Molecular analyses of the chemokine fractalkine and its receptor CX3C-R1 in the rat brain have revealed a striking polarization: fractalkine is expressed constitutively in neurons and is up-regulated by TNF-α and IL-1β in astrocytes. Expression of its specific receptor, CX3C-R1, is restricted to astrocytes and microglia. We have analyzed the functional correlates of this expression and demonstrate that fractalkine induces microglial cell migration and activation. However, the activity of this chemokine on astrocytes may also be highly relevant in inducing astrocyte-microglia cell interactions through cytokine/mediator release leading to microglial activation.

Chemokines are a superfamily of proinflammatory proteins thought to be central to the trafficking of immune cells (1). The chemokine superfamily consists of four subfamilies, typified by the conservation of cysteine residues in the N-terminal sequence. Members of the CXC (α chemokines) subfamily, such as IL-8 and stromal cell-derived factor-1α, exhibit separation of the first two cysteines by a single amino acid, while members of the CC (β subfamily), such as monocyte chemoattractant proteins, RANTES, and eotaxin, have the first two cysteines adjacent to each other. Two other subfamilies exist for which there are, thus far, only single members: the C family (lymphotactin) and the CX3C family (fractalkine). While considerable overlap exists in the migration-inducing potential of these molecules for specific leukocyte subtypes, patterns begin to emerge, delineating hierarchies of potency and efficacy. Additionally, specific pathologies reveal restricted expression patterns of the ligands and receptors on infiltrating leukocytes; thus, the concept of redundancy is likely outmoded.

Fractalkine, the CX3C chemokine, exists as membrane-anchored and potentially soluble forms, suggesting a capacity for localized and spatial effects (2). Fractalkine has been shown to be produced by IL-1-stimulated endothelial cells and to mediate the migration of monocytes and T lymphocytes in vitro and in vivo (3), suggesting its relevance as an inflammatory mediator. The existence of a naturally tethered chemokine at the endothelial cell surface may be relevant to the stimulation of haptotactic migration in vivo, thus fractalkine has emerged as an important candidate mediating leukocyte trafficking. Studies have also demonstrated adhesion-promoting effects of fractalkine (4), linking two of the most important features in the multistep cascade of leukocyte emigration to the periphery (5).

While chemokines have long been implicated in leukocyte trafficking and the inflammation arena, their role(s) in vivo may extend into most physiological and pathological states (1). This has been typified more recently by demonstrations of abundant levels of fractalkine in neurons and robust expression of fractalkine receptor CX3C-R1 on microglial cells in normal CNS tissue (6). In an animal model of peripheral nerve injury (facial motor nerve axotomy), the release of small molecular mass forms of fractalkine was induced, and there was a profound increase in CX3C-R1-expressing microglia in the facial motor nucleus. It is thus apparent that this chemokine may fulfill important roles both in CNS normal physiology and pathology. We have undertaken a study to address these issues by analyzing the effects elicited by fractalkine on microglia and astrocytes in vitro. We observe different patterns of activation cascades depending on fractalkine concentrations. Fractalkine may thus exist as a cell regulator when low concentrations of membrane-bound chemokine can function in a juxtacrine manner, while increased concentrations (including de novo release) may fulfill a proinflammatory role.

Recombinant human fractalkine, both full-length and chemokine domain, as well as antifractalkine Ab were purchased from R&D Systems (Minneapolis, MN). A mAb directed against rat CX3CR-1 was a gift from Dr. L. Feng, (Scripps Research Institute, La Jolla, CA).

Mixed glial cell cultures were established as previously described (7). Briefly cortexes from newborn rats were isolated, mechanically dissociated, and plated at a density of one brain/72-cm2 flask in 20 ml DMEM containing 10% FCS. Once confluent, the cells were left for 5–7 days without medium change to favor microglia proliferation. The mixed glial cells were then shaken for 6–20 h at 225 rpm. The supernatant, containing an enriched population of microglia, was passed through a 70-μm sieve and spun down, and the cells were replated on petri dishes at a density of 1.25 × 105 cells/cm2 in DMEM + 10% FCS. Then, 2 h later, the cells were manually vigorously shaken, and the medium was replaced with DMEM + 10% FCS containing 200 U/ml GM-CSF and M-CSF. The cells (>95% pure microglia) were grown for 2 or more days before assaying.

