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*
Laboratory of Tumor Immunology, Division of Oncology, and
Department of Neurosurgery, University Hospital, Geneva, Switzerland; and
Laboratory of Immunopathology, Faculty of Medicine, Marseille, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, resistant astrocytes became
sensitive to CD95-mediated death. These data suggest that
microenvironmental factors can influence the consequences of CD95
ligation on astrocytes. Therefore, we propose that CD95 expressed by
human astrocytes plays a pivotal role in the regulation of astrocyte
life and death and may be a key factor in inflammatory processes in the
brain, such as reactive gliosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apoptosis (active programmed cell death) can be induced by diverse
stimuli. A key molecule is CD95 (Fas/Apo-1), a transmembrane protein
belonging to the TNF receptor family that transduces apoptotic signals
after ligation by agonistic Abs or by its natural ligand (CD95L/FasL),
a member of the TNF family 11 . Here, we examine whether CD95 may
contribute to human astrocyte apoptosis by using cultured astrocytes
derived from human embryos. In this culture system, a high level of
purity is obtained after three passages, allowing the study of
functional parameters on glial fibrillary acidic protein (GFAP)
positive astrocytes 12, 13, 14 . We show that human astrocytes express
CD95 that can transduce an apoptotic signal or an activation signal,
leading to proinflammatory cytokine release. The particular outcome is
governed by the presence of IFN-, indicating that in vivo,
microenvironmental factors and cellular interactions can alter the
course and duration of inflammatory brain diseases by effects on the
CD95 pathway of astrocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mAbs against human GFAP (6F2, mouse IgG1) and CD3 (4B5, mouse
IgG1, negative control for GFAP staining), anti-GFAP polyclonal
antiserum, FITC-conjugated anti-human Bcl-2 mAb (124, mouse IgG1),
FITC-conjugated mouse IgG1 (negative control mAb for Bcl-2 detection),
and an FITC-conjugated F(ab')2 of goat anti-mouse Ig
were obtained from Dako (Zug, Switzerland). The lytic anti-human
CD95 mAb (CH11, mouse IgM), phycoerythrin (PE)-conjugated
anti-human CD95 mAb (UB2, mouse IgG1), and Cy3-conjugated
F(ab')2 of goat anti-rabbit Ig were obtained from
Immunotech (Marseille, France). Mouse IgM anti-human CD57 mAb HNK-1
(ATCC TIB200) and PE-conjugated anti-human CD8 mAb (mouse IgG1;
Serotec, Oxford, U.K.) were used as isotype-matched negative control
mAb for CD95 ligation and CD95 staining, respectively. Recombinant
CD95L was obtained from supernatant of CD95L-transfected Neuro-2a cells
15 . Supernatant from mock-transfected Neuro-2a cells was used as
control. These cells were provided by A. Fontana (Zurich, Switzerland).
Caspase inhibitors z-VAD-fmk
(benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) and YVAD-cmk
(acetyl-Tyr-Val-Ala-Asp-chloromethylketone) were obtained from Bachem
Biosciences (Budendorf, Switzerland). Human rIFN- and neutralizing
anti-human IL-8 mAb (mouse IgG1) were obtained from R & D Systems
(Abingdon, U.K.). Anti-human CD3 mAb (mouse IgG, OKT3), provided by
Drs. P. Tiberghien and E. Robinet (Besançon, France), was used as
an isotype control for anti-IL8 mAb. Human rTNF-
was provided by
C. Ruegg (Lausanne, Switzerland).
Established cell lines
Astrocytoma cell lines LN18, LN215, and LN229 were provided by N. de Tribolet and E. G. Van Meir (Lausanne, Switzerland). The human cell line Jurkat (ATCC TIB152) was obtained from the American Type Culture Collection (Manassas, VA). The human pancreatic cancer cell line Colo357 cDNA was provided by H. Kalthoff and H. Ungefroren (Kiel, Germany).
