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of June 18, 2017.
The Chemokine CX3CL1 Reduces Migration
and Increases Adhesion of Neurons with
Mechanisms Dependent on the β1 Integrin
Subunit
Clotilde Lauro, Myriam Catalano, Flavia Trettel, Fabrizio
Mainiero, Maria Teresa Ciotti, Fabrizio Eusebi and Cristina
Limatola
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2006 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2006; 177:7599-7606; ;
doi: 10.4049/jimmunol.177.11.7599
http://www.jimmunol.org/content/177/11/7599
The Journal of Immunology
The Chemokine CX3CL1 Reduces Migration and Increases
Adhesion of Neurons with Mechanisms Dependent
on the ␤1 Integrin Subunit1
Clotilde Lauro,2* Myriam Catalano,2*† Flavia Trettel,*‡ Fabrizio Mainiero,§
Maria Teresa Ciotti,¶ Fabrizio Eusebi,*†‡ and Cristina Limatola3*†‡
T
he chemokine CX3CL1 and its specific receptor CX3CR1
are abundantly expressed in the CNS where they mediate
microglia-neuron interaction under physiological and
pathological conditions (1). In contrast with earlier studies, where
the expression of CX3CL1 and CX3CR1 was considered to be the
exclusive prerogative, respectively, of neuronal and glial cells (2–
5), it is now documented that, in different species and conditions,
both neurons and glial cells express CX3CL1 and its receptor (6 –
11). CX3CL1 is present on cells as a transmembrane molecule,
which is transformed into the soluble form upon extracellular
shedding, mediated by the activation of the constitutive or inducible metalloproteinases ADAM10 and ADAM17 (12–14). Several
pieces of evidence describe that toxic insults and nerve injuries
induce an increase of CX3CL1 expression and its release from
neurons with microglial recruitment (2, 11, 13, 15–17). The cytokine milieu locally generated in different chronic and acute neu-
*Istituto Pasteur-Fondazione Cenci Bolognetti and Dipartimento di Fisiologia Umana
e Farmacologia, Centro di Eccellenza, Università La Sapienza, Rome, Italy; †Neuromed, Pozzilli, Italy; ‡Istituto di Medicina e Scienza dello Sport, Comitato Olimpico
Nazionale Italiano, Rome, Italy; §Dipartimento di Medicina Sperimentale e Patologia,
Università La Sapienza, Rome, Italy; and ¶Istituto di Neurobiologia, Consiglio Nazionale delle Ricerche, Rome, Italy
Received for publication June 1, 2006. Accepted for publication September 12, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Ministero dell’Università e della
Ricerca (to F.E. and C.Li.).
2
C.La. and M.C. contributed equally.
3
Address correspondence and reprint requests to Dr. Cristina Limatola, Dipartimento
di Fisiologia Umana e Farmacologia, Università La Sapienza, I-00185 Rome, Italy.
E-mail address: [email protected]
Copyright © 2006 by The American Association of Immunologists, Inc.
roinflammatory conditions may influence glial expression of
CX3CL1 and CX3CR1 (10). The CX3CL1 released plays a direct
neuroprotective role, reducing the neuronal damage caused by
toxic insults (7, 11, 18, 19), and impairing IFN-␥- and LPS-induced microglial activation (5, 19). Although CX3CR1 is expressed both in neurons and glial cells, its chemotactic activity
develops only in microglial cells (20, 21). In different cell types, it
is reported that CX3CL1-mediated chemotaxis depends on receptor-activated signal transduction pathways (20, 22, 23), while the
adhesive properties have been explained both by intrinsic adhesive
function of the CX3CL1/CX3CR1 molecules and the activation of
intracellular signaling (24 –28). Another issue of debate is the role
of integrins in mediating the adhesive properties of CX3CL1: it
has been reported that CX3CL1 regulates integrin avidity, similarly to what has been shown for other chemokines (26), but conflicting data are reported (24, 25).
During development, chemokine expression is functional to correct localization of neural precursors to their final destination; in
particular, CXCR4 is essential for proper neural migration in the
hippocampus, cerebellum, neocortex (29 –31), and in the spinal
cord (32). Furthermore, it has been reported that CX3CL1, together with other chemokines, is able to modulate mesenchymal
stem cell migration in the brain (33), where these cells could differentiate toward a neural phenotype (34 –36). CX3CL1 is also
expressed by embryonic and adult neural progenitor cells (37), and
has trophic effects on neural precursors (38).