The adherent mixed glial cells remaining after shaking were replenished with medium and incubated for another week before shaking again. After three to four shakes, they were depleted of microglia and constituted >95% pure astrocytes.

Dispersed cortical and hypothalamic cultures were derived from 15-day-old fetal Sprague Dawley rats (Charles River, Boston, MA). Dissected tissue was collected in HBSS, incubated for 10 min at 37°C with 0.9% trypsin, and dissociated in DMEM containing 0.6% glucose, 0.2% l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10% horse serum, and 10% FCS. The cell suspension was then filtered through a 70-μm nylon sieve, and plated on poly-l-lysine/laminin-coated 24-well trays at a density of 1.25 × 105 cells/cm2. The next day, two-thirds of the medium was replaced by DMEM + B27 containing 10 μm (final) deoxy-fluoro-uridine. This procedure was then repeated every 2–3 days, and the cultures were used after 6 days in vitro.

Cells were stimulated for 24 h and total RNA extracted using RNAzol for analysis by RPA, as previously described (8). Briefly, 5 μg of each RNA were hybridized with 105 cpm of [32P]UTP-labeled antisense riboprobe at 55°C for at least 10 h. The unhybridized RNA was then digested with RNase T1 and RNase A. The RNA hybrids were isolated on a sequencing gel, dried, and scanned. The radioactivity was quantitated on the Ambis radioanalytic imaging system. (Ambis Systems, San Diego, CA). The housekeeping rat ribosomal gene L32 was used as loading reference.

Chemotaxis assay was performed as described using the 48-well chamber apparatus (Neuroprobe, Cabin John, MD). Briefly, microglial cultures were detached with Versene (Life Technologies, Grand Island, NY) and resuspended in DMEM containing 1% BSA at a density of 4 × 106 cells/ml. Various dilutions of the chemokines were prepared in DMEM + 1% BSA. Aliquots of 26 μl were distributed in quadruplicates in the lower wells. An 8-μm pore size polycarbonate filter, coated with poly-d-lysine on the lower surface to favor microglial adhesion, separated the upper wells containing 50 μl of cell suspension. The chamber was incubated for 2 h at 37°C in a moist 5% CO2 atmosphere. After incubation, the nonmigrating cells adherent to the upper surface of the filter were washed and scraped. The filter was subsequently fixed in methanol, stained with Diffquick (Dade, Aguada, PR), and dried on a glass slide. The number of migrating cell was counted at ×400 or ×1000 magnification, according to their density. At least five high power fields were examined in each well. The results are expressed as the mean cell number ± SD.

Analyses of chemokine-induced actin rearrangement were assessed in microglia grown in 8-well glass slides, according to standard protocols. After incubation with chemokines, the cells were fixed for 10 min in 3.7% formaldehyde, permeabilized with 0.1% Triton X-100, and stained with rhodamine-labeled phalloidin according to the manufacturer’s protocol. (Molecular Probes, Eugene, OR)

Pure astrocytes or microglia cells were labeled with 3 μM indo-1AM (Molecular Probes) for 45 min at room temperature. The cells were then resuspended in 1 ml HBSS containing 1% BSA. Measurement of calcium flux was performed as previously described (9) using a PTI (South Brunswick, NJ) fluorometer.

