| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Neurocrine Biosciences, Inc., San Diego, CA 92121
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
or IL-1ß
in astrocytes (5, 6). The fractalkine receptor,
CX3CR-1, is highly expressed by microglia
(5, 7, 8, 9). It has been shown to induce cell migration in
primary microglial cells, as well as activate the p42/p44
mitogen-activated protein kinase and phosphatidylinositol-3
(PI-3)3 kinase/protein
kinase B (PKB) signal transduction pathways (6).
Microglia have been shown to express both Fas (CD95) and Fas ligand
(CD95L), and Fas-mediated apoptosis has been implicated in the
pathogenesis of various CNS diseases, such as ischemia-reperfusion
injury (10, 11), multiple sclerosis (12, 13, 14),
and its rodent counterpart, experimental autoimmune encephalomyelitis
(15, 16, 17). However, the exact role of Fas/Fas
ligand in the pathology of multiple sclerosis/experimental autoimmune
encephalomyelitis is unclear. Thus, as the Fas/Fas ligand
interaction plays a role in maintaining hemopoietic homeostasis, it may
also contribute to the pathogenesis of various disease processes
(18). The binding of Fas ligand to the cell surface
receptor Fas initiates a cascade of events leading to activation of
various caspases in conjunction with modulation of Bcl-2 family
proteins (19). The latter results in compromised
mitochondrial function and integrity, which may contribute directly to
the apoptotic process or be a result of caspase 8 and caspase 3
activation (19, 20). Specifically, the proapoptotic Bcl-2
family member BAD has the ability to complex with either Bcl-2 or
Bcl-xL and antagonize their antiapoptotic
function (21). This process is regulated in part by
PKB/Akt, which can phosphorylate BAD at serine 136, rendering this
proapoptotic protein inactive (22, 23, 24, 25). Additionally,
PKB/Akt can act via NF-
B to block apoptosis (26, 27).
The proapoptotic protein BID is cleaved upon Fas ligand binding,
allowing heterodimerization with Bcl-2 or Bcl-xL,
and provides another pathway resulting in the abrogation of Bcl-2 or
Bcl-xL antiapoptotic function
(28).
The expression of fractalkine by neurons, and CX3CR-1 by microglial cells suggests that a paracrine interaction exists; however, no definitive CNS function has yet been elucidated. We have recently shown that fractalkine activates the PKB/Akt signaling pathway in primary microglia (6). As PKB/Akt has been shown to be a survival factor in a number of systems, we asked whether fractalkine stimulation could promote survival of primary brain microglia, and furthermore block Fas-mediated microglial cell death. We show in this work for the first time that the chemokine fractalkine can increase microglial cell survival and block Fas-induced programmed cell death of primary cells. This novel function provides crucial insight into the role fractalkine plays in regulating CNS homeostasis, as well as demonstrating evidence of neuron/microglia cross-talk.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
All reagents were obtained from Sigma (St. Louis, MO), unless otherwise specified. Animal experimentation was approved by the Institutional Animal Care and Use Committee (IACUC) before implementation.
Primary microglial cell cultures
Microglial cell cultures were established as previously
described (6). Briefly, cortexes from newborn Sprague
Dawley rats (Charles River, Boston, MA) were isolated, mechanically
dissociated, and plated at a density of one brain/T75 flask (Costar,
Charlotte, NC) in DMEM (Mediatech, Tustin, CA) containing 10% FCS
(HyClone, Logan, UT). Once confluent, the cells were left for 5–7 days
without changing the media 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 pelleted and the
cells were replated in DMEM + 10% FCS. After 2 h, the cells were
manually shaken and the medium was replaced with DMEM + 10% FCS
containing 200 U/ml of both GM-CSF (R&D Systems, Minneapolis, MN) and
M-CSF (R&D Systems). The adherent cells (>95% pure microglia) were
grown for an additional 48 h before assaying. At this point, they
were detached with Versene (Life Technologies, Gaithersburg, MD),
replated in DMEM + 10% FCS, and cultured in experiments, as described.
