Abstract

Background

Chemokines are key mediators controlling infiltration of leukocytes to inflamed areas. They consist of a class of related proteins that exert chemotactic properties on leukocytes via interactions with select members of the G-protein coupled, cell membrane-spanning receptors (GPCRs). The so far 50 identified chemokines are divided into 4 subgroups, the XC, CC, CXC and CX₃C chemokines, and the corresponding GPCRs are accordingly denominated XCR (presently one member), CCR (11 deorphanized members), CXCR (6 members) and CX₃CR (one single member). Synthesis of chemokines is rapidly induced in damaged or infected tissues and the cell-specific expression of chemokine receptors combined with the situation-specific production of their chemokine ligands provide cues to attract appropriate cell populations to combat invading organisms and neoplastic cells and to clear and repair damaged tissues. Chemokines are also thought to drive chronic inflammatory processes and this have fuelled hopes that pharmacological intervention of ligand-triggered activation of chemokine receptors may serve to reduce clinical manifestations in disorders with inflammatory components [1].

CX₃CL1 (alternative names: fractalkine or neurotactin) was identified in 1997 as a chemokine of 373 amino acids with, for the chemokine family, an atypical structure, a chemokine domain tethered on top of a mucin-like domain which is followed by a single transmembrane spanning domain and a short cytoplasmic tail [2-4]. CX₃CL1 is expressed within the brain, heart, lung, kidney, muscle and testis [4-8] where it interacts with a single GPCR, CX₃CR1 [9,10] to trigger chemotaxis and adhesion [10-13] of CX₃CR1 expressing cells, including neutrophils, monocytes, NK cells and Th-1 polarized T cells [10,14]. Studies of expression profiles, functional role in in vitro and in vivo systems and genetic associations to diseases have provided promising clues to a potential role of CX₃CL1 and its receptor in, among others, rheumatoid arthritis [15-17], allograft rejections [18] and atherosclerosis [19-21].

The physiological role of CX₃CL1 and its receptor in the brain, however, is less clear. Expression of CX₃CL1 within the brain is localized to neurons [22-25], whereas CX₃CR1 is expressed by brain microglia [6,24-26]. Axotomy of the facial nerve trigger increased expression of CX₃CL1 among the severed motor neurons in the facial nucleus [6] and a similar response was recently observed following intraparenchymal injection of prion proteins [22]. Induction of experimental autoimmune encephalomyelitis (EAE; an animal model for multiple sclerosis) in the mouse has also been associated with CX₃CL1-like immunoreactivity in blood vessels within the inflammatory brain lesions [3]. This finding is complemented by the increased levels of CX₃CR1 mRNA in the spinal cord of rats with EAE, as demonstrated by RNAse protection assay [27]. CX₃CL1 has moreover been shown to regulate microglia functions, including CX₃CL1-induced mobilization of intracellular Ca²⁺, chemotaxis and the inhibition of Fas-mediated apoptosis in vitro [6,23,28]. This is reflected in the activation of microglia following injection of CX₃CL1 in vivo to the rat brain parenchyma [22], indicating a potential role for CX₃CL1 and CX₃CR1 in mediating neuronal-microglial cross talk under normal and pathological conditions. In addition, previous claims of CX₃CL1 expression in endothelial cells [3,15,16,29-32], combined with CX₃CR1 on monocytes and Th-1 cells [5,14-16,33,34] may indicate a role in attracting pathogenic cells to sites of neuroinflammation, as well.

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS. It is associated with local activation of microglia and astroglia, infiltration of activated macrophages and T cells, active degradation of myelin and damage to axons and neurons. The disease often develops from isolated self-limiting episodes with various neurological manifestations, including paralysis, to persistent, accentuated loss of neurological functions. The neuroinflammatory component of the disease is, at least in initial phases, thought to mediate important aspects of the clinical manifestations. The proposed role for CX₃CL1 in the control of microglia activation and leukocyte infiltration places this chemokine/receptor pair in a potentially strategic position to control key aspects in the pathological events that are associated with development of brain lesions in MS. In this study, we examine this hypothesis further by analyzing the distribution, kinetics, regulation and cellular origin of CX₃CL1 and CX₃CR1 mRNA expression in the CNS of rats with MOG-induced EAE. The expression of CX₃CL1 and its receptor was studied with in situ hybridization histochemical detection of their mRNA with radio labeled cRNA probes in combination with immunohistochemical staining of phenotypic cell markers. Our findings suggest that CX₃CL1 and its receptor may control aspects of the neuroinflammatory processes in MOG-induced EAE, and possibly also MS.