Human astrocyte culture
Primary human astrocyte cultures were prepared as previously described 12, 13, 14 from fetal abortions (7–9 wk). Tissue was procured in accordance with institutional regulations. Independent cultures were established from each brain specimen. The myelencephalon and mesencephalon, telencephalon, or spinal cord were isolated under sterile conditions. After removal of meninges, the tissue was dissociated by trypsinization and mechanical disruption. The cells were seeded on poly-L-lysine (10 µg/ml; Sigma, St. Louis, MO)-coated flasks at a density of 2 x 103 cells/cm2 and grown at 37°C in a humidified atmosphere with 5% CO2 in MEM D-valine (Life Technologies, Grand Island, NY) supplemented with 5% FCS, 2 mM L-glutamine (Life Technologies), 20 ng/ml EGF (Sigma), 50 µg/ml apotransferrin (Sigma), 5 µg/ml insulin (Sigma), 100 IU/ml penicillin (Life Technologies), and 100 µg/ml streptomycin (Life Technologies). After 1 mo of culture, a confluent layer of GFAP-positive cells was obtained. Cells were then passaged using 0.25% trypsin-EDTA (Life Technologies) every 3 wk and were replated at a 1/3 dilution. The expression of GFAP and the absence of mycoplasma (ELISA detection kit, Boehringer Mannheim, Rotkreuz, Switzerland) were confirmed for all cultures.
Flow cytometry
Analysis of astrocytes was performed on a FACScan (Becton Dickinson, Mountain View, CA) using LYSYS II software. Cells were detached from culture vessels using brief exposure to trypsin-EDTA and were stained with the appropriate mAb or isotype-matched control mAb. Dead cells were excluded by forward and side scatter or propidium iodide (Sigma) gating. For intracytoplasmic staining, cells were fixed in 0.25% paraformaldehyde (Fluka, Buchs, Switzerland) in PBS and permeabilized in 70% cold ethanol before the staining procedure.
RT-PCR analysis
Total RNAs isolated from astrocytes using TRIzol reagent (Life Technologies) were converted to cDNA by standard methods using reverse transcriptase (Life Technologies) and an oligo(dT) primer (Life Technologies). These cDNA were amplified in nonsaturating PCR conditions using specific primers. Primer sequences (sense primers are indicated first) are as follows: ß-actin, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' (positions 541–569) and 5'-CTAGAAGCATTTGCGGTGGACGACGATGGAGGG-3' (positions 1172–1201); CD95, 5'-ATGCTGGGCATCTGCACCCT-3' and 5'-TCTAGACCAAGCTTTGGATTTC-3' 16 ; Fas-associated phosphatase-1 (FAP-1), 5'-GAATACGAGTGTCAGACATGG-3' and 5'-AGGTCTGCAGAGAAGCAAGAATAC-3' 17, 18 ; and Bcl-x, 5'-GGAGTCAGTTTAGTGATGTG-3' (positions 205–224) and 5'-CTGCATTGTTCCCATAGAGT-3' (positions 713–732). After amplification, PCR products were separated by electrophoresis on agarose gel containing ethidium bromide and visualized by UV light illumination.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay
Astrocytes obtained from mesencephalon/myelencephalon were
seeded into poly-L-lysine-coated wells of 24-well plates at
4 x 104/well and incubated for 16 h to obtain
cell adherence. Then, cells were incubated with lytic anti-CD95 mAb
CH11 or control IgM, TNF-, C2 ceramide (Sigma), t-butyl
hydroperoxide (Sigma), or neutralizing anti-IL-8 mAb (at the
indicated concentrations) for 24 h. Cell viability was determined
by the MTT assay, which determines mitochondrial activity in living
cells 19 . Results are expressed as the percentage of dead cells
= 100 - [(anti-CD95 mAb-treated astrocyte
OD550/control IgM-treated astrocyte OD550) x
100].
Detection of apoptosis by cell cycle analysis
Apoptotic cells were detected by propidium iodide staining and flow cytometry as previously reported 20 . Briefly, astrocytes were fixed in 1% paraformaldehyde (Fluka) in PBS and permeabilized in 70% cold ethanol before the staining procedure. Then, cells were incubated with propidium iodide (50 µg/ml) and RNase A (50 µg/ml; Boehringer Mannheim, Indianapolis, IN) and were analyzed by flow cytometry.