Analysis of mitogen-activated protein kinase (MAPK) phosphorylation was assessed in astrocyte and microglia cells. A total of 106 cells in 500 μl were used per sample and stimulated with the indicated concentrations of chemokine for up to 15 min. Following stimulation, cells were rapidly centrifuged and the pellets lysed (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.25% Na deoxycholate, 5 mM EDTA, containing protease and phosphatase inhibitors (1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM sodium orthovanadate, 1 mM EGTA, 100 μg/ml β-glycerophosphate, 10 mM sodium fluoride, 1 mM tetrasodium pyrophosphate)) on ice for 15 min with periodic vortexing. For analysis of whole cell lysate phosphotyrosine incorporation, 50 μg of total protein was loaded per lane on a 12% Tris-glycine gel (Novex, San Diego, CA). Resolved proteins were transferred onto polyvinylidene difluoride membranes (Novex) and the Western blots stained with rabbit polyclonal anti-active MAPK Ab (Promega, Madison, WI). Blots were washed three times (TBS, 0.5% Nonidet P-40) then stained with secondary Ab (donkey anti-rabbit IgG-HR; Pierce, Rockford, IL). The Western blots were then washed three times and phosphorylated species visualized using enhanced chemiluminescence reagent and Biomax MR autoradiography film (Eastman Kodak, Rochester, NY). Subsequently, blots were stripped (0.2 M glycine, 0.5% SDS (pH 2.5); 2 h at 65°C) then restained with polyclonal anti-ERK1/2 to assess equal loading.

Assay for PKB activity was performed as described (10). Astrocytes or microglia were stimulated with chemokines in the presence or absence of the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 (11) for the indicated times. Cells were then lyzed (1% Triton X-100, 10% glycerol, 137 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM sodium orthovanadate, 1 mM EGTA, 100 μg/ml β-glycerophosphate, 10 mM sodium fluoride, 1 mM tetrasodium pyrophosphate) and PKB immunoprecipitated using anti-rat PKBα polyclonal Ab (Upstate Biotechnology, Lake Placid, NY), coupled to Protein G (Pharmacia, Uppsala, Sweden). Immunoprecipitates were washed three times in lysis buffer, once in water, and finally once in kinase reaction buffer (20 mM HEPES (pH 7.4), 10 mM MgCl2, 10 mM MnCl2). Kinase reactions were performed in 20-μl volumes (kinase reaction buffer containing 0.05 mg/ml Histone 2B, 5 μM ATP, 1 mM DTT, and 10 μCi [γ-32P]ATP) for 30 min at 30°C. Reactions were stopped by addition of 2× Laemmli sample buffer and boiling. Histone was resolved on 16% Tris-glycine gels (Novex) and visualized by autoradiography. PKB equal loading was detected by Western blot analysis as described above.

Cell death was assessed by the liberation of lactate dehydrogenase (LDH) in the culture medium using a cytotoxicity detection kid (Boehringer Mannheim, Indianapolis, IN).

Proliferative activity was assayed by measuring [3H]thymidine (Amersham, Arlington Heights, IL) incorporation after 24–48 h incubation in the presence or absence of different chemokines.

Statistical analysis was done using a two-tailed Student’s t test.

RPA was performed on RNA extracted from primary cultures of astrocytes, hypothalamic and cortical neurons, and microglia. Fig. 1,A shows a blot of cells stimulated with cytokines or nerve growth factor (NGF) for 24 h. Astrocytes showed little, if any, constitutive expression of fractalkine, but a robust expression when stimulated with 10 ng/ml TNF-α. IL-1β (10 ng/ml) also increased fractalkine expression, but within a different time course, eliciting a response as early as 2 h and peaking between 4 and 8 h, while TNF-α induction started after 12 h and was maximal at 24 h (L. Feng, unpublished observations). In contrast, both cortical and hypothalamic neurons showed constitutive expression of fractalkine. This expression could not be increased by stimulation with cytokines or with a range of agents inducing toxic conditions (serum deprivation, glucose deprivation, 10 μM β-amyloid, 500 μM glutamate, 20 ng/ml TNF-α, 1 μg/ml LPS), compounds inducing reactive oxygen species (10 μM menadione, 100 μM tert-butyl-hyperoxyde, 25 μM FeSO4), as well as agents promoting growth and differentiation. (100 nM PMA, 250 μM IBMX, 25 μM Forskolin, 10 ng/ml NGF) (Fig. 1 B). None of the stimuli significantly affected fractalkine mRNA expression when compared with expression of L-32. However, fractalkine message was slightly increased in hypothalamic neurons when treated with NGF. Microglia showed no expression of fractalkine mRNA under basal or cytokine-stimulated conditions.

FIGURE 1.