Methods

Results

Expression of mRNA encoding CX₃CL1 and its receptor in the normal rat brain and spinal cord

Radio labeled antisense cRNA probes transcribed from cDNA encoding rat CX₃CL1 (Fig. ​(Fig.1)1) and its receptor, CX₃CR1 (data not shown), were initially hybridized in situ to 5 μm tissue section obtained throughout the entire rat brain and spinal cord. In the forebrain, cells expressing high levels of CX₃CL1 mRNA were detected throughout the olfactory bulb, cerebral cortex, amygdala, globus-pallidus and thalamus (Fig. ​(Fig.1).1). These cells manifested, with few exceptions, a neuronal phenotype and co-distributed with cells staining positive for the pan-neuronal marker NeuN (exemplified in Fig. 5a, b at the spinal cord level). High levels of CX₃CL1 mRNA expression were also detected in all pyramidal cells within the hippocampal formation (Fig. ​(Fig.1).1). Significantly lower, but still clearly detectable, levels of CX₃CL1 mRNA were observed within all aspects of the hypothalamus (Fig ​(Fig1),1), with neurons within the ventromedial nucleus displaying the strongest labeling. In the mesencephalon, pons, medulla oblongata and the spinal cord low to medium expression levels were uniformly detected in neuronal-like cells. A notable exception was the cerebellum, where very low levels of expression were detected within granule and pyramidal cells whereas neuronal like cells in the deep cerebellar nuclei expressed CX₃CL1 mRNA at medium levels (Fig. ​(Fig.1).1). Tissue sections hybridized in parallel with a radio labeled cRNA probe transcribed in the sense orientation did not reveal any hybridization signal above background levels (data not shown).

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Figure 1

Distribution of CX₃CL1 mRNA expressing cells in the normal rat brain. Coronal sections sampled at regular intervals throughout the rostro-caudal extent of the normal rat brain hybridized with a 35S-labeled antisense-CRNA probe encoding rat CX₃CL1. Cells expressing mRNA encoding the chemokine are visualized as accumulations of white silver grain in this microscopic darkfield illumination at low magnification. The letters in each subfigure refer to the approximate levels according to the Paxinos stereotaxic brain atlas [61]. The highest levels of CX₃CL1 mRNA were detected exclusively within the grey matter of the cerebral cortex (A:a, B:a, C:a, D:a), hippocampus (C:b), septum (B:c), thalamus (C:d) and striatum (B:e). Medium to low level expression was detected in the hypothalamus (C:f), pons (E:g), mesencephalon (D:h), medulla oblongata (not shown) and spinal cord (F:i). The cerebellum (E:j) was devoid of CX₃CL1 expression, except for a low level of expression in the deep cerebellar nuclei. Parallel sections hybridized with a sense-transcribed CX₃CL1 cRNA probe of equal specific activity did not reveal signals above background levels.

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Figure 5

Phenotyping of CX₃CL1 and CX₃CR1 expressing cells. CX₃CL1 mRNA expressing cells (upper left). Immunohistochemical staining for the neuronal marker NeuN (upper right) performed on a parallel tissue section. Dual staining for CX₃CL1 mRNA and the astrocyte marker GFAP (lower left). Combined staining for CX₃CR1 mRNA and the microglial marker GSI-B4 isolectin (lower right).

In addition to the expression of CX₃CL1 mRNA among neuronal-like cells within the rat brain and spinal cord, we also detected low-to medium expression of CX₃CL1 in a few, solitary cells dispersed throughout the white matter areas. These cells manifested a flattened, elongated morphology (data not shown) and were not positively stained in dual labeling experiments for CX₃CL1 mRNA with phenotypic markers for macrophages/microglias or astrocytes. No expression of CX₃CL1 mRNA was detected in cells associated with the cerebral vasculature and the meninges, including the endothelial cells themselves.