Caspase activity assay
Cells were treated for 6 h (defined in Fig. 3
) and lysed in
ice-cold hypotonic solution consisting of 20 mM Tris-HCl (pH 7.2), 1 mM
EDTA (Sigma), and 10 µg/ml trypsin inhibitor (Sigma). Then,
homogenates were clarified by centrifugation at 13,000 rpm at 4°C,
and supernatants were collected and diluted in 50 mM Tris-HCl (pH 7.5),
0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic, and
10 mM DTT (Boehringer Mannheim). Enzymatic reactions were conducted in
triplicate in a total volume of 200 µl containing lysate from
105 cells and 100 µM DEVD-p-nitroaniline
(succinyl-Asp-Glu-Val-Asp-pNA) or 100 µM YVAD-pNA
(acetyl-Tyr-Val-Ala-Asp-pNA) (Alexis Corp., Laufelfingen, Switzerland)
in the presence or the absence of the specific inhibitor DEVD-CHO
(succinyl-Asp-Glu-Val-Asp-aldehyde) or YVAD-CHO
(acetyl-Tyr-Val-Ala-Asp-aldehyde; Alexis Corp.). The release of pNA was
determined by measuring the OD at 405 nm.
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One million astrocytes were lysed in ice-cold hypotonic lysis buffer as previously described 21 . Proteins were separated by SDS-PAGE under reducing conditions, then transferred to nitrocellulose. CPP32/Caspase-3 was detected with a specific mAb (mouse IgG2a, Transduction Laboratories, Exeter, U.K.) followed by peroxidase-conjugated rabbit anti-mouse mAb (Dianova, Hamburg, Germany) and visualized by chemiluminescence reaction using the ECL system (Amersham, Aylesbury, U.K.).
IL-8 production
Astrocytes obtained from mesencephalon/myelencephalon were stimulated with CH11 mAb or control IgM for 24 h. Then, supernatant was collected, and IL-8 production was measured using an ELISA kit (R & D Systems) according to the manufacturer’s instructions. Data were analyzed using Wilcoxon’s test for paired samples 22 .
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells derived from mesencephalon and myelencephalon showed typical
astrocytic morphology, with >than 90% of the cells expressing the
astrocyte-specific marker GFAP (Fig. 1,
A and B). CD95 expression was first analyzed by
RT-PCR. A PCR product of 1008 bp corresponding to the full-length CD95
cDNA and a smaller transcript of 945 bp corresponding to CD95 splicing
variant mRNA (called Fas
TM in 23 were clearly identified in
astrocytes at all passages tested (Fig. 1C). Since the
expression pattern of cell surface glycoproteins may be heterogeneous
in astrocytes isolated from different brain regions 24 , CD95 cell
surface expression was analyzed in astrocytes derived from the
mesencephalon/myelencephalon, the telencephalon, and the spinal cord.
There was a conservation of CD95 expression not only in all astrocyte
cultures tested (Fig. 1D), but also throughout the culture
period (passages 3–12) (data not shown).
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To test the outcome of CD95 ligation, the percentage of dead cells
among early passage astrocytes (<7 passages) incubated with
lytic anti-CD95 mAb or recombinant CD95L was assessed in an MTT
cell viability assay. These cells were totally resistant, even with
increasing mAb concentrations (Fig. 2) or
prolonged time of exposure (until 72 h). This resistance is an
active process, since astrocytes pretreated with cycloheximide (CHx; a
protein synthesis inhibitor that did not affect CD95 expression or
astrocyte viability; data not shown) were highly sensitive to CD95
mAb-induced death (Fig. 3
A).
After 8–10 passages in culture (late passage), astrocytes became
sensitive to anti-CD95 mAb-mediated cell death (Fig. 2). Similar
results were obtained with recombinant CD95L rather than CH11 mAb (data
not shown). However, these cells remained resistant to TNF- or
oxidative stress (data not shown).
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The same experiments using CD95-sensitive astrocytes or
CHx-treated astrocytes were repeated in the presence of caspase
inhibitors with different specificities. While YVAD-cmk preferentially
inhibits protease of the ICE/caspase-1 subfamily, DEVD-CHO is a
selective inhibitor of the CPP32/caspase-3 subfamily, and z-VAD-fmk is
an inhibitor with broader activity 11, 25 . CD95-induced death of
astrocytes was totally suppressed in the presence of z-VAD-fmk or
DEVD-CHO, but persisted with YVAD-cmk (Fig. 3A). This
suggests a preferential involvement of the CPP32/caspase-3 subfamily in
CD95 astrocyte death. To extend these data, we measured
CPP32/caspase-3-like enzymatic activity in the cell lysates of early
passage astrocytes treated under different conditions (Fig. 3B). There
was no significant cleavage of the colorimetric substrate DEVD-pNA by
lysates of either unstimulated astrocytes (data not shown) or cells
incubated with CD95 mAb or CHx plus control IgM. In contrast,
significant cleavage was observed in lysates from dying astrocytes
(i.e., those cultured in the presence of CD95 mAb and CHx), an effect
totally abrogated by DEVD-CHO but not by YVAD-CHO. The direct
involvement of CPP32/caspase-3 in the CD95-mediated death of astrocytes
was confirmed by Western blot analysis (Fig. 3C). A
colorimetric assay using ICE/caspase-1 subfamily-specific substrate
YVAD-pNA was also performed, but no activation of ICE/caspase-1 was
detected in lysates of cells pretreated with CHx and CD95 mAb (data not
shown).