RPA of fractalkine expression in cultures grown for 24 h in various conditions. A, Fractalkine expression in astrocytes, cortical and hypothalamic neurons, and microglia. B, Fractalkine expression in hypothalamic neurons. Rat ribosomal L32 gene was used as a housekeeping gene. This figure represents one typical experiment reproduced three times with similar results.

FIGURE 1.

RPA of fractalkine expression in cultures grown for 24 h in various conditions. A, Fractalkine expression in astrocytes, cortical and hypothalamic neurons, and microglia. B, Fractalkine expression in hypothalamic neurons. Rat ribosomal L32 gene was used as a housekeeping gene. This figure represents one typical experiment reproduced three times with similar results.

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Fractalkine receptor CX3CR-1 mRNA was not detected under any conditions of stimulation in the neuronal populations (data not shown). The high expression of CX3C-R1 receptor has previously been demonstrated on microglia (6). We now demonstrate that CX3C-R1 is also expressed on astrocytes but at a much lower level (Fig. 2). RPA shows the presence of receptor on unstimulated astrocytes at low levels (Fig. 2,A). Expression was increased in the presence of 10 ng/ml TNF-α and IL-1β. To confirm the presence of the receptor on the cell surface, FACS analysis using a specific CX3CR-1 Ab was performed. Fig. 2,D shows significant expression of the fractalkine receptor, which is only modestly increased upon stimulation with IL-1β. Equilibrium-binding analyses using 125I-labeled fractalkine reveal specific and saturable binding kinetics (Kd of 0.143 nM, corresponding to 8000 sites/cell) (Fig. 2 B). Little, if any, difference was observed when assays were performed using the chemokine domain alone or the full-length fractalkine (C), and, in all cases, equilibrium binding was inhibited when cells were preincubated with anti-CX3CR-1 Ab for 30 min before assay (data not shown). Neurons did not exhibit any significant binding of 125I-fractalkine (data not shown).

FIGURE 2.

Expression of CX3CR-1 on rat astrocyte cultures. A, RPA of CX3CR-1 expression in astrocyte cultures treated for 24 h with IL-1β (10 ng/ml) or TNF-α (10 ng/ml). B, Saturation binding analysis. C, Competition of 125I-labeled fractalkine binding with unlabeled chemokine domain (▪) or full-length (▴) fractalkine. D, FACS analysis using a specific rat CX3CR-1 Ab. Open trace, isotype control; shaded area, untreated astrocytes labeled with anti-CX3CR-1; fat trace, astrocytes pretreated with IL-1β (10 ng/ml).

FIGURE 2.

Expression of CX3CR-1 on rat astrocyte cultures. A, RPA of CX3CR-1 expression in astrocyte cultures treated for 24 h with IL-1β (10 ng/ml) or TNF-α (10 ng/ml). B, Saturation binding analysis. C, Competition of 125I-labeled fractalkine binding with unlabeled chemokine domain (▪) or full-length (▴) fractalkine. D, FACS analysis using a specific rat CX3CR-1 Ab. Open trace, isotype control; shaded area, untreated astrocytes labeled with anti-CX3CR-1; fat trace, astrocytes pretreated with IL-1β (10 ng/ml).

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The presence of functional receptors on both microglia and astrocytes was further verified by the induction of Ca2+ mobilization following stimulation with fractalkine (Fig. 3). Although microglial cell responses have already been demonstrated (6), we were able to demonstrate that astrocytes also express a signaling receptor for fractalkine (Fig. 3,B). The response in all cases was robust, prolonged, and inhibited by preincubation of cells with pertussis toxin (PTX) (100 ng/ml) (Fig. 3 C). Intracellular calcium mobilization was also inhibited in cells pretreated with the neutralizing anti-CX3CR-1 Ab (data not shown) demonstrating specificity of the chemokine-induced response.

FIGURE 3.

Effect of fractalkine (10 nM) on intracellular calcium mobilization in primary cultures. A, Representative traces of microglial cell Ca2+ mobilization in the presence or absence of PTX (100 ng/ml) and rCX3CR-1 Ab. B and C, Representative traces of astrocytes in the presence or absence of PTX (100 ng/ml).