It should be noted that no exogenous growth factors were added during
the experimental procedures, except where indicated (Fig. 1
).
|
A total of 5 x 104 microglia/well was seeded into a 96-well flat-bottom plate (Costar) following the described conditions, and each condition was done in triplicate. Before the addition of cells, tissue culture plate wells were coated by the addition of the indicated concentration of fractalkine (chemokine portion only; R&D Systems) diluted in PBS (Mediatech), and incubated at 4°C overnight. Control wells received PBS. Following incubation, the wells were washed once with PBS; the cells were then added and cultured for the indicated time periods. Soluble Fas ligand was obtained from Upstate Biotechnology (Lake Placid, NY). Eighteen hours postplating, the cells were harvested, and the oligosomal DNA was quantitated using the Cell Death Detection ELISA (Roche Diagnostics, Indianapolis, IN), according to the manufacturer’s protocol.
Photomicroscopy
Microglial cells were cultured in 24-well plates at 5 x 105 cells/well for 18 h under the various conditions. They were then isolated using Versene, pelleted onto slides, and fixed using 4% paraformaldehyde, followed by a -20°C methanol wash. The cells were then stained with DAPI (Calbiochem, La Jolla, CA) at a 1 µg/ml concentration for 15 min, and rinsed in PBS. Photomicrographs were taken using a Nikon Eclipse TE300 microscope.
Western blot analysis
Microglia were seeded into 24-well plates at a concentration of
5 x 105/well at the described conditions.
Following an 18-h incubation, the microglia were harvested with Versene
and washed twice with PBS. The cells were subsequently suspended in
lysis solution (1% Nonidet P-40, 50 mM Tris-HCl (pH 8), 150 mM NaCl,
0.25% deoxycholate, and 5 mM EDTA) containing protease and phosphatase
inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, 1 mM
sodium orthovanadate, 1 mM EGTA, 100 µM ß-glycerophosphate, 10 mM
sodium flouride, and 1 mM tetrasodium phosphate). Cells were incubated
on ice for 30 min and centrifuged to pellet insoluble material, and the
protein was quantified using the bicinchoninic acid assay (Pierce,
Rockford, IL). Western blot analysis was conducted as previously
described, using Tris-glycine acrylamide gels (Novex, San Diego, CA)
(29). Hybridized proteins were visualized using enhanced
chemiluminescence reagents (Pierce) and Biomax MR autoradiography film
(Eastman Kodak, Rochester, NY). The following primary Abs were used to
probe the Western blots: anti-PKB polyclonal Ab (Upstate
Biotechnology), anti-BAD Ab (New England Biolabs, Beverly, MA),
anti-phosphoserine Ab (Zymed Laboratories, South San Francisco,
CA), anti-Bcl-xL (Transduction Laboratories,
Lexington, KY), anti-Bax Ab (Upstate Biotechnology), and
anti-BID polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA).
Equal amounts of protein (25 µg) were loaded into each lane.
Pulldown experiments
Microglial cell lysates were collected as described above and used to carry out the pulldown experiments. One microgram of Bcl-2-GST fusion protein coupled to agarose beads (Upstate Biotechnology), or 1 µg anti-BAD Abs and protein G-coated agarose beads (Santa Cruz Biotechnology) were added to 5 µg of fresh cell lysates, and the immunoprecipitation was conducted overnight at 4°C on an orbital rocker. The agarose beads were subsequently pelleted by centrifugation, washed twice with lysis buffer containing the protease and phosphatase inhibitors, and analyzed by Western blotting.