CPP32/caspase-3 is responsible for proteolysis of substrates involved
in DNA repair. This cleavage contributes to nuclear alterations
associated with apoptosis 25 . Thus, apoptosis was analyzed using
propidium iodide staining and flow cytometry (Fig. 4, A–C). Significant DNA
fragmentation was found in astrocytes treated for 14 h with CD95
mAb CH11 or rCD95L in the presence of CHx. Subdiploid nuclei
(corresponding to apoptotic cells) were reduced when astrocytes were
pretreated by the CPP32/caspase-3 family-specific inhibitor DEVD-CHO
(Fig. 4C). Thus, DNA fragmentation in sensitive astrocytes
is induced after CD95 ligation and is subsequent to CPP32/caspase-3
activation.
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Possible mechanisms that may contribute to the resistance of early
passage astrocytes to CD95-mediated death were then investigated. A
first hypothesis was the secretion of a functional recombinant CD95
molecule (FasTM) that could neutralize CD95 agonists 16, 23 , since
the 945-bp transcript that codes for this form was clearly identified
in all cultured astrocytes (Fig. 1C). Supernatants collected
from early or late passage astrocytes did not protect CD95-sensitive
Jurkat cells from death (data not shown), making this hypothesis
unlikely. Another possibility was a differential expression of Bcl-2
family members that have been implicated in the control of cell death
in the astrocyte lineage 26, 27 . In the human astrocyte cultures
tested here, Bcl-2 was expressed, but there was no correlation between
expression level and CD95 resistance (Fig. 5A). The mRNA expression of
both Bcl-xL and Bcl-xS isoforms was similar in
all astrocytes tested (Fig. 5B). A particularly interesting
candidate to further test in CD95-resistant cells was FAP-1, since this
molecule directly and specifically interacts with the negative
cytoplasmic domain of CD95 17 . FAP-1 transcript was highly expressed
in CD95-resistant (early passage) astrocytes, whereas only a faint
RT-PCR signal was obtained in CD95-sensitive (late passage) astrocytes
(Fig. 5C). The present data suggest that FAP-1 mRNA
expression is correlated with astrocyte resistance to CD95-induced
death.
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In addition to apoptosis, CD95 can deliver activation signals
28, 29, 30 . Cultured astrocytes refractory to CD95-induced death were
therefore attractive candidates to test for other consequences of CD95
ligation. Basal levels of IL-8 were detected in the supernatants of
unstimulated astrocytes, as previously described 13 . CD95 ligation
using CH11 mAb significantly (p < 0.001)
enhanced IL-8 secretion (Fig. 6), with
similar results obtained using recombinant CD95L (data not shown).
Interestingly, an increase in IL-8 secretion was detected only in early
passage astrocytes (i.e., CD95 resistant), and astrocytes pretreated
with neutralizing anti-IL-8 mAb became sensitive to CD95-mediated
death (Fig. 7). This strongly suggests
that IL-8 contributes to astrocyte resistance to CD95-induced
apoptosis. Overall, these data demonstrate that the same receptor
(CD95) can mediate different cellular functions (i.e., apoptosis or
cytokine secretion) depending upon the status of the astrocytes tested.