In this study, we compare the chemotactic effects of CX3CL1 on
neurons and glial cells obtained from either the cerebellum or the
hippocampus of newborn rats and report for the first time that
soluble CX3CL1 specifically reduces basal neuronal motility and
increases neuron adhesion to the extracellular substrate laminin.
0022-1767/06/$02.00
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Fractalkine/CX3CL1 and its specific receptor CX3CR1 are constitutively expressed in several regions of the CNS and are reported
to mediate neuron-microglial interaction, synaptic transmission, and neuronal protection from toxic insults. CX3CL1 is released
both by neuronal and astrocytic cells, whereas CX3CR1 is mainly expressed by microglial cells and neurons. Microglial cells
efficiently migrate in response to CX3CL1, whereas no evidence is reported to date on CX3CL1-induced neuronal migration. For
this reason, we have investigated in vitro the effects of CX3CL1 on basal migration of neurons and of the microglial and astrocytic
populations, all these cells being obtained from the hippocampus and the cerebellum of newborn rats. We report that CX3CL1
stimulates microglial cell migration but efficiently reduces basal neuronal movement, regardless of the brain source. The effect of
CX3CL1 is pertussis toxin (PTX) sensitive and PI3K dependent on hippocampal neurons, while it is PTX sensitive, PI3K dependent, and ERK dependent on cerebellar granules. Interestingly, CX3CL1 also increases neuron adhesion to the extracellular
matrix component laminin, with mechanisms dependent on PTX-sensitive G proteins, and on the ERK and PI3K pathways. Both
the reduction of migration and the increase of neuron adhesion require the activation of the ␤1 and ␣6 integrin subunits with the
exception of cerebellar neuron migration, which is only dependent on the ␤1 subunit. More importantly, in neurons, CX3CL1/
CXCL12 cotreatment abolished the effect mediated by a single chemokine on chemotaxis and adhesion. In conclusion, our findings
indicate that CX3CL1 reduces neuronal migration by increasing cell adhesion through integrin-dependent mechanisms in hippocampal and cerebellar neurons. The Journal of Immunology, 2006, 177: 7599 –7606.
7600
We demonstrate that these effects are both dependent on receptor
signal transduction and are mediated by the activation of the integrin subunits ␤1 and ␣6, with minor differences observed between neurons obtained from different brain sources. Furthermore,
we report that neuron cotreatment with CX3CL1 and CXCL12, a
chemokine specific for the CXCR4 receptor, counteracts the effects of a single chemokine on chemotaxis and adhesion. The fact
that both CX3CL1 and CXCL12 are constitutively expressed in the
brain, together with the observation that both their expression and
those of their receptors may increase upon neuroinflammation or
traumatic injuries, strongly indicates that the simultaneous stimulation of cells with these chemokines may have both physiological
and pathological implications.
Materials and Methods
Materials
Neuronal cultures
Hippocampal neuronal cultures were prepared from 1- or 2-day-old (p1-p2)
Sprague Dawley rats. In brief, after careful dissection from diencephalic
structures, the meninges were removed and tissues were chopped and digested in 0.25% trypsin for 15 min at 37°C. Cells were dissociated and
either immediately used for chemotaxis and adhesion assays or were plated
at a density of 8 ⫻ 105 in poly-L-lysine coated 35-mm dishes in MEM with
Earl’s salts and GlutaMAX containing 10% dialyzed and heat-inactivated
FBS, 100 ␮g/ml gentamicin, and 25 mM KCl, and maintained at 37°C in
5% CO2. After 24 h, cytosine-␤-D-arabinofuranoside was added at a final
concentration of 5 ␮M to prevent glial cell proliferation. Cells were used
for experiments after 7 days. Primary cultures of cerebellar granule cells
were obtained from p7-p8 rats, as already described (39). The use of animals in this study follows the protocols approved by the institutional animal care and use committee of the University of Rome La Sapienza.