In consecutive tissue sections, in situ hybridization with a radio labeled antisense cRNA probe encoding rat CX₃CR1 revealed a low-to-medium expression within cells uniformly distributed throughout the entire neuraxis (data not shown). The morphology of these cells, as well as their positive labeling with GSI-B4 (Griffornia simplifolica isolectin B4; stains microglia, macrophages and endothelial cells) identified them as being inactive microglia (data not shown). No detectable expression was observed over other cells, including neurons and perivascular and meningeal macrophages. Hybridization with a sense cRNA probe transcribed from the same cDNA did not reveal a signal above background levels (data not shown).

Expression of mRNA encoding CX₃CL1 and its receptor in the CNS of rats with MOG-EAE

To explore the role of CX₃CL1 and its receptor in the control of inflammatory cascades in MS, we examined their expression and regulation in the CNS of rats with MOG-induced EAE. In inbred DA rats, this disease manifested a mostly relapsing-remitting disease course with an initial paralytic episode, commencing around day 9–13 followed by a partial or complete remission and then a relapse of paresis (Fig. ​(Fig.2).2). In some rats the initial (acute) paresis progressed directly into a prolonged paralysis without any intervening remission of symptoms. In a minority of the rats the acute episode spontaneously resolved without further clinical signs of disease. The acute phase of paresis was characterized histopathologically by astroglial and microglial activation and perivascular and submeningeal infiltration of lymphocytes, macrophages and granulocytes. The inflammatory lesions were mostly confined to the spinal cord and, in some rats, the optic nerve and the cerebellar white matter. In rats that exhibited a clinical relapse the inflammation followed generally the pattern observed during the acute phase with submeningeal and perivascular lesions. However, the inflammatory reaction was often more extensive with numerous confluent lesions that covered substantial areas of the spinal white matter (Fig. ​(Fig.4),4), sometimes extending into the grey matter areas. Inflammatory lesions were at this stage closely associated with a marked demyelination as well as axonal degeneration and loss of axonal density. There was a distinct transition in cellular composition observed within the inflammatory lesions, from predominantly lymphocytes and granulocytes in the acute phase lesions to an overwhelming presence of macrophages and microglia with only a few granulocytes throughout the inflammatory lesions during the clinical relapse. These phagocytic cells exhibited obvious signs of uptake and degradation of myelin components. This pattern of paresis and histopathological alterations was generally in accordance with previous studies [37,42], to which interested readers are referred for further details on the disease pathology.

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Figure 2

Sampling of rats from various clinical stages of MOG-EAE. Female DA rats (n = 20) were immunized with mineral oil-emulsified MOG and evaluated daily for severity of paralysis. The arrows indicate selected time point at which subsequent kinetic analyses were performed. Rats (n = ≥ 3/group) which conformed in the clinical score curve above were perfused transcardially, tissues were dissected out and subjected to histopathological analysis of encephalitis and distribution of CX₃CL1 and CX₃CR1 expressing cells.

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Figure 4

Sublesional distribution of CX₃CR1 expressing cells. Cellular infiltrations in the MOG-EAE rat brain (early relapse phase; day 21 post immunization) are observed with hematoxylin-eosin staining (upper left). Areas of active demyelination are revealed with Luxol fast blue/PAS staining (upper right). Actively phagocytozing cells are detected with immunohistochemical staining for ED-1 (lower left). This figure exemplifies the uneven distribution of CX₃CR1 mRNA expressing cells (lower right) within the inflammatory aggregates.

For the following studies we selected rats that conformed to the typical relapsing-remitting disease phenotype (Fig. ​(Fig.2).2). In situ hybridization experiments revealed an uncompromised neuronal expression of CX₃CL1 mRNA throughout the entire CNS at all time points examined, except for a tendency to a reduced level of expression in grey matter regions which were notably infiltrated with inflammatory cells (data not shown). In addition, increased expression of CX₃CL1 mRNA was evident in a small number of non-neuronal cells within the inflammatory lesions. Double-labeling experiments showed those cells to stain positive for the astroglial marker glial fibrillary acidic protein, GFAP (Fig. ​(Fig.5).5). No hybridization signal above background levels was detected in cells staining positive for GSI-B4 (e.g. macrophages/microglia and endothelial cells). Tissue sections hybridized in parallel with a sense-transcribed CX₃CL1 cRNA probes did not generate hybridization signals above background levels (data not shown).

