes may be activated.     

CX3CL1/CX3CR1-mediated microglia activation plays a detrimental role in ischemic mice brain via p38MAPK/PKC pathway

The exact roles of activated microglia and fractalkine (CX3CL1)/fractalkine receptor (CX3CR1) signaling are not fully understood in brain ischemic injury and the findings reported are controversial. Here, we investigated the effects of CX3CR1 siRNA on the expression of CX3CR1, p38 mitogen-activated protein kinase (p38MAPK), Protein Kinase C (PKC) and inflammatory cytokines, microglia activation, white matter lesions, and cognitive function in mice treated with bilateral common carotid artery stenosis (BCAS) in vivo as well as effects of exogenous CX3CL1, CX3CR1 siRNA, and SB2035080 on expression of inflammatory cytokines in BV2 microglia treated with oxygen–glucose deprivation (OGD) in vitro. We showed that CX3CR1 siRNA significantly inhibited the increased expression of CX3CR1, p38MAPK, PKC as well as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, and also attenuated microglia activation, white matter lesions, and cognitive deficits induced by BCAS in mice brain. We also showed that exogenous CX3CL1 could induce a further enhancement in TNF-α and IL-1β expression, which could be suppressed by CX3CR1 siRNA or by the p38MAPK inhibitor in OGD-treated BV2 microglial cells in vitro. Our findings indicated that CX3CL1/CX3CR1-mediated microglial activation plays a detrimental role in ischemic brain via p38MAPK/PKC signaling and also suggested that CX3CL1/CX3CR1 axis might be a putative therapeutic target to disrupt the cascade of deleterious events that lead to brain ischemic injury.     

Fractalkine (CX3CL1) and brain inflammation: Implications for HIV-1-associated dementia

Leukocyte migration and activation play an important role in immune surveillance and the pathogenesis of a variety of neurodegenerative disorders, including human immunodeficiency virus (HIV)-1-associated dementia (HAD). A novel chemokine named fractalkine (FKN, CX3CL1), which exists in both membrane-anchored and soluble isoforms, has been proposed to participate in the generation and progression of inflammatory brain disorders. Upon binding to the CX3C receptor one (CX3CR1), FKN induces adhesion, chemoattraction, and activation of leukocytes, including brain macrophages and microglia (MP). Constitutively expressed in the central nervous system (CNS), mainly by neurons, FKN is up-regulated and released in response to proinflammatory stimuli. Importantly, FKN is up-regulated in the brain tissue and cerebrospinal fluid (CSF) of HAD patients. Together, these observations suggest that FKN and its receptor have a unique role in regulating the neuroinflammatory events underlying disease. This review will examine how FKN contributes to the recruitment and activation of CX3CR1-expressing MP, which are critical events in the neuropathogenesis of HAD.     

CX3 chemokine receptor 1 deficiency leads to reduced dendritic complexity and delayed maturation of newborn neurons in the adult mouse hippocampus

Previous studies have shown that microglia impact the proliferation and differentiation of neurons during hippocampal neurogenesis via the fractalkine/CX3 chemokine receptor 1 (CX3CR1) signaling pathway. However, whether microglia can influence the maturation and dendritic growth of newborn neurons during hippocampal neurogenesis remains unclear. In the present study, we found that the number of doublecortin-positive cells in the hippocampus was decreased, and the dendritic length and number of intersections in newborn neurons in the hippocampus were reduced in transgenic adult mice with CX3CR1 deficiency (CX3CR1^GFP/GFP). Furthermore, after experimental seizures were induced with kainic acid in these CX3CR1-deficient mice, the expression of c-fos, a marker of neuronal activity, was reduced compared with wild-type mice. Collectively, the experimental findings indicate that the functional maturation of newborn neurons during hippocampal neurogenesis in adult mice is delayed by CX3CR1 deficiency.     

An initial investigation of spinal mechanisms underlying pain enhancement induced by fractalkine, a neuronally released chemokine

Fractalkine is a chemokine that is tethered to the extracellular surface of neurons. Fractalkine can be released, forming a diffusible signal. Spinal fractalkine (CX3CL1) is expressed by sensory afferents and intrinsic neurons, whereas its receptor (CX3CR1) is predominantly expressed by microglia. Pain enhancement occurs in response both to intrathecally administered fractalkine and to spinal fractalkine endogenously released by peripheral neuropathy. The present experiments examine whether fractalkine-induced pain enhancement is altered by a microglial inhibitor (minocycline) and/or by antagonists/inhibitors of three putative glial products implicated in pain enhancement: interleukin-1 (IL1), interleukin-6 (IL6) and nitric oxide (NO). In addition, it extends a prior study that demonstrated that intrathecal fractalkine-induced mechanical allodynia is blocked by a neutralizing antibody to the rat fractalkine receptor, CX3CR1. Here, intrathecal anti-CX3CR1 also blocked fractalkine-induced thermal hyperalgesia. Furthermore, blockade of microglial activation with minocycline prevented both fractalkine-induced mechanical allodynia (von Frey test) and thermal hyperalgesia (Hargreaves test). Microglial activation appears to lead to the release of IL1, given that pretreatment with IL1 receptor antagonist blocked both fractalkine-induced mechanical allodynia and thermal hyperalgesia. IL1 is not the only proinflammatory cytokine implicated, as a neutralizing antibody to rat IL6 also blocked fractalkine-induced pain facilitation. Lastly, NO appears to be importantly involved, as l-NAME, a broad-spectrum NO synthase inhibitor, also blocked fractalkine-induced effects. Taken together, these data support that neuronally released fractalkine enhances pain via activation of spinal cord glia. Thus, fractalkine may be a neuron-to-glia signal triggering pain facilitation.     