(microglial cells) treated 12-mm transwells (3-␮m pore size polycarbonate filters for neurons and 8-␮m pore size for microglia and glia
cells; 5 ⫻ 105 cells/well). The lower chambers contained CX3CL1 alone or
together with CXCL12 (at concentrations ranging from 0.05 to 100 nM),
prepared in the same medium. For chemokinetic assay, CX3CL1 was
present both in the lower and upper chambers. When necessary, cells were
preincubated at 37°C for 2 h with PTX (1 ␮g/ml), anti-rat CX3CR1
(TP501, 3 ␮g/ml), rabbit preimmune Igs (3 ␮g/ml), or for 15 min with
PD98059 (30 ␮M) and LY294002 (25 ␮M), or with blocking mAbs for ␤1,
␣6, or MHC class I (10 ␮g/ml, 30 min, on ice) that were also present in the
upper chamber during the assay. In some experiments, lower doses of
PD98059 (10 ␮M) and LY294002 (10 ␮M) were tested. The chambers
were incubated for 2 h at 37°C in a moist 5% CO2 atmosphere. After
incubation, cells were treated with 10% trichloroacetic acid on ice for 10
min and the nonmigrating cells adhering to the upper face of the filters
were scraped off, while cells on the lower side were stained with a solution
containing 50% isopropanol, 1% formic acid, and 0.5% (w/v) brilliant blue
R250 and dried on a glass slide. The number of migrating cells was counted
in 20 fields with a ⫻63 objective. The results were expressed as the mean
cell number ⫾ SE and the chemotactic index was obtained by the ratio
between chemokine-treated vs untreated cells.
L-lysine-
Adhesion assay
For adhesion assay, freshly obtained neurons or microglial cells (3 ⫻ 104
cells/well) were plated on laminin- (50 ␮g/ml) or poly-L-lysine- (100 ␮g/
ml) coated 96-well plates, respectively. When necessary, cell suspension
was preincubated with PTX (1 ␮g/ml, 2 h), TP501 (3 ␮g/ml, 2 h),
PD98059 (30 ␮M, 15 min), or LY294002 (25 ␮M, 15 min) at 37°C, or with
blocking Abs for ␤1, ␣6, or MHC class I (10 ␮g/ml, 30 min, on ice). Cells
were seeded in serum-free medium containing 0.1% BSA and allowed to
adhere for 30 min at 37°C. Cells were then treated with CXCL12 in the
presence or in the absence of CX3CL1 (both 5 nM) or water, as control, for
different times (from 2 to 20 min). Treatment with CX3CL1 was performed
backwards from 20 to 2 min to stop the adhesion at the same moment in the
whole plate. Inverting the plates pulled off nonadhering cells and, after two
washes with PBS, adherent cells were treated with lysis buffer and nuclei
were counted in a hemocytometer, as described (41). Nonspecific attachment to BSA alone (0.01% in PBS), ranging from 2 to 3 ⫻ 104, was
subtracted from values obtained for specific adhesion at each time point.
Each assay was performed at least in triplicate. In preliminary experiments,
the adhesion assay was performed by labeling cells with calcein AM (10
␮M, 15 min; Molecular Probes) and by analyzing the fluorescence of adherent cells upon chemokine stimulation. Because similar results were obtained with the two methods, we decided to use the first one for practical
reasons.
Results
Glia and microglia cell cultures
CX3CL1 specifically reduces neuronal cell motility
Mixed glial cell cultures were prepared from hippocampi and cerebella of
newborn and p7 rats. In brief, the hippocampi and cerebella were isolated,
mechanically dissociated, and plated at low density (3 ⫻ 106 cells/90-mm
dish) and cultured in basal, supplemented with 10% heat-inactivated FCS
and 5 mM KCl, for 20 days. Once confluent, the cells were left for 5–7 days
without medium changes to favor microglial proliferation. The mixed glial
cells were then gently shaken, centrifuged, and the supernatant, containing
an enriched population of microglia, was collected (40).
We decided to investigate the effect of CX3CL1 on neuronal migration because it has been demonstrated that only microglial cells,
in the brain, respond to CX3CL1 in the classical chemotactic assay
(2, 13, 20). For this purpose, hippocampal and CGN neurons,
freshly dissociated from either p2 or p7 rats, respectively, were
treated as described in Materials and Methods and analyzed for
chemotaxis vs a chemokine gradient. The results reported in Fig. 1
show that CX3CL1 is not chemotactic for neurons, yet, interestingly, it induces a dose-dependent inhibition of neuronal cell
movement, both on hippocampal- (A) and cerebellar- (B) derived
cells, with maximal effect obtained at 0.1 nM CX3CL1 for both
cell types. Inhibition of cell movement was observed both with
poly-L-lysine and laminin used as substrate, with similar dose dependence. All successive chemotaxis experiments were performed
on laminin, it being the physiological ligand for neuronal-expressed integrins. To verify whether the results obtained could
reflect a change in chemokinetic activity, CX3CL1 was simultaneously put in both the upper and lower chambers to eliminate the
gradient. Under these conditions, no significant differences in cell
movement were observed for both cell types (Table I, n ⫽ 5).