In situ hybridization with an antisense cRNA probe for CX₃CR1 demonstrated a low, constitutive expression of the receptor in cells evenly distributed throughout the spinal cord of healthy, non-immunized control rats (n = 3) and presymptomatic rats (day 8 p.i, n = 3) (Fig. ​(Fig.3).3). The morphology and positive staining for GSI-B4 identified these cells as being inactive microglial cells. Rats examined at various stages following onset of clinically manifest MOG-induced EAE (day 13 p.i, acute phase, n = 3; day 18 p.i, remission phase, n = 3; day 21 p.i, early relapse phase, n = 3; day 24 p.i, mid relapse phase, n = 3; day 29 p.i, late relapse phase, n = 3) demonstrated a clear visual increase in CX₃CR1 mRNA levels per cell, as well as a notably increased density of CX₃CR1 mRNA expressing cells, within the inflammatory areas (Fig. ​(Fig.33 and ​and4).4). These aggregates of CX₃CR1 mRNA expressing cells were closely overlapping with the inflammatory lesions, being prominent in the perivascular and submeningeal lesions during the acute phase and with a few remaining lesions detectable during the remission phase. In rats examined at various time points of the relapse phase the aggregation of cells expressing high levels of CX₃CR1 mRNA closely followed the areas of expanding lesions. The great majority of the CX₃CR1 mRNA expressing cells were at all stages of disease positively stained with GSI-B4 isolectin as well as with the marker for active phagocytosis, ED-1. While all GSI-B4 positive, CX₃CR1 mRNA expressing cells in PPWM regions displayed abundant ramified processes identifying them as resident microglia, the morphology of the GSI-B4 positive, CX₃CR1 mRNA expressing cells within EA, LA and DM lesions was consistent with both macrophages and microglia in an activated, phagocytic state (Fig. ​(Fig.5).5). Occasional GSI-B4 negative, CX₃CR1 mRNA expressing cells were identified within the inflammatory lesions. These cells had rounded or slightly elongated nuclei and were of lymphocyte-like size. However, the great majority of lymphocytes within the lesions did not express CX₃CR1mRNA (data not shown).

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Figure 3

Distribution of CX₃CR1 mRNA expressing cells in spinal cord of EAE rats. In situ hybridization with a radiolabeled antisense cRNA probe encoding rat CX₃CR1 to coronal sections from the lumbar segment of spinal cord of rats with MOG-EAE. Cells expressing CX₃CR1 mRNA are visualized by darkfield illumination of the photoemulsion-dipped slides.

Quantification of CX₃CR1 mRNA-expressing cells in relation to the stage of demyelinating activity

To further characterize the distribution of CX₃CR1 mRNA expressing cells in MOG-induced EAE, we performed a quantitative analysis in defined time-staged lesions. The strongest expression of CX₃CR1 mRNA was found in LA areas, which contained a significantly higher density of silver grains per square unit compared to EA areas (p < 0.05), DM areas (p < 0.05), PPWM regions (p < 0.001) and NWM (p < 0.001) (Table ​(Table1,1, Figure ​Figure6a).6a). In EA and DM areas, the expression of CX₃CR1 mRNA was significantly higher compared to PPWM (p < 0.001) and NWM (p < 0.001). Although PPWM regions tended to show a higher density of silver grains per square unit compared to NWM, this difference was not statistically significant (Table ​(Table1,1, Figure ​Figure6a).6a). The actual number of CX₃CR1 mRNA expressing cells was three-fold higher in lesional areas compared to PPWM and NWM but did not differ significantly between EA, LA and DM. The overwhelming majority of the CX₃CR1 mRNA expressing cells stained positive for GSI-B4 isolectin, identifying them as macrophages/microglia (Table ​(Table1,1, Figure ​Figure6b).6b). Although not all GSI-B4+ cells expressed CX₃CR1 mRNA, there was a significant (p < 0.0001) correlation between the total number of GSI-B4+ cells and the density of silver grains per square unit (Table ​(Table1,1, Figure ​Figure6c6c).