FIGURE 3.

Effect of fractalkine (10 nM) on intracellular calcium mobilization in primary cultures. A, Representative traces of microglial cell Ca2+ mobilization in the presence or absence of PTX (100 ng/ml) and rCX3CR-1 Ab. B and C, Representative traces of astrocytes in the presence or absence of PTX (100 ng/ml).

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Analyses of intracellular signaling have revealed that fractalkine, in a manner similar to the actions of many other chemokines on leukocytes, induces the activation of MAPK in microglia. This activation was both time- and dose-dependent, being maximal at 10 nM and following 1 min of stimulation (Fig. 4,A). This activity occurred through a standard cascade, since the specific MAPK kinase (MEK) inhibitor PD98059 completely reduced the phosphotyrosine incorporation (Fig. 4 A, lane 8, top panel). Surprisingly, however, little, if any, phosphotyrosine incorporation into MAPK was observed in astrocytes, nor was there any measurable activation of p38 MAPK or JNK in either cell type (data not shown). As a control, astrocytes were stimulated with other chemokines for which receptors are highly expressed, such as stromal cell-derived factor-1α. In a similar manner, little, if any, changes in phosphorylation or MAPK activity were observed in response to other chemokines (data not shown).

FIGURE 4.

Activation of intracellular signaling in microglia. Western blot analysis of whole cell lysates obtained from microglia treated with increasing doses of fractalkine. Also shown is a time response with 10 nM fractalkine. A, Rabbit polyclonal anti-active MAPK Ab was used. Equal loading of protein was determined using polyclonal p42/p44 MAPK Abs. The phosphorylated species were visualized using enhanced chemiluminescence reagent and Biomax MR autoradiography film. B, PKB immunoprecipitates from the various cell lysates were subjected to kinase reactions using histone 2B as an exogenous substrate, resolved on 16% Tris-glycine gels, and visualized by autoradiography.

FIGURE 4.

Activation of intracellular signaling in microglia. Western blot analysis of whole cell lysates obtained from microglia treated with increasing doses of fractalkine. Also shown is a time response with 10 nM fractalkine. A, Rabbit polyclonal anti-active MAPK Ab was used. Equal loading of protein was determined using polyclonal p42/p44 MAPK Abs. The phosphorylated species were visualized using enhanced chemiluminescence reagent and Biomax MR autoradiography film. B, PKB immunoprecipitates from the various cell lysates were subjected to kinase reactions using histone 2B as an exogenous substrate, resolved on 16% Tris-glycine gels, and visualized by autoradiography.

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Since constituents of the PI3-K pathway have been implicated in both cellular activation through 7-transmembrane receptors (12) and as a prominent signaling event for the migratory process of certain chemokines (13), we analyzed whether there was additional activation of another substrate of PI3-K signaling, protein kinase B/Akt. Fig. 4 B reveals that significant PKB activation, as measured by phosphorylation of histone 2B, is induced in microglia in response to fractalkine. The response was clearly time- and dose-dependent, with significant increases in phosphorylation occurring after 5 min of stimulation and with 10 nM fractalkine. We were, however, unable to demonstrate similar activity in astrocytes.

Chemotaxis assays were performed on both astrocytes and microglia. Microglia exhibited strong migratory activity in response to fractalkine. Maximum migration was obtained with 3 nM fractalkine (p < 0.01; Fig. 5,A). The decrease in cell number at higher doses is a typical feature of these in vitro assays, suggesting a greater adhesive effect at these elevated concentrations, thus lower migration to the opposite side of the filter. Significant differences in migration were observed as early as 1 h of assay (Fig. 5,B). Prolonged incubations resulted in decreased signal to noise ratio, and human and rat fractalkine were equally active (data not shown). The photomicrograph details the response of microglia to fractalkine after 1 h (Fig. 5,C). Of relevance to note is the rounded morphology of the cells on the filter, with little spreading of the cytoplasmic processes normally observed in culture dishes with these cells. Using the chemokine module or the whole fractalkine elicited similar migratory activity (Fig. 5,E). Astrocytes showed no significant migration in the presence of the chemokine module of fractalkine alone or the protein containing the mucin stalk (Fig. 5 D). In addition, preincubation of astrocytes with TNF-α or IL-1β or coating both forms of fractalkine on the filter were not effective in inducing migration of astrocytes.