The inhibitory effect on cell movement was specific for neurons,
because when microglial cells obtained either from hippocampus
or cerebellum were tested for chemotaxis in response to CX3CL1
Measurement of ERK1/2 and Akt phosphorylation
Cerebellar granule neurons (CGN) and hippocampal cultures, cultured for
7 days, were incubated for 2 h in Locke’s buffer and stimulated with
CX3CL1 (100 nM) for different times, from 1 to 15 min. Corresponding
cellular lysates were quantified for protein content and ⬃20 ␮g of total
proteins were analyzed by SDS-PAGE and Western blot with phospho-Akt
and phospho-ERK1/2-specific Abs. Parallel blots loaded with the same
samples were analyzed for total Akt and ERK2 content as further control
of equal protein loading.
Chemotactic and chemokinetic assays
Chemotaxis assay was performed on neurons, microglia, and astrocytes,
freshly obtained from rat hippocampi and cerebella. Cells were resuspended in serum-free medium and plated onto laminin- (neurons) or poly4
Abbreviations used in this paper: PTX, pertussis toxin; CGN, cerebellar granule
neuron.
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Polyclonal Abs to rat (TP501) and human (TP502) CX3CR1 were obtained
from Torrey Pines Biolabs; anti-phospho-Akt (Ser473), anti-Akt, antiphospho-ERK1/2 (Thr202/Tyr204), and anti-ERK2 Abs were obtained from
New England Biolabs. mAbs to ␣6 were obtained from Beckman Coulter,
anti-␤1 was obtained from Immunological Sciences, and anti-MHC class I
was obtained from Serotec. Transwell cell culture inserts were obtained
from BD Labware; pertussin toxin (PTX)4 and recombinant rat CX3CL1
were obtained from Calbiochem; all culture media were obtained from
Invitrogen Life Technologies. LY294002 and PD98059 were obtained
from Alexis Italia; laminin (from Engelbreth-Holm-Swarm, murine sarcoma) was obtained from Sigma-Aldrich.
CX3CL1 REDUCES NEURON CELL MIGRATION
The Journal of Immunology
7601
FIGURE 1. CX3CL1 dose-dependent reduction of basal neural movement. Freshly dissociated hippocampal (A) and cerebellar (B) neurons, respectively, from p2 and p7 rats, were treated as described in Materials and
Methods for chemotaxis assay being plated on laminin-coated filters and
exposed to different doses of CX3CL1, as indicated. After 2 h incubation
at 37°C, filters were fixed, stained, and migrated cells were counted in at
least 10 different fields at original magnification of ⫻40. Results are the
mean ⫾ SE, from six to nine different experiments, and are expressed as
percentage of cells migrated upon CX3CL1 stimulation vs untreated cells
(0 dose). Numbers of migrated cells in untreated samples were 115.3 ⫾ 7.1
and 43.8 ⫾ 4.7 for optical field, respectively, for hippocampal and cerebellar neurons. Student’s t test: data sets significantly different from respective control ⴱⴱ, p ⱕ 0.001; ⴱ, p ⱕ 0.005.
(3 nM), we observed cell migration (Fig. 2, right), while mixed
glial populations–mainly containing astrocytes, obtained from the
same brain regions–were unresponsive to CX3CL1 (Fig. 2, left).
To verify whether the inhibitory effect of CX3CL1 on neuronal
migration was mediated by chemokine interaction with its specific
receptor CX3CR1, the same experiments were performed in the
presence of the blocking Ab specific for rat CX3CR1, TP501.
When hippocampal (Fig. 3A) or cerebellar (Fig. 3B) neurons were
preincubated with TP501 (3 ␮g/ml) for 2 h, the effect of CX3CL1
(0.1 nM) on motility was completely abolished; similar neuron
treatment with preimmune rabbit Igs (3 ␮g/ml, 2 h pretreatment)
failed to abolish the CX3CL1-mediated effect (data not shown).