Table 1

Quantification of CX₃CR1 mRNA expressing cells per square unit in rat EAE lesions (mean ± SEM)

	NWM	PPWM	EA	LA	DM
Density of silver grains* per 1.9 × 104 μm2	39.4 ± 8.2	70.8 ± 7.2	220.3 ± 17.4a	276.3 ± 14.8b	208.4 ± 18.3a
Total number of GSI-B4+ cells per 1.9 × 104 μm2	11.0 ± 1.5	18.6 ± 1.6	65.3 ± 5.3a	68.1 ± 3.3a	59.8 ± 2.4a
Number of GSI-B4+/CX3CR1+ cells per 1.9 × 104 μm2	5 ± 0.9	12.4 ± 1.3	46.1 ± 7.4a	50.7 ± 3.3a	46.0 ± 3.8a
Number of GSI-B4-/CX3CR1+ cells per 1.9 × 104 μm2	0 ± 0	0 ± 0	0.8 ± 0.3	2.1 ± 0.7a	0.8 ± 0.4

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*Silver grains: defined as areas exceeding the mean background level + 11 SD

^a Statistically significant against NWM and PPWM

^bStatistically significant against NWM, PPWM, EA and DM

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Figure 6

Quantification of CX₃CR1 mRNA expressing cells in defined lesional stages. (a) Density of silver grains (defined as areas exceeding the mean background + 11 SD) per square unit. (b) Number of CX₃CR1 mRNA-expressing cells positively stained with GSI-B4 isolectin. (c) Correlation between total number of GSI-B4 cells and intensity of in situ hybridization signal (density of silver grains per square unit).

Discussion

This study was designed to provide further insights into the mechanisms of proinflammatory cell trafficking into the CNS in MS. Our results show that the chemokine CX₃CL1 is expressed, and actively regulated, within inflammatory CNS lesions in rats with MOG-induced EAE, a rodent model of MS. In addition, we demonstrate that cells expressing the CX₃CR1 receptor, the majority of which are potentially pathogenic phagocytic cells (i.e. macrophages and/or microglia), are densely accumulating within the inflammatory lesions. This is consistent with the hypothesis for a role of CX₃CL1-CX₃CR1 in the local control of leukocyte infiltration into CNS lesions in MOG-EAE rats, and possibly also MS. The constitutive CX₃CL1 and CX₃CR1 expression by neurons and microglia, respectively, even in the healthy rat brain, indicates a potential mechanism whereby neurons control microglia functions in the intact brain tissue, as well.

Our present findings of constitutive expression of CX₃CL1 within the normal rat brain are in agreement with previous reports where CX₃CL1 mRNA [24,25,43] and protein [25,43] was demonstrated in most CNS neurons of healthy rats. Similar results have also been obtained in the mouse [4], rhesus monkey [25] and human [44] brain. It is interesting to note that the levels of CX₃CL1 mRNA differed greatly between different CNS regions, possibly indicating a more pronounced role for neuronal CX₃CL1 in many forebrain structures, i.e. cerebral cortex, hippocampus and striatum. Our demonstration that CX₃CL1 mRNA expression is induced in astrocytes within EAE lesions is novel although the data are in accordance with study of humans with HIV-associated dementia where CX₃CL1 immunoreactivity was detected in astrocytes [45]. Induced expression of CX₃CL1 in astrocytes was also observed following intraparenchymal administration of prion protein to the rat brain [22]. Co- cultivation of human astrocytes and HIV-infected macrophages also resulted in increased CX₃CL1 immunoreactivity in astrocytes [45] and stimulation of rat [23] and human [46] astrocytes with TNF-α and IL-1β or INF-γ in vitro has been shown to up regulate CX₃CL1 mRNA levels. A recent immunohistochemical study of MS- and normal brain tissue demonstrates a constituent CX₃CL1 expression in astrocytes and an increased expression in human adult astrocyte cultures stimulated with pro-inflammatory cytokines, but fail to demonstrate an upregulation of CX₃CL1 in MS patients [47]. However, these data collectively indicate that astrocytes are capable of upregulating CX₃CL1 expression in inflamed or injured brain tissues.