FIGURE 5.

: Chemotaxis of microglia in response to fractalkine. Each value represents the mean ± SD cell number in five high-power fields from two to four experiments performed in quadruplicate. A, Dose-response. B, Time-response. C, Bright field micrograph (400×) showing migrating microglia in the absence (left panel) or presence (right panel) of 10 nM fractalkine. D, Significance of fractalkine structure and presentation (cFrac, chemokine domain of fractalkine; wfrac, full-length fractalkine). Chemotaxis of astrocytes is also shown. E, Inhibition of fractalkine-induced chemotaxis by 500 ng/ml Pertussis (PTX) or rCX3CR-1 Ab (∗, p < 0.05; ∗∗, p < 0.01).

FIGURE 5.

: Chemotaxis of microglia in response to fractalkine. Each value represents the mean ± SD cell number in five high-power fields from two to four experiments performed in quadruplicate. A, Dose-response. B, Time-response. C, Bright field micrograph (400×) showing migrating microglia in the absence (left panel) or presence (right panel) of 10 nM fractalkine. D, Significance of fractalkine structure and presentation (cFrac, chemokine domain of fractalkine; wfrac, full-length fractalkine). Chemotaxis of astrocytes is also shown. E, Inhibition of fractalkine-induced chemotaxis by 500 ng/ml Pertussis (PTX) or rCX3CR-1 Ab (∗, p < 0.05; ∗∗, p < 0.01).

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CX3CR-1 is a 7-transmembrane-spanning, G-protein-coupled receptor that stimulates intracellular calcium mobilization following ligation (4). It is thought that the receptor couples minimally to Gαi family G-proteins (PTX-sensitive) in stimulating the hydrolysis of phosphoinositides, leading eventually to calcium mobilization and PKC activation. This initial calcium transient induces a cascade of intracellular signaling, leading to cytoskeletal changes and actin rearrangements, ultimately leading to the migratory response.

To test the specificity of the migratory response, cells were preincubated with anti-CX3CR-1 (1:100) Ab and PTX (500 ng/ml). Fig. 5 E shows that migration could be completely blocked by incubating the cells with anti-CX3CR-1 Ab, or diminished in the presence of PTX. Immobilizing fractalkine (both chemokine domain alone and whole chemokine) by coating it on the lower side of the filter did not change the migratory activity (data not shown). Resting microglia (isolated and plated for 2–3 days) showed less migratory activity than cells freshly isolated from astrocytes or shaken for 2 h in astrocyte-conditioned medium before the chemotaxis assay (data not shown).

Microglia exhibit a migratory response when in the presence of fractalkine. To follow the reorganization of the actin cytoskeleton in response to the chemokine, we stained the actin skeleton of microglia that had been exposed to 10 nM fractalkine over a time course. The unstimulated cells exhibit diffusely arranged actin, with some organization in very thin cables and irregular actin bundles at the periphery (Fig. 6,A). As early as 10 min after the addition of fractalkine, the cells begin rounding up and reducing cytoplasmic spreading, displaying a concentrated peripheral band of actin bundles with some retraction fibers, characteristic of motile cells (Fig. 6,B). These changes are totally inhibited in the presence of the anti-CX3CR1 Ab and, additionally, following preincubation of the cells with the specific Rho inhibitor, Botulinum toxin C3 exoenzyme (14) (Fig. 6, C and D).

FIGURE 6.

Actin reorganization induced by fractalkine. Microglial cells incubated for 1 h either with control medium (A), 10 nM fractalkine (B), 10 nM fractalkine + rCX3CR-1 Ab (1:100 dilution) (C), or preincubated with 200 ng/ml botulinum toxin C3 exoenzyme 2 h before10 nM fractalkine addition (D). The cells were fixed and stained with TRITC-labeled phalloidin, then viewed under red fluorescence (400×) using a Zeiss axiophot microscope.