When both kinds of neurons were preincubated with PTX (1 ␮g/
ml, 2 h) before chemokine treatment, a block of the CX3CL1 effect
Table I. Comparison of the chemotactic and chemokinetic activities of
CX3CL1 on neuronsa
CX3CL1
Hippocampus
Cerebellum
None
Lower
Lower/upper
100
46.9 ⫾ 3.1**
101.2 ⫾ 4.3
100
44.4 ⫾ 3.8**
88.7 ⫾ 8.9
a
Hippocampal and cerebellar neurons, freshly prepared from p2 and p7 rats, were
treated as described in Materials and Methods and CX3CL1 was present either in the
lower, or both in the lower and upper wells, as indicated. Results are expressed as
percentage of cells migrated in the lower part of the filter in the different conditions
vs untreated (none) cells, and are the means ⫾ SE of five independent determinations.
Statistical significance, ⴱⴱ, p ⬍ 0.0001.
was observed, indicative of receptor coupling to Gi protein subtypes in both cell types, as already shown in hippocampal neurons
(Refs. 7 and 11, Fig. 3). Because it has been reported that CX3CL1
induces the activation and the phosphorylation of ERK1/2 and of
the PI3K substrate Akt in neurons (6, 7, 11) and we have confirmed these data in our cellular systems (11, Fig. 3), we also tested
the involvement of the ERK1/2 and PI3K signaling on the
CX3CL1-mediated effect, using their specific pharmacological inhibitors, PD98059 (30 ␮M, 15 min) and LY294002 (25 ␮M, 15
min). In previous studies, we already provided evidence that in
neuronal primary cultures, these drugs, at the indicated doses, are
fully effective in inhibiting, respectively, ERK1/2 and Akt phosphorylation (42, 43). Besides, even if we were aware that these
doses were far above the reported IC50 (44), no toxic effects were
observed for LY294002 and PD98059 at the time points indicated
in several experimental paradigms (11, 43, 45). The results shown
in Fig. 3 indicate that the inhibitory effect on migration of hippocampal neurons was independent of ERK1/2 and dependent on
the PI3K pathway, while both pathways were involved in the modulation of CGN migration. When, in some experiments, lower
doses of PD98059 (10 ␮M, 15 min) and LY294002 (10 ␮M, 15
min) were used, we obtained results similar to those reported
above (data not shown). Nevertheless, in successive experiments,
we preferred to use the higher doses already tested for their efficacy in the inhibition of the specific kinase activities (11, 43, 45).
CX3CL1/CXCL12 cotreatment abolishes the effect of single
chemokine on neuronal movement
Both cerebellar and hippocampal neurons express functional
CXCR4 receptors and migrate in response to their specific stimulation (29, 43, 46 – 47; Fig. 4). Because we demonstrated that
CX3CR1 activation in these cells reduced cell motility, we analyzed the effect of the simultaneous stimulation of CX3CR1 and
CXCR4 receptors in these neurons. The results reported in Fig. 4
demonstrate that, in the presence of CX3CL1, CXCL12 was still
able to induce chemotaxis ( p ⫽ 0.039 and p ⫽ 0.0004, respectively, for hippocampal and cerebellar neurons) and, in the presence of CXCL12, CX3CL1 was still able to reduce cell movement
( p ⫽ 0.025 and 0.00027, respectively, for hippocampal and cerebellar neurons). Nevertheless, the cotreatment with the two chemokines CX3CL1/CXCL12 did not produce any change in cell
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FIGURE 2. CX3CL1-induced migration of microglial cells. Cultured
glial or microglial cells, obtained from cerebellar (cbl) or hippocampal
(hipp) tissues, were analyzed for chemotaxis after plating on poly-L-lysinecoated filters and exposure for 2 h to CX3CL1 (3 nM). Filters (8-␮m pore
size) were fixed, stained, and migrated cells were counted in at least 10
different fields at original magnification of ⫻40. Results are the mean ⫾ SE
of three different experiments, and are expressed as cells migrated as the
percentage of untreated, control cells (C). Statistical significance always
calculated between C and CX3CL1-treated samples for each type of cells,
as indicated. ⴱ, p ⱕ 0.005.
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CX3CL1 REDUCES NEURON CELL MIGRATION
migration rate compared with untreated neurons, thus abolishing
the single chemokine effects reported above.