In contrast to the observed expression of CX₃CL1 in neurons and astrocytes, we were not able to confirm previous findings in EAE-afflicted mice [2] where endothelial immunoreactivity for CX₃CL1 was demonstrated within inflamed brain lesions. Our findings are corroborated by a recent study by Schwaeble et al. [25] in which no CX₃CL1 mRNA expression was detected within the endothelium of EAE rats and mice. Possible explanations for the contradictory results are differences in the sensitivity of the detection methods used or a potential non-specific, immunohistochemical cross-reactivity to other proteins within the endothelium in the study by Bazan et al. [2]. An alternative explanation would be if the secreted form of CX₃CL1, which is produced within the inflamed brain parenchyma by neurons and astrocytes, is transcytosed in an abluminal-to-luminal direction for presentation to blood leukocytes in a similar manner to what previously have been described for IL-8 and RANTES [48]. It is, however, important to note that endothelial cells outside the CNS have convincingly been demonstrated to express CX₃CL1 mRNA and protein [6,8,29-32]. CX₃CL1 expression by the brain endothelium, which may be below the detection limit of our assay, therefore remains a tantalizing possibility by which blood leukocytes may become attracted to migrate across the blood-brain barrier into inflammatory CNS lesions of MOG EAE rats.

Our present studies of CX₃CR1 expression in the rat brain confirm previous findings by Harrison et al [43], Nishiyori et al [24] and others by demonstrating that microglia express CX₃CR1 mRNA constitutively within the normal rat brain. These findings are also consistent with previous demonstrations that CX₃CL increases microglial migration [43], proliferation [49], survival [28], intracellular recruitment of calcium [43] and secretion of cytokines and metalloproteases [50]. In addition, we demonstrate that CX₃CR1 mRNA expressing cells are rapidly accumulating at high densities within the inflammatory CNS lesions of rats with MOG-induced EAE, confirming previous observations [41]. Interestingly, those cells amassed throughout most kinetic stages of the lesions, with the most conspicuous densities detected in the late active stages. It is tempting to speculate about a down-regulatory role of CX₃CR1 expression in these late lesional stages, based on the possible induction of nonsignaling CX₃CR1 receptors [51]. The overwhelming majority of the CX₃CR1 mRNA expressing cells within the lesional areas were phagocytic cells (i.e. macrophages and/or microglias) as demonstrated in double-labeling protocols with the GSI-B4 isolectin or the ED-1 antibody. However, a few additional, non-GSI B4 labeled, CX₃CR1 mRNA expressing cells were also detected within the CNS lesions. These cells may be speculated to correspond to infiltrating blood leukocytes, such as NK cells, γδ T cells and/or terminally differentiated CD4⁺ and CD8⁺ T cells, which previously have been described to express CX₃CL1 receptors and/or to respond functionally upon CX₃CL1 stimulation [14,15,33,52,53]. However, the majority of the lymphocyte-like cells within the inflammatory lesions of the present rats did not express CX₃CR1 mRNA. Notably, no expression of CX₃CR1 mRNA was detected in GFAP-labeled astrocytes (data not shown) or neuronal-like cells. This contrasts to earlier findings of CX₃CR1-like immunoreactivity within neurons of a rat model for prion disease [22], in humans with HIV encephalitis [44] and in vitro cultivated hippocampal neurons [54]. Hulshof et al. [47] found weak to moderate neuronal CX₃CR1-like immunoreactivity in the cortical grey matter, depending on the tissue sample observed. Those data suggest that some populations of neurons may indeed have the capacity to express CX₃CR1 under certain conditions.