FIGURE 6.

Actin reorganization induced by fractalkine. Microglial cells incubated for 1 h either with control medium (A), 10 nM fractalkine (B), 10 nM fractalkine + rCX3CR-1 Ab (1:100 dilution) (C), or preincubated with 200 ng/ml botulinum toxin C3 exoenzyme 2 h before10 nM fractalkine addition (D). The cells were fixed and stained with TRITC-labeled phalloidin, then viewed under red fluorescence (400×) using a Zeiss axiophot microscope.

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Microglia grown for 24–48 h in the presence of 1–100 nM soluble or immobilized fractalkine did not display signs of reduced viability, as measured with LDH. To verify whether the chemokine induced proliferation, we measured [3H]thymidine incorporation. Fractalkine did not significantly increase [3H]thymidine incorporation, while M/GM-CSF doubled the [3H]thymidine uptake (data not shown). When testing fractalkine effect on astrocytes, we also did not observe any effect on cell death (LDH), or proliferation ([3H]thymidine) under any experimental conditions.

We further investigated whether fractalkine-treated, astrocyte-conditioned medium could influence microglial cell proliferation. Pure microglia were grown for 48 h in the conditioned medium of astrocytes previously plated and grown for 24 h in wells coated with 100 nM full-length fractalkine. As shown in Fig. 7,A, microglia grown in this medium (fractalkine-astrocytes conditioned medium (F-ACM)) showed a 2-fold increase in [3H]thymidine incorporation when compared with those grown in unstimulated ACM. This effect was nearly as robust as the effect of M/GM-CSF. The increase in thymidine incorporation was dose-dependent, with a maximum increase of 3.5-fold over control with astrocytes grown in the presence of 250 nM whole fractalkine (Fig. 7,B, bars 5–7). This effect was not observed when increasing doses of soluble fractalkine were added to control astrocyte medium (Fig. 7 B, bars 2–4) or when Abs directed against fractalkine or CX3C-R1 were added to the astrocytes.

FIGURE 7.

A, [3H]Thymidine incorporation in microglia grown for 48 h in control (untreated) ACM, F-ACM, or regular DMEM + medium with or without 200 U/ml M-CSF and GM-CSF. B, Dose-response curve with increasing concentrations of fractalkine. Controls consist of untreated ACM with or without increasing doses of fractalkine. Abs (1:100 dilution) against fractalkine (αfrac) and rCX3CR-1 (αCX3CR-1) were used to inhibit microglial proliferation in F-ACM. Results are the mean ± SD of quadruplicate determinations from one representative experiment repeated three times with similar results (∗, p < 0.05; ∗∗, p < 0.01).

FIGURE 7.

A, [3H]Thymidine incorporation in microglia grown for 48 h in control (untreated) ACM, F-ACM, or regular DMEM + medium with or without 200 U/ml M-CSF and GM-CSF. B, Dose-response curve with increasing concentrations of fractalkine. Controls consist of untreated ACM with or without increasing doses of fractalkine. Abs (1:100 dilution) against fractalkine (αfrac) and rCX3CR-1 (αCX3CR-1) were used to inhibit microglial proliferation in F-ACM. Results are the mean ± SD of quadruplicate determinations from one representative experiment repeated three times with similar results (∗, p < 0.05; ∗∗, p < 0.01).

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In this study, we have reconfirmed that mRNA for fractalkine is expressed constitutively by neurons. Unlike our previous study, we have extended these findings by additionally investigating the potential of inflammatory cytokines, growth factors, and noxious stimuli to regulate fractalkine message. Only NGF slightly increased fractalkine message expression in hypothalamic neurons. Interestingly, the regulation was only observed in hypothalamic neurons, suggesting that differences may exist between cell types expressing this chemokine. A range of stimuli usually present in inflammatory conditions had no effect on fractalkine mRNA expression, suggesting that fractalkine expression in neurons is rather linked to homeostasis and ontogeny.