CX₃CR1 deficiency exacerbates neuronal loss and impairs early regenerative responses in the target-ablated olfactory epithelium

The olfactory epithelium is a site of sustained adult neurogenesis where olfactory sensory neurons are continuously replaced from endogenous stem/progenitor cells. Epithelial macrophages have been implicated in the phagocytosis of degenerating cells but the molecular mechanisms allowing for their recruitment and activation while maintaining a neurogenic microenvironment are poorly understood. We have previously shown that the chemokine fractalkine (CX?CL1) is expressed by olfactory sensory neurons and ensheathing cells in the olfactory epithelium. In turn, the fractalkine receptor, CX?CR1, is expressed on macrophages and dendritic cells within the olfactory epithelium. We report that a selective cell death of olfactory sensory neurons in the epithelium of CX?CR1-deficient mice via target ablation (i.e. olfactory bulbectomy) results in an exacerbated loss of olfactory sensory neurons compared to wild-type mice. In addition, reduced proliferation of intraepithelial stem/progenitor cells was observed in lesioned CX?CR1-deficient mice, suggesting an impaired regenerative response. Importantly, a lack of CX?CL1-signaling caused increased recruitment of macrophages into the olfactory epithelium, which in turn contained higher levels of pro-inflammatory cytokines (e.g. TNF-α and IL-6) as determined by qPCR. We also present novel data showing that, relative to wild-type, CX?CR1-deficient macrophages have diminished phagocytic activity following stimulation with CX?CL1. Collectively, our data indicate that signaling through the CX?CR1 receptor modulates macrophage activity, resulting in an environment conducive to olfactory sensory neuron clearance and targeted replacement from endogenous stem/progenitor cells.     

CX3CR1 Disruption Differentially Influences Dopaminergic Neuron Degeneration in Parkinsonian Mice Depending on the Neurotoxin and Route of Administration

Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic neurons accompanied by an inflammatory reaction. The neuron-derived chemokine fractalkine (CX3CL1) is an exclusive ligand for the receptor CX3CR1 expressed on microglia. The CX3CL1/CX3CR1 signaling is important for sustaining microglial activity. Using a recently developed PD model, in which the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin is delivered intranasally, we hypothesized that CX3CR1 could play a role in neurotoxicity and glial activation. For this, we used CX3CR1 knock-in mice and compared results with those obtained using the classical PD models through intraperitonal MPTP or intrastriatal 6-hydroxydopamine (6-OHDA). The striatum from all genotypes (CX3CR1^+/+, CX3CR1^+/GFP and CX3CR1-deficient mice) showed a significant dopaminergic depletion after intranasal MPTP inoculation. In contrast to that, we could not see differences in the number of dopaminergic neurons in the substantia nigra of CX3CR1-deficient animals. Similarly, after 6-OHDA infusion, the CX3CR1 deletion decreased the amphetamine-induced turning behavior observed in CX3CR1^+/GFP mice. After the 6-OHDA inoculation, a minor dopaminergic neuronal loss was observed in the substantia nigra from CX3CR1-deficient mice. Distinctly, a more extensive neuronal cell loss was observed in the substantia nigra after the intraperitoneal MPTP injection in CX3CR1 disrupted animals, corroborating previous results. Intranasal and intraperitoneal MPTP inoculation induced a similar microgliosis in CX3CR1-deficient mice but a dissimilar change in the astrocyte proliferation in the substantia nigra. Nigral astrocyte proliferation was observed only after intraperitoneal MPTP inoculation. In conclusion, intranasal MPTP and 6-OHDA lesion in CX3CR1-deficient mice yield no nigral dopaminergic neuron loss, linked to the absence of astroglial proliferation.     

CX3CL1 and CX3CR1 expression in human brain tissue: noninflammatory control versus multiple sclerosis

An important role for CX3CL1 in neuroinflammation and neurodegeneration has been suggested in recent publications. In this study, we compared the expression of CX3CL1 and its receptor CX3CR1 in human brain tissue derived from control patients without neurological complications and in multiple sclerosis (MS) patients. Results from this study demonstrate that CX3CL1 is constitutively expressed in human central nervous system (CNS) astrocytes in vivo and under basal conditions in human adult astrocyte cultures. CX3CR1 is expressed on astrocytes and microglial cells both in vivo and in vitro. Chemotaxis assay shows a functional response upon CX3CR1 signaling in microglial cells. Although CX3CL1 expression is upregulated in cultured astrocytes in response to proinflammatory cytokines, no evidence for expression differences of CX3CL1 between control patients and MS patients was found. Our data suggest that CX3CL1 has more general physiological functions, which occur also in the absence of proinflammatory conditions.     

Role of fractalkine (CX3CL1) in regulating neuron-microglia interactions: development of viral-based CX3CR1 antagonists

Blocking the effects of fractalkine therapeutically may regulate microglia cell activation and provide neuroprotection in the AD brain. A human herpesvirus 8-encoded chemokine, termed vMIP-II is a non-selective chemokine receptor antagonist (binding multiple chemokine receptors, including CX3CR1). By comparing vMIP-II and FKN, we have generated molecules that selectively antagonize CX3CR1 activation. The results from these studies will guide future development of therapeutic agents designed to modulate microglial activation with the goal of preventing or slowing the progression of AD.