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*
Neurocrine Biosciences Inc., San Diego, CA 92121; and
Department of Immunology, Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1
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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chemokines) subfamily, such as IL-8 and stromal cell-derived
factor-1, exhibit separation of the first two cysteines by a single
amino acid, while members of the CC ( 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Glial cell cultures
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 x 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.
Neuronal cultures
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 x 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.
RNase protection assay (RPA)2
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
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 x 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 x400 or x1000 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.
Actin staining
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)
Calcium mobilization
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.
Western blot analyses
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 -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.
Measurement of Akt/protein kinase B (PKB) activity
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
-glycerophosphate, 10 mM sodium fluoride, 1 mM tetrasodium
pyrophosphate) and PKB immunoprecipitated using anti-rat PKB
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
[
-32P]ATP) for 30 min at 30°C. Reactions
were stopped by addition of 2x 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 survival, death, and proliferation
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
Statistical analysis was done using a two-tailed Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-. IL-1 (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- 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 -amyloid, 500 µM glutamate, 20 ng/ml TNF-,
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. 1B). 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.
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). RPA shows the
presence of receptor on unstimulated astrocytes at low levels (Fig. 2A). Expression was increased in the presence of 10 ng/ml
TNF- and IL-1. To confirm the presence of the receptor on the
cell surface, FACS analysis using a specific
CX3CR-1 Ab was performed. Fig. 2D
shows significant expression of the fractalkine receptor, which is only
modestly increased upon stimulation with IL-1. Equilibrium-binding
analyses using 125I-labeled fractalkine reveal
specific and saturable binding kinetics
(Kd of 0.143 nM, corresponding to 8000
sites/cell) (Fig. 2B). 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).
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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. 3B). The response in all cases was
robust, prolonged, and inhibited by preincubation of cells with
pertussis toxin (PTX) (100 ng/ml) (Fig. 3C). 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.
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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. 4A). This activity
occurred through a standard cascade, since the specific MAPK kinase
(MEK) inhibitor PD98059 completely reduced the phosphotyrosine
incorporation (Fig. 4A, 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-1. In a similar manner, little, if any, changes in
phosphorylation or MAPK activity were observed in response to other
chemokines (data not shown).
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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.
Fractalkine-induced migration of microglia
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. 5A). 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. 5B). 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. 5C). 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. 5E). Astrocytes showed no significant migration in the
presence of the chemokine module of fractalkine alone or the protein
containing the mucin stalk (Fig. 5D). In addition,
preincubation of astrocytes with TNF- or IL-1 or coating both
forms of fractalkine on the filter were not effective in inducing
migration of astrocytes.
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i 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. 5E 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).
Fractalkine induces actin rearrangement and shape change in microglia
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. 6A). 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. 6B). 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).
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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.
Fractalkine-activated astrocytes promote microglial cell proliferation
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. 7A, 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. 7B, bars 5–7). This effect was not
observed when increasing doses of soluble fractalkine were added to
control astrocyte medium (Fig. 7B, bars 2–4) or when Abs
directed against fractalkine or CX3C-R1 were
added to the astrocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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- and IL1-. 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.
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Acknowledgments |
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Footnotes |
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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.
Received for publication March 19, 1999. Accepted for publication May 25, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3(IV) collagen gene: differential expression of mRNA transcripts that predicts three protein variants with distinct carboxyl regions. J. Biol. Chem. 269:2342. induces migration and activation of human thymocytes. Blood 91:2905.-chemokine, stromal cell-derived factor-1, binds to the transmembrane G-protein-coupled CXCR-4 receptor and activates multiple signal transduction pathways. J. Biol. Chem. 272:23169.
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 C. Habasque, A.-P. Satie, F. Aubry, B. Jegou, and M. Samson Expression of fractalkine in the rat testis: molecular cloning of a novel alternative transcript of its gene that is differentially regulated by pro-inflammatory cytokines Mol. Hum. Reprod., August 1, 2003; 9(8): 449 - 455. [Abstract] [Full Text] [PDF]
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 R. A. Tripp, A. Dakhama, L. P. Jones, A. Barskey, E. W. Gelfand, and L. J. Anderson The G Glycoprotein of Respiratory Syncytial Virus Depresses Respiratory Rates through the CX3C Motif and Substance P J. Virol., June 1, 2003; 77(11): 6580 - 6584. [Abstract] [Full Text] [PDF]
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T. Harada, C. Harada, S. Kohsaka, E. Wada, K. Yoshida, S. Ohno, H. Mamada, K. Tanaka, L. F. Parada, and K. Wada Microglia-Muller Glia Cell Interactions Control Neurotrophic Factor Production during Light-Induced Retinal Degeneration J. Neurosci., November 1, 2002; 22(21): 9228 - 9236. [Abstract] [Full Text] [PDF]
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A. M. Fong, S. M. Alam, T. Imai, B. Haribabu, and D. D. Patel CX3CR1 Tyrosine Sulfation Enhances Fractalkine-induced Cell Adhesion J. Biol. Chem., May 24, 2002; 277(22): 19418 - 19423. [Abstract] [Full Text] [PDF]
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 K. Fujimoto, T. Imaizumi, H. Yoshida, S. Takanashi, K. Okumura, and K. Satoh Interferon-gamma Stimulates Fractalkine Expression in Human Bronchial Epithelial Cells and Regulates Mononuclear Cell Adherence Am. J. Respir. Cell Mol. Biol., August 1, 2001; 25(2): 233 - 238. [Abstract] [Full Text] [PDF]
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 D. N. Cook, S.-C. Chen, L. M. Sullivan, D. J. Manfra, M. T. Wiekowski, D. M. Prosser, G. Vassileva, and S. A. Lira Generation and Analysis of Mice Lacking the Chemokine Fractalkine Mol. Cell. Biol., May 1, 2001; 21(9): 3159 - 3165. [Abstract] [Full Text]
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L. A. Robinson, C. Nataraj, D. W. Thomas, D. N. Howell, R. Griffiths, V. Bautch, D. D. Patel, L. Feng, and T. M. Coffman A Role for Fractalkine and Its Receptor (CX3CR1) in Cardiac Allograft Rejection J. Immunol., December 1, 2000; 165(11): 6067 - 6072. [Abstract] [Full Text] [PDF]
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C. Sauder, W. Hallensleben, A. Pagenstecher, S. Schneckenburger, L. Biro, D. Pertlik, J. Hausmann, M. Suter, and P. Staeheli Chemokine Gene Expression in Astrocytes of Borna Disease Virus-Infected Rats and Mice in the Absence of Inflammation J. Virol., October 1, 2000; 74(19): 9267 - 9280. [Abstract] [Full Text]
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S. A. Boehme, F. M. Lio, D. Maciejewski-Lenoir, K. B. Bacon, and P. J. Conlon The Chemokine Fractalkine Inhibits Fas-Mediated Cell Death of Brain Microglia J. Immunol., July 1, 2000; 165(1): 397 - 403. [Abstract] [Full Text] [PDF]
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A. M. Fong, H. P. Erickson, J. P. Zachariah, S. Poon, N. J. Schamberg, T. Imai, and D. D. Patel Ultrastructure and Function of the Fractalkine Mucin Domain in CX3C Chemokine Domain Presentation J. Biol. Chem., February 11, 2000; 275(6): 3781 - 3786. [Abstract] [Full Text] [PDF] |
) 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-1