CX3CL1 enhances neuron cell adhesion to the extracellular
matrix protein laminin
sine was preferred to laminin, because it has been reported that
microglial cells require the presence of cytokines for adhesion to
laminin (48). The results obtained on neurons are shown in Fig. 5
and indicate that CX3CL1 stimulates a time-dependent increase of
We next addressed the experiments to see whether the different
effects of CX3CL1 on the migration of neuronal and glial cells
could be explained by modulation of cell adhesion to molecules of
the extracellular matrix. For this reason, experiments of cell adhesion were performed on laminin substrates for neurons, and on
poly-L-lysine for microglial cells. For microglial cells, poly-L-ly-
FIGURE 4. Effect of CX3CL1/CXCL12 cotreatment on neuronal
movement. Hippocampal (left) and cerebellar (right) neurons were plated
on laminin-coated filters in the presence of the indicated chemokines in the
lower wells, both 5 nM, and analyzed for migration after 2 h. Results are
the mean ⫾ SE from four to eight different experiments and are expressed
as the percentage of untreated cells. Statistical significance shown is analyzed between the control of vehicle-treated cells and all the other conditions for each cell type; ⴱⴱ, p ⱕ 0.001. Statistical significance for CXCL12/
CX3CL1 vs CX3CL1: ⴱ, p ⱕ 0.05 for hippocampal and ⴱⴱ, p ⱕ 0.001 for
cerebellar neurons.
FIGURE 5. Time course of CX3CL1-induced neuronal adhesion to
laminin. Hippocampal (A) and cerebellar (B) neurons were analyzed for
adhesion to laminin substrate, as described in Materials and Methods, and
treated with CX3CL1 (5 nM) for the indicated times. Cells adhering to the
substrate after extensive washing were counted and the results are expressed as the mean ⫾ SE from three to five different triplicate experiments. Student’s t test: all data sets significantly different (ⴱ, p ⱕ 0.001)
from control (time 0).
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FIGURE 3. Modulation of CX3CL1-induced neuronal movement by inhibitors of different signaling pathways. Freshly dissociated hippocampal (A) and cerebellar (B) neurons were preincubated with the indicated
substances (TP501, 2 ␮g/ml, 2 h; PTX, 1 ␮g/ml, 2 h;
PD98059, 30 ␮M, 15 min; and LY294002, 25 ␮M, 15
min), and analyzed for migration as described in Fig. 1,
on laminin-coated filters, in the presence of the indicated substances and CX3CL1 (0.1 nM). Results, expressed as in Fig. 1, from four to six different experiments. Statistical significance is analyzed between
CX3CL1-stimulated vs untreated cells (C) for each
group of treatments, as indicated in the abscissa. ⴱⴱ, p ⱕ
0.001. Right portions of A and B, Western blots from
one representative experiment (n ⫽ 3) where neurons
were stimulated with CX3CL1 (100 nM) for the indicated time and analyzed for Akt and ERK1/2 phosphorylation. Total Akt and ERK2 were analyzed to verify
equal protein loading between different samples.
The Journal of Immunology
FIGURE 7. Effect of integrin inhibition on CX3CL1-mediated cell adhesion. Hippocampal (A) and cerebellar (B) neurons were preincubated
with the indicated blocking mAbs for 30 min on ice and analyzed for
adhesion to laminin. Results are the mean ⫾ SE from five to six different
triplicate experiments. Statistical significance for both cell types is calculated between C and CX3CL1-treated cells for each different condition.
ⴱⴱ, p ⱕ 0.001; ⴱ, p ⱕ 0.005.
vented. A similar drastic block or reduction of cell adhesion was observed, respectively, on the hippocampal and cerebellar neurons, with
an anti-␣6 mAb (Fig. 7). As control, an anti-MHC class I mAb was
used under the same experimental conditions: the results shown in
Fig. 7 demonstrate that this mAb was ineffective in reducing
hippocampal (A) and cerebellar (B) neuron adhesion to laminin; in
each set of experiments, a control with vehicle alone was included
at the longer time point and the number of adhering cells was not
significantly different from those indicated at time 0. Microglial
cells adhesion was reduced by 10-min CX3CL1 treatment (5 nM)
to 53 ⫾ 10% of control (n ⫽ 4 triplicate experiments). The increased adhesion of neurons was drastically reduced or abolished
by TP501 or PTX treatment (Fig. 6, left panels). Preimmune rabbit
Igs (3 ␮g/ml, 2 h pretreatment), used as control for TP501 specificity, were ineffective (data not shown). The CX3CL1-mediated
increase of cell adhesion was also completely abolished by
PD98059 and LY294002 treatment (Fig. 6, right panels), indicating the involvement of the ERK and PI3K pathways in CX3CL1mediated neuronal cell adhesion.
Role of ␤1 and ␣6 integrins on CX3CL1-mediated cell adhesion
and movement
To investigate the specificity of the effect of CX3CL1 on celllaminin interaction, the hippocampal and cerebellar neurons were
pretreated with a blocking anti-integrin ␤1 mAb, because ␤1 integrins are the main targets of laminin together with ␣6␤4. The laminin we used for our assay contains as major constituent laminin-1,
which is preferentially recognized by ␣6␤1 integrin (49, 50). The results shown in Fig. 7 indicate that when hippocampal (A) and cerebellar (B) neurons were preincubated with anti-␤1 mAb (30 min, on
ice), the effect of CX3CL1 on cell adhesion was completely pre-
FIGURE 8. Effect of integrin inhibition on CX3CL1-induced modulation of cell migration. Hippocampal (A) and cerebellar (B) neurons were
preincubated with the indicated blocking mAbs for 30 min on ice and
analyzed for migration on laminin upon CX3CL1 treatment. Results are the
mean ⫾ SE from five different triplicate experiments. Statistical significance for both cell types is calculated between C and CX3CL1-treated cells
for each different condition. ⴱⴱ, p ⱕ 0.001; ⴱ, p ⱕ 0.005.
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FIGURE 6. Modulation of CX3CL1-mediated cell adhesion by inhibitors of different signaling pathways. Hippocampal (A) and cerebellar (B)
neurons were preincubated with the indicated substances (TP501, 2 ␮g/ml,
2 h; PTX, 1 ␮g/ml, 2 h; PD98059, 30 ␮M, 15 min; and LY294002, 25 ␮M,
15 min), and analyzed for adhesion to laminin in the presence of CX3CL1
(5 nM, 10 min). Two independent sets of controls are shown for each brain
tissue because of a minor difference in basal movement between different
sets of experiments. Each point was performed in triplicate and nonspecific
binding to BSA was subtracted. Results are expressed as the mean ⫾ SE
from four to six different experiments. Statistical significance for both cell
types is calculated between C and CX3CL1-treated cells for each different
condition. ⴱⴱ, p ⱕ 0.001; ⴱ, p ⱕ 0.05. Statistical difference between
CX3CL1 and PTX/CX3CL1: p ⱕ 0.005.
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7604
CX3CL1-mediated cell adhesion, confirming the specificity of anti-␤1
and anti-␣6 blocking mAbs.
When the same mAbs were used to investigate the role of integrin subunits in mediating the CX3CL1 inhibitory effect on cell
movement, we observed a similar complete block by anti-␤1 mAb
on neurons from the hippocampus (Fig. 8A) and cerebellum (Fig.
8B) while anti-␣6 mAb was slightly inhibitory for hippocampal
neuron movement and without effect on cerebellar neurons. MHC
class I blocking mAb was again ineffective on CX3CL1-mediated
reduction of movement on both neuronal cell types (Fig. 8).
Effect of CXCL12/CX3CL1 cotreatment on neuron adhesion
Because we observed mutual inhibitory effects on neuronal movement between CXCL12 and CX3CL1, we wondered whether analogous effects were obtained for cell adhesion. For this reason, we
decided to investigate whether CXCL12/CX3CL1 cotreatment
could modulate CX3CL1-mediated cell adhesion. The results
shown in Fig. 9 indicate that CXCL12 did not increase neuron
adhesion to laminin for the time points analyzed (from 2 to 20
min), a slight increase being observed for CGN only at 20 min.
Interestingly, however, neuron cotreatment with CXCL12/
CX3CL1 blocked CX3CL1-mediated cell adhesion at all time
points analyzed. Comparable results were obtained on hippocampal and cerebellar neurons (Fig. 9).
Discussion
The level of CX3CL1 in the CNS has been often found increased
in association with traumatic and/or pathological events and it is
considered responsible for microglial recruitment to the injured
sites (2) and neuronal protection from toxic stimuli (7, 11, 18, 19).
In this respect, several reports have described the chemotactic activity of neuron-released CX3CL1 on microglial cells and the in-
creased expression of CX3CL1 and CX3CR1 on astrocytes only
after their treatment with inflammatory cytokines (21). In contrast,
data of CX3CL1 effects on neuron movement are completely lacking. The movement of neural precursors may have physiological
reasons during development and repairing functions upon pathological insults; in this respect, many recent studies have focused on
the role of chemokines in regulating the correct migration of neural
progenitors both in the developing brain (1, 29, 31, 51) and to sites
of neuroinflammation (52). In this study, we show that CX3CL1
specifically reduces neuron movement, being chemotactic for microglial and ineffective on astroglial cells, regardless of the brain
sources. This effect on neurons is already maximal at CX3CL1
concentrations of 0.1 nM, a dose that is compatible with other
described physiological effects (11).