We could not demonstrate the presence of CX3CR-1 on our neurons in any of the conditions tested. This is in contrast to the results of Meucci et al. (15), who describe the presence of CX3CR-1 on hippocampal neurons. Again, this suggests differences between neuronal types, and/or potentially, conditions used in isolation and culture of these cells.

We have also demonstrated the expression of mRNA for fractalkine and its receptor, CX3CR-1, in astrocyte populations, the mRNA of which is up-regulated in both cases upon stimulation of cells by TNF-α and IL1-β. While the implications for such expression and regulation are unclear, we have begun to analyze their relevance by investigating the direct and indirect actions of fractalkine on astrocyte and microglia cell population and any “cross-talk” that may exist. Fractalkine receptor activation in astrocytes does not seem to mediate chemotaxis, since we did not observe any effect on astrocyte migration.

In contrast, fractalkine stimulates the directed migration of microglia. Fractalkine mediates immediate increases in intracellular calcium mobilization in both cell types and a robust program of protein phosphorylation and enzyme activation only in microglia. Fractalkine had no direct effect on cell proliferation in either cell type; however, the activation of the kinase Akt/PKB suggests a survival role (16). Akt/PKB is known to directly activate cAMP response element binding protein and its binding of CREB binding protein (17), leading to the expression of target genes required for cell survival. The robust stimulation by fractalkine in mediating this enzyme activity may infer an important functional correlate. Indeed, assays using MTT (our unpublished observations) suggest that microglia have greater metabolic function following fractalkine stimulation.

The significance of increased MAPK activity awaits further experimentation. Since there are no obvious direct proliferative signals, the functional significance of MAPK activation in microglia is unclear. While a significant number of chemokines mediate MAPK activation in vitro, and in some cases this may be linked to the migratory events (18, 19, 20), microglial cell migration in response to fractalkine is sensitive to PTX pretreatment. It is possible however that this kinase cascade is stimulated as a direct consequence of G-protein-mediated phospholipase C or PI3-K activity (21) and serves as an amplification signal for the dynamic of the migratory response.

When we focused our efforts at the level of the astrocytes, we observed that there were no survival or proliferative signals activated in this cell type by fractalkine. However, one of the major effects of fractalkine on astrocytes was the stimulation of the expression of a factor that promotes microglial cell proliferation. Only higher doses of fractalkine (100 nM) elicit this release, suggesting that this is part of a response to a change in the cell homeostasis, most probably in response to inflammatory signals. Further assays in the presence of neutralizing Abs will be critical to our understanding of the mechanism of this indirect proliferative response. In recent publications, Fong et al. (22) and Haskell et al. (23) showed that fractalkine could mediate cell adhesion in the absence of signal transduction, suggesting that this chemokine mediates additional functions to those mediated by cellular signaling. Since we could not find intracellular signaling other than calcium mobilization with fractalkine in astrocytes, nor any obvious functionality, it is possible that the presence of CX3CR-1 on astrocytes serves more limited roles. It is interesting to speculate that this chemokine can fulfill selective and specific roles in signaling and activation, depending on the cell phenotype.

We have demonstrated that complex regulatory patterns of fractalkine mRNA exist in neurons and astrocytes. In addition to potential autocrine effects of this chemokine on astrocytes, it has the capability to activate kinase and survival signals in microglia, although the most obvious function is the induction of microglial cell migration. What is intriguing is the potential of fractalkine to induce the release of soluble mediators of microglial cell proliferation from astrocytes. Although these factors remain to be identified, as does the source and the nature of the fractalkine (released or membrane-anchored), our findings begin to dissect an important interaction between astrocytes, neurons, and microglia, hinting at possible physiological mechanisms for tissue repair in injurious or inflammatory situations.

We thank Ahn Tucker for her skillful technical assistance in preparing the cell cultures, and Lee Ellingson for his expert help with the graphics.

2

Abbreviations used in this paper: RPA, RNase protection assay; MAPK, mitogen-activated protein kinase; PKB, protein kinase B; LDH, lactate dehydrogenase; NGF, nerve growth factor; PTX, pertussis toxin; F-ACM, fractalkine-astrocyte conditioned medium; PI3-K, phosphatidylinositol 3-kinase.

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