Measurement of Akt/PKB activity
Assay for Akt/PKB activity was performed as previously described
(6). Microglial cells were stimulated with fractalkine and
the PI-3 kinase inhibitor LY294002 (Calbiochem) at the indicated
concentrations. PKB was immunoprecipitated using an anti-rat PKB
polyclonal Ab (Santa Cruz Biotechnology) and precipitated with protein
G agarose beads (Santa Cruz Biotechnology). Kinase reactions were
performed in kinase reaction buffer (20 mM HEPES (pH 7.4), 10 mM
MgCl2, 10 mM MnCl2, 0.05
mg/ml histone 2B, 5 µM ATP, 1 mM DTT, and 10 µCi
[
-32P]ATP) for 30 min at 30°C. The
reactions were halted by the addition of an equal volume of 2x Laemmli
sample buffer and boiling. Histone 2B phosphorylation was resolved
using a 16% Tris glycine gel (Novex) and visualized by
autoradiography. Equal loading of Akt/PKB was detected by Western
analysis, as described above.
Cell viability and proliferation
Microglia were cultured in 96-well plates (5 x 104 cells/well) and were either untreated, seeded onto wells coated with 100 nM fractalkine, or cultured in the presence of 200 U/ml M-CSF and GM-CSF (R&D Systems). Mitochondrial function was used as a measure of cell survival, and after 44 h of culture, an MTT assay was performed, according to the manufacturer’s instructions (Roche Diagnostics). After 24 h, parallel cultures were pulsed with 1 µCi of [3H]TdR (6.7 mCi/mmol; New England Nuclear, Boston, MA), and [3H]TdR incorporation was measured by betaplate scintigraphy at 48 h. Each experimental point was done in quadruplicate, and five independent experiments were performed. Dr. Lili Feng (Scripps Research Institute, La Jolla, CA) kindly provided the neutralizing anti-CX3CR-1 Ab.
Statistical analysis
Two-tailed Student’s t test was used to determine whether differences observed were significant. A p value <0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In analyses of fractalkine effects on primary rat microglial cells
in vitro, we observed that cells grown in the presence of immobilized
fractalkine (as described in Materials and Methods) showed
increased viability over time when compared with unstimulated cells by
MTT assay (Fig. 1
). Both microglial cell
survival and mitochondrial function were enhanced in the presence of
fractalkine, and a specific blocking Ab to the receptor,
CX3CR-1, abrogated this effect. No comparable
effects were seen with 20 other chemokines tested (data not shown). In
contrast to microglia treated with both M-CSF/GM-CSF,
fractalkine-treated cells proliferated minimally (Fig. 1;
[3H]thymidine). These data suggest that
fractalkine serves a unique function compared with other chemokines,
that of a survival factor for microglial cells in vitro.
Fractalkine can block Fas-mediated apoptosis of microglia
To further analyze the survival-promoting effects of fractalkine,
we asked whether fractalkine could antagonize Fas-induced programmed
cell death. Microglia were stimulated with plate-bound fractalkine
(immobilized chemokine domain alone) and various doses of soluble Fas
ligand. After an 18-h incubation period (determined through time-course
experiments), cell viability was assessed. Microscopic examination of
the microglia revealed extensive cell death in samples treated only
with Fas ligand, as evidenced by cell aggregation and condensed
cytoplasm (data not shown). DAPI staining of the cells showed that Fas
ligand-treated microglia had undergone chromatin condensation and
nuclear fragmentation (Fig. 2A). In contrast, untreated,
fractalkine-treated or fractalkine and Fas ligand-treated microglia had
substantially fewer cells displaying condensed chromatin. In parallel,
microglial cells that did not receive fractalkine treatment had an
approximate 4–5-fold increase in the amount of oligosomal DNA, which
is indicative of apoptosis, and this effect was dependent on the dose
of Fas ligand added (Fig. 2B). A dose titration of
fractalkine revealed that 10 nM was sufficient to effectively inhibit
Fas-mediated apoptosis (Fig. 2C). Additional evidence that
this effect was unique to fractalkine was obtained using eotaxin.
Functional CCR-3 receptors are widely expressed on microglia
(30), and both fractalkine and eotaxin stimulate
phosphorylation of extracellular signal-related kinase 1 and 2,
demonstrating an overlap in signal transduction pathways (Ref.