Our present data are thus consistent with a role for CX₃CL1 in the rat CNS, both during healthy and inflammatory conditions. The constitutive expression of CX₃CL1 and its receptor on neurons and microglia respectively demonstrates that this chemokine-receptor pair is not normally promoting inflammation. Their function may under these conditions possibly be inert but CX₃CL1, which is normally linked physically to the neuronal membrane via a spacer domain and a transmembrane spanning motif [2], may alternatively mediate a direct contact between neurons and neighboring microglia via its proadhesive properties [2]. Microglia are highly reactive to insults to brain tissue and may inflict permanent damage if not kept in check. A CX₃CL1-mediated interaction between neurons and surrounding microglias may under healthy conditions provide a mechanism by which neurons subdue the proinflammatory and neurotoxic capacity of microglia. This notion is supported by recent findings from in vitro studies where CX₃CL1 inhibited neuronal death following the stimulation in vitro of cocultured microglia and hippocampal neurons with lipopolysaccharide, LPS [55]. In this study, CX₃CL1 was also demonstrated to inhibit LPS stimulated activation of microglia and associated TNF-α synthesis [55]. Moreover, endogenous CX₃CL1 has also been shown to inhibit the increased levels of TNF-α and 8-isoprostane in the hippocampus and cerebrospinal fluid following intracerebroventricular injections of LPS [56]. In another study, CX₃CL1 protected neurons in vitro from the neurotoxic properties of platelet activating factor or the HIV envelope protein, Tat [44]. In contrast, under conditions where neurons and their axonal and dendritic projections are injured, as is frequently the case in EAE and MS, CX₃CL1 is likely to be cleaved and released from the neuronal membrane by locally acting metalloproteases [57]. This would create a chemotactic gradient that, in combination with other locally active proinflammatory mediators, may subserve the extensive accumulation and activation of microglia within injured brain sites as observed in the present study. In addition, the soluble form of CX₃CL1 may also contribute to the infiltration of CX₃CR1-expressing leukocytes, including macrophages and effector T and NK cells. These cell types have crucial roles in EAE as mediators of tissue destruction and/or disease regulation [58]. A functional significance of the soluble CX₃CL1 / CX₃CR1-pathway is suggested by the present demonstration of CX₃CR1 expression in macrophage-like cells and a small fraction of lymphocyte like cells. Interestingly, concurrent studies have provided functional evidences that CX₃CR1 and its ligand serve as important mediators of inflammation and pathology in animal models models for atherosclerosis, transplant rejections, glomerulonephritis and stroke [12,19,29,59]. Studies in mice with targeted deletions of the CX₃CL1 or CX₃CR1 genes have, however, failed to provide information regarding a contributory function for this chemokine-receptor pair in EAE. CX₃CL1 ^-/- mice [60] or CX₃CR1 ^-/- mice [18] developed EAE in a manner not significantly deviating from wildtype mice. Furthermore, CX₃CR1 ^-/- mice did not manifest alterations in the microglia responses around injured/dying motor neurons in a peripheral axotomy model [53]. These studies suggest a redundant compensatory mechanism by other chemotactic factors or a more subtle role of this chemokine-receptor pair in these mouse models for MS. The aforementioned studies, which proposed a neuroprotective role for CX₃CL1, would also warrant a more thorough investigation of the long-term outcome as regards paralysis and neuronal injuries in EAE-challenged CX₃CL1, or CX₃CR1, deficient mice. A definite assignment of the role of CX₃CL1 and its receptor in normal and inflamed or injured CNS conditions will await conceptual testing in animal disease models using more informative tools such as selective immunoneutralizing antisera, peptide antagonists or non-peptidergic CX₃CR1 antagonists delivered at various stages of the disease.

Conclusion

• We have provided data to demonstrate constitutive and regulated expression of the chemokine CX₃CL1 and its receptor CX₃CR1 by neurons/astrocytes and microglia, respectively, within the normal and inflamed rat brain.

• Our findings propose a mechanism by which neurons and reactive astrocytes may control migration and function of the surrounding microglia.

• In addition, the accumulation of CX₃CR1 expressing cells other than microglia within the inflammatory brain lesions indicate a possible role for CX₃CL1 in controlling invasion of peripheral leucocytes to the brain.

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

Design of studies (DS, SE, EW, TO, HL, AE-D), cloning of plasmid construct (AE-D), experimental induction of EAE and preparation of tissues (SE, DS, MN), in situ hybridization and immunohistochemical histochemical stainings (DS, MN), analysis of data (DS, SE, HL, A-LB, AED), writing/reviewing of manuscript (all authors). All authors have read and approved the final manuscript.

Acknowledgements

References