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as a switch factor that influences the astrocyte response
to CD95
To determine whether the outcome of CD95 ligation may be
influenced by microenvironmental factors, we examined the effects of
IFN-. This cytokine was chosen because it can potentiate
CD95-mediated apoptosis in certain cell types and increase CD95
expression on the cell surface 11 . IFN- alone did not modify the
viability of astrocytes, but preincubation (100 IU/ml, 24 h)
rendered them sensitive to CD95-mediated apoptosis (Figs. 4D
and 8A). This effect was
abrogated by incubation with z-VAD-fmk (Fig. 8A). In
addition, CPP32/caspase-3 cleavage was detected by immunoblot analysis
after IFN- pretreatment and CD95 ligation (Fig. 3C),
demonstrating that this caspase is required for CD95-induced death of
IFN--treated cells. Whether the sensitizing effect of IFN- is
restricted to the CD95 pathway or is applicable to other apoptotic
stimuli to which astrocytes in culture show resistance 31, 32, 33 was
another question to address. After IFN- treatment, astrocytes
remained totally insensitive to TNF- or oxidative stress-induced
death, and only a small augmentation of ceramide-mediated death was
noted (Fig. 8B). Why IFN- preferentially operates on the
CD95 pathway is not known. An increase in cell surface CD95 expression
was observed after IFN- treatment (Fig. 8C). However, it
is unlikely that this is the sole mechanism involved, since IFN-
also induces a slight augmentation of type 1 TNF- receptor
(TNFR1/CD120a) expression (data not shown) without inducing sensitivity
to TNF--mediated death.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or
IFN- with enhanced expression of adhesion molecules 12, 36 and
acquisition of MHC class II expression, suggesting that they may
potentially function as APCs 34 . Here, we report that human cultured
astrocytes derived from different areas of the brain express
significant levels of CD95. This receptor can mediate either death via
CPP32/caspase-3 cleavage or proinflammatory cytokine release depending
upon the astrocyte activation state. The final outcome of the astrocyte
response to CD95 ligation can be influenced by microenvironmental
factors, since IFN- rendered resistant astrocytes susceptible to
CD95-mediated death. Our results are consistent with CD95 expression by
astrocytes being important in the regulation of inflammatory responses
associated with many brain diseases.
Until now, most of our understanding about the role of the CD95 system
and its signaling pathway has been obtained with cells of the immune
system. However, it is clear that information concerning one cell type
cannot be directly applied to other cells. Indeed, intracellular events
conferring resistance to CD95 triggering are cell specific and can be
modified for example after differentiation 37 . In the same way, the
second messengers responsible for the execution phase of apoptosis may
also depend upon cell type 38, 39, 40, 41 , but no information was available
to date for astrocytes. Here we show that astrocytes resistant to death
mediated by CD95 ligation express abundant FAP-1, whereas FAP-1 was
barely detectable in sensitive astrocytes. Such a correlation between
FAP-1 mRNA expression and resistance to CD95-induced apoptosis was also
recently reported in Th1 and Th2 lymphocytes 37 and in some
CD95-positive tumor cell lines 17, 18 . The present data suggest that
this mechanism also occurs in astrocytes, although FAP-1 may not be the
sole molecule involved, since FAP-1 mRNA expression was not
significantly modified after IFN- treatment (data not shown).
Furthermore, we clearly show the involvement of CPP32/caspase-3 in
CD95-mediated astrocyte death. This observation is particularly
relevant, since CPP32/caspase-3 deficient mice die at 1–3 wk of age,
with accumulation of neurons and GFAP-positive cells due to defective
cell death 42 . An increase in brain cell number has not been reported
to date for lpr or gld mice (i.e., mice with a
nonfunctional CD95/CD95L system), suggesting that multiple mechanisms
converging through CPP32/caspase-3 exist in the CNS to control
astrocyte death during development. However, MRL/lpr
mice present behavioral dysfunctions 43, 44 , suggesting a role for
the CD95 signaling pathway during CNS development.
In certain cell types, CD95 ligation delivers not only a death signal
but also activation signals 28, 29, 30 , although no data exist for
resident cells of the brain. One potentially important function of
astrocytes is the release of cytokines and chemokines, among which IL-8
is of particular interest. Indeed, IL-8 may promote inflammation 45
and angiogenesis 46 in the CNS and may also support neuronal survival
47 following brain injury. However, the activation signals augmenting
IL-8 release by astrocytes are not fully defined. Previous studies have
reported that IL-1ß and TNF-, but not LPS, induced IL-8 production
by human cultured astrocytes 13, 48 . Here, we demonstrate that CD95
ligation significantly enhanced IL-8 secretion by astrocytes. Although
the production of IL-8 through CD95 stimulation has been previously
reported for other cell types 29, 49 , implication of this