6 ; and S. A. Boehme and K. B. Bacon, unpublished
observations). Unlike fractalkine, eotaxin was unable to rescue
Fas-induced cell death (Fig. 2B).
|
). A total of 4.2% of fractalkine and
fas ligand-treated cells had a hypodiploid DNA content contrasted with
20.6% of the Fas ligand-treated cells. These data are consistent with
our results measuring oligosomal DNA, and are not due to
fractalkine-induced down-regulation of Fas from the microglial cell
surface (data not shown). Additionally, this analysis further
demonstrated fractalkine’s survival factor effect on microglia, as
7.21% of untreated microglia had hypodiploid DNA content compared with
just 1.93% of the fractalkine-treated cells (Table I). DNA content
analysis also suggested that fractalkine may be inducing a
G1 cell cycle block in microglia, as a
substantially greater number of cells were in the
G1 phase of the cell cycle following 18 h of
fractalkine stimulation compared with controls (Table I). Collectively,
these results demonstrated that under the conditions tested,
fractalkine stimulation of microglia had a pronounced survival
effect.
|
The significance of these effects on cell survival correlates with
in vitro biochemical signaling. Previously, we demonstrated that
fractalkine activates the PI-3 kinase/PKB pathway in primary microglia
(6), and this might account for fractalkine acting as a
survival factor. Therefore, we tested whether blockade of this pathway
by the PI-3 kinase inhibitor LY294002 could reverse the protective
effect of fractalkine (31). The addition of 1 µM
LY294002 to microglial cell cultures effectively blocked the kinase
activity of PKB (Fig. 3A);
however, it only partially antagonized the protective effect of
fractalkine (Fig. 3B). Similar effects were observed using
another PI-3 kinase antagonist, wortmannin, at several concentrations
(data not shown). This observation illustrates that activation of the
PI-3 kinase/PKB pathway plays a role in fractalkine’s ability to block
Fas-mediated apoptosis, although only partial protection was seen under
various doses, suggesting multiple pathways mediate fractalkine’s
protective effect.
|
A). This
revealed that microglial cells treated with fractalkine had
substantially higher levels of phosphoserine-BAD protein, indicative of
inhibition of its proapoptotic function. When the immunoblot was
reprobed, comparable levels of BAD protein were found under all
conditions tested (Fig. 4B). Additionally, we tested whether
BAD could heterodimerize with Bcl-2 under these conditions; Western
blots of pulldown experiments using a Bcl-2-GST fusion protein were
probed for BAD. The levels of BAD protein precipitated with the Bcl-2
fusion protein were reduced in untreated, fractalkine-treated, or
fractalkine and Fas ligand-treated microglia, compared with Fas
ligand-stimulated microglia, further suggesting that BAD had been
phosphorylated and lost its ability to heterodimerize (Fig. 4C).
|
As the regulatory pathway of BAD phosphorylation did not appear to
be the sole regulator of apoptosis (Fig. 3B), the role of
other Bcl-2 family proteins was explored by measuring levels of the
antiapoptotic protein Bcl-xL and the proapoptotic
protein Bax. The intracellular ratio of these proteins is thought to
dictate cell fate (32). Our results show that
Bcl-xL is up-regulated in microglia treated with
fractalkine for 18 h; conversely, Bax protein levels are reduced
(Fig. 5, A and B).
Additionally, levels of the proapoptotic protein BID were examined
(28). BID has been shown to be a critical mediator in the
mitochondrial amplification loop, resulting in Fas-induced apoptosis
(33). We observed BID to be cleaved and activated in Fas
ligand-treated cultures of microglia; however, the amount of activated
protein was significantly reduced in cells stimulated with fractalkine
(Fig. 5C). These results illustrate that fractalkine
inhibition of Fas-induced microglial cell death is mediated at least in
part through specific antiapoptotic signaling. Taken together with the
survival effect measured by mitochondrial function (MTT assay, Fig. 1),
these observations suggest that one mechanism by which fractalkine is
acting to suppress Fas-mediated apoptosis may be by
maintaining mitochondrial integrity.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The Fas ligand/Fas pathway has been shown to be involved in various CNS
diseases (10, 11, 12, 13, 14, 15, 16, 17). Normally, Fas is only weakly expressed
in the brain, but is up-regulated in brain tissues from stroke,
multiple sclerosis, and Alzheimer’s patients, suggesting it may play a
role in various CNS pathological states (10, 38). In these
diseases, there is a concomitant release of Fas ligand into the CNS
environment by various cell types, including microglia (10, 14). We show in this work that fractalkine can inhibit
Fas-mediated death of primary microglial cells in vitro. Although
fractalkine is constitutively expressed in the brain, deleterious
stimuli further induce fractalkine expression by neurons
(5). Additionally, the proinflammatory cytokines TNF-
and IL-1ß trigger fractal-kine expression by astrocytes
(6). Taken together, these observations suggest a critical
role of fractalkine in various CNS disease states, and one function may
be to promote microglial cell survival.
Microglia are brain-resident macrophage, and they perform a critical role as phagocytic cells. Furthermore, their anatomic location close to the blood brain barrier, and their ability to secrete inflammatory cytokines, reactive oxygen intermediates, NO, and Fas ligand allow microglia to serve as critical regulators of CNS inflammation (34). Thus, microglia play a key role mediating CNS tissue damage, as this cell type has the ability to both amplify and control CNS pathology, both in the early stages of inflammation and the later phase of tissue repair.
In light of microglial function in the CNS both in homeostasis and disease states, our results demonstrate a novel and potentially critical function of fractalkine. These results illustrate a physiologically relevant role for this constitutively expressed chemokine in the brain. In summary, the data presented demonstrate a novel biological precedent for a chemokine, and provide insight into basic cellular interactions between neurons and microglia in both normal and pathological conditions within the CNS. These observations may, in turn, open up new avenues for therapeutic gain in reducing CNS damage in various diseases by modulating the fractalkine/CX3CR-1 interaction.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Bayer Yakuhin, Ltd., Research Center Kyoto, Soraku-gun, Kyoto 619-0216, Japan.
3 Abbreviations used in this paper: PI-3, phosphatidylinositol-3; BID, BH3-interacting domain death agonist; DAPI, 4',6'-diamidino-2-phenylindole; PKB, protein kinase B.
Received for publication January 24, 2000. Accepted for publication April 12, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B activation by tumor necrosis factor requires the AKT serine-threonine kinase. Nature 401:82.[Medline]
B is a target of AKT in anti-apoptotic PDGF signalling. Nature 401:86.[Medline]
This article has been cited by other articles:
|
|
 |
|
  L. Landsman, L. Bar-On, A. Zernecke, K.-W. Kim, R. Krauthgamer, E. Shagdarsuren, S. A. Lira, I. L. Weissman, C. Weber, and S. Jung CX3CR1 is required for monocyte homeostasis and atherogenesis by promoting cell survival Blood, January 22, 2009; 113(4): 963 - 972. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. G. McComb, M. Ranganathan, X. H. Liu, J. M. Pilewski, P. Ray, S. C. Watkins, A. M.K. Choi, and J. S. Lee CX3CL1 Up-Regulation Is Associated with Recruitment of CX3CR1+ Mononuclear Phagocytes and T Lymphocytes in the Lungs during Cigarette Smoke-Induced Emphysema Am. J. Pathol., October 1, 2008; 173(4): 949 - 961. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Zhao, A. De, Z. Hu, J. Li, S. M. Mulders, M. D. Sollewijn Gelpke, E.-K. Duan, and A. J. W. Hsueh Gonadotropin Stimulation of Ovarian Fractalkine Expression and Fractalkine Augmentation of Progesterone Biosynthesis by Luteinizing Granulosa Cells Endocrinology, June 1, 2008; 149(6): 2782 - 2789. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Lauro, S. Di Angelantonio, R. Cipriani, F. Sobrero, L. Antonilli, V. Brusadin, D. Ragozzino, and C. Limatola Activity of Adenosine Receptors Type 1 Is Required for CX3CL1-Mediated Neuroprotection and Neuromodulation in Hippocampal Neurons J. Immunol., June 1, 2008; 180(11): 7590 - 7596. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ishida, J.-L. Gao, and P. M. Murphy Chemokine Receptor CX3CR1 Mediates Skin Wound Healing by Promoting Macrophage and Fibroblast Accumulation and Function J. Immunol., January 1, 2008; 180(1): 569 - 579. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D.-J. Oh, B. Dursun, Z. He, L. Lu, T. S. Hoke, D. Ljubanovic, S. Faubel, and C. L. Edelstein Fractalkine receptor (CX3CR1) inhibition is protective against ischemic acute renal failure in mice Am J Physiol Renal Physiol, January 1, 2008; 294(1): F264 - F271. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Mizutani, T. Sakurai, T. Shibata, K. Uchida, J. Fujita, R. Kawashima, Y. I. Kawamura, N. Toyama-Sorimachi, T. Imai, and T. Dohi Dose-Dependent Differential Regulation of Cytokine Secretion from Macrophages by Fractalkine J. Immunol., December 1, 2007; 179(11): 7478 - 7487. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-S. Kim, M. S. Kim, A. L. Hancock, J. C. P. Harper, J. Y. Park, G. Poy, A. O. Perantoni, M. Cam, K. Malik, and S. B. Lee Identification of Novel Wilms' Tumor Suppressor Gene Target Genes Implicated in Kidney Development J. Biol. Chem., June 1, 2007; 282(22): 16278 - 16287. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ueha, M. Murai, H. Yoneyama, M. Kitabatake, T. Imai, T. Shimaoka, S. Yonehara, S. Ishikawa, and K. Matsushima Intervention of MAdCAM-1 or fractalkine alleviates graft-versus-host reaction associated intestinal injury while preserving graft-versus-tumor effects J. Leukoc. Biol., January 1, 2007; 81(1): 176 - 185. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. T. Murooka, M. M. Wong, R. Rahbar, B. Majchrzak-Kita, A. E. I. Proudfoot, and E. N. Fish CCL5-CCR5-mediated Apoptosis in T Cells: REQUIREMENT FOR GLYCOSAMINOGLYCAN BINDING AND CCL5 AGGREGATION J. Biol. Chem., September 1, 2006; 281(35): 25184 - 25194. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. R. Wallace, R. W. Vaughan, E. Kondeatis, R. Mathew, Y. Chen, E. M. Graham, and M. R. Stanford A CX3CR1 Genotype Associated with Retinal Vasculitis in Patients in the United Kingdom. Invest. Ophthalmol. Vis. Sci., July 1, 2006; 47(7): 2966 - 2970. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Sonnet, P. Lafuste, L. Arnold, M. Brigitte, F. Poron, F. Authier, F. Chretien, R. K. Gherardi, and B. Chazaud Human macrophages rescue myoblasts and myotubes from apoptosis through a set of adhesion molecular systems J. Cell Sci., June 15, 2006; 119(12): 2497 - 2507. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. N. Davis and J. K. Harrison Proline 326 in the C Terminus of Murine CX3CR1 Prevents G-Protein and Phosphatidylinositol 3-Kinase-Dependent Stimulation of Akt and Extracellular Signal-Regulated Kinase in Chinese Hamster Ovary Cells J. Pharmacol. Exp. Ther., January 1, 2006; 316(1): 356 - 363. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. N. Davis, V. Zujovic, and J. K. Harrison Viral Macrophage Inflammatory Protein-II and Fractalkine (CX3CL1) Chimeras Identify Molecular Determinants of Affinity, Efficacy, and Selectivity at CX3CR1 Mol. Pharmacol., December 1, 2004; 66(6): 1431 - 1439. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Garin, N. Tarantino, S. Faure, M. Daoudi, C. Lecureuil, A. Bourdais, P. Debre, P. Deterre, and C. Combadiere Two Novel Fully Functional Isoforms of CX3CR1 Are Potent HIV Coreceptors J. Immunol., November 15, 2003; 171(10): 5305 - 5312. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Murakami, A. R. Cardones, S. E. Finkelstein, N. P. Restifo, B. A. Klaunberg, F. O. Nestle, S. S. Castillo, P. A. Dennis, and S. T. Hwang Immune Evasion by Murine Melanoma Mediated through CC Chemokine Receptor-10 J. Exp. Med., November 3, 2003; 198(9): 1337 - 1347. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Kari, A. Loboda, M. Nebozhyn, A. H. Rook, E. C. Vonderheid, C. Nichols, D. Virok, C. Chang, W.-H. Horng, J. Johnston, et al. Classification and Prediction of Survival in Patients with the Leukemic Phase of Cutaneous T Cell Lymphoma J. Exp. Med., June 2, 2003; 197(11): 1477 - 1488. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Banas, M. Wornle, T. Berger, P. J. Nelson, C. D. Cohen, M. Kretzler, J. Pfirstinger, M. Mack, M. Lipp, H.-J. Grone, et al. Roles of SLC/CCL21 and CCR7 in Human Kidney for Mesangial Proliferation, Migration, Apoptosis, and Tissue Homeostasis J. Immunol., May 1, 2002; 168(9): 4301 - 4307. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-J. Grone, C. D. Cohen, E. Grone, C. Schmidt, M. Kretzler, D. Schlondorff, and P. J. Nelson Spatial and Temporally Restricted Expression of Chemokines and Chemokine Receptors in the Developing Human Kidney J. Am. Soc. Nephrol., April 1, 2002; 13(4): 957 - 967. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
V. Kansra, C. Groves, J. C. Gutierrez-Ramos, and R. D. Polakiewicz Phosphatidylinositol 3-Kinase-dependent Extracellular Calcium Influx Is Essential for CX3CR1-mediated Activation of the Mitogen-activated Protein Kinase Cascade J. Biol. Chem., August 17, 2001; 276(34): 31831 - 31838. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. S. Mizoue, S. K. Sullivan, D. S. King, T. N. Kledal, T. W. Schwartz, K. B. Bacon, and T. M. Handel Molecular Determinants of Receptor Binding and Signaling by the CX3C Chemokine Fractalkine J. Biol. Chem., August 31, 2001; 276(36): 33906 - 33914. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
)
and [3H]thymidine incorporation (
) measuring cell
viability and proliferation, respectively. Microglia were either
untreated (control) or treated for 48 h with 100 nM fractalkine
(chemokine domain), or 200 U/ml M-CSF and GM-CSF in the presence or
absence of neutralizing concentrations of CX3CR-1 Ab. MTT
was added for the last 4 h of the experiment to measure
mitochondrial metabolic function as an indicator of cell survival.
Cells were pulsed after 24 h, and [3H]thymidine
incorporation was measured at 48 h to assess proliferation. All
results have been normalized to control levels (100%) ± SEM, and
a representative of five separate experiments is shown. Statistical
differences were significant between the various samples; *, denotes
p < 0.001, and **, indicates
p < 0.005.
), 10 nM plate-bound fractalkine (chemokine
domain; 
), or soluble 10 nM eotaxin (
) for 18 h. The results
shown are the mean of four independent experiments ± SEM.
c, Fractalkine protection of Fas ligand-treated
microglia is dose dependent. Microglia were treated as above with
various doses of plate-bound fractalkine, and oligosomal DNA content
was determined after 18 h. Representative results from three
independent experiments are shown ± SEM.