In addition, we show that CX3CL1 reduces the movement of
neurons and increases their adhesion to the extracellular substrate
laminin with mechanisms that require the ␤1 and in part also the ␣6
integrins, the PTX-dependent G proteins, and the activation of the
PI3K and ERK pathways. The involvement of intracellular signaling in mediating CX3CL1 inhibition of neuron movement and increase of adhesion indicates that these effects are not caused by
CX3CL1/CX3CR1 acting as adhesion molecules, as described in
different cell types (24, 25). In particular, the contribution of the
ERK1/2 and PI3K pathways to CX3CL1-induced neuron adhesion
is comparable to that reported for human monocytes (27), even if
the involvement of other more downstream targets has not yet been
proved.
The role of ␣6␤1 integrin in mediating neuronal cell migration
and adhesion has been already partially investigated and it has
been shown that both ␣6 and ␤1null mice show abnormalities in the
migration rate of specific neuron populations during development
(53, 54); similar results are also reported with the inhibition of
␣3␤1 integrin (55). However, the involvement of integrins in
CX3CL1-mediated cell adhesion is somewhat controversial: the
full-length membrane form of CX3CL1 is reported to mediate cellcell adhesion by virtue of its adhesive properties, with mechanisms
independent of receptor activation or modulation of integrin avidity for the extracellular matrix substrates (22, 24, 25), but dependent on integrins and receptor signal transduction in different cell
types (26, 28). The soluble chemokine domain of CX3CL1 is chemotactic on leukocytes (28, 56, 57), but is also reported to exert
adhesive properties on monocytes (27, 58). The general view is
that both soluble and membrane-anchored forms of CX3CL1 may
have chemotactic action on most leukocytes but exert adhesive
properties on monocytes; the reasons for these differences are not
clear, but it has been hypothesized that CX3CR1 couples to different G proteins in different cell types (59). This scenario is quite
similar to what we describe in the nervous system, where CX3CL1
specifically increases neuron adhesion to the extracellular substrate
laminin, being chemotactic for microglial cells, indicating that the
effect of CX3CL1 is cell-type specific in different tissues.
It is interesting to note that, at least in the two neuronal systems
we used in this study, the simultaneous stimulation of the CX3CR1
and CXCR4 receptors has no net effects with respect to cell adhesion and migration (see Figs. 4 and 9), as if receptor costimulation would result in their mutual functional inactivation, likely
due to their reciprocal neutralization. Nevertheless, we cannot exclude that the block of neuron adhesion and migration we observe
upon CX3CR1/CXCR4 costimulation is indirectly mediated
through the modulation of multiple signal transduction pathways.
Among them, of particular interest, are the ERK1/2 and PI3K/Akt
pathways, which are activated by both chemokines in neurons
(Refs. 6, 7, and 43 and this study) and the small G protein rac or
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FIGURE 9. Effect of CX3CL1/CXCL12 cotreatment on cell adhesion.
Experiments were performed on hippocampal (A) and cerebellar (B) neurons as described in Fig. 2 and incubated in the presence of CX3CL1,
CXCL12, or both (5 nM), for 0, 2, 10, and 20 min, and analyzed for
adhesion to laminin. Results are the mean ⫾ SE from five independent
triplicate experiments. Statistical significances shown refer to C vs all the
other conditions. ⴱⴱ, p ⱕ 0.001. For both cell types, no significant differences were observed between CXCL12 and CXCL12/CX3CL1-treated
cells, at all the time points analyzed.
CX3CL1 REDUCES NEURON CELL MIGRATION
The Journal of Immunology
the MAPK p38, known to be involved in the regulation of cell
movement and adhesion.
Our observation that CX3CL1/CXCL12 cotreatment abolished
the net chemotactic activity of CXCL12 on neurons is analogous to
the CX3CL1-mediated reduction of MCP-1-stimulated chemotaxis
in monocytes (58). These data may have physiological implications, because it is known that CXCL12 has important roles in
regulating neural progenitor migration during CNS development
(29 –31) and in the peripheral nervous system (32) and we speculate that the simultaneous presence of CX3CL1, or temporally
regulated CX3CL1 expression, may contribute to this modulation
and is worthy of further investigation.
Disclosures
The authors have no financial conflict of interest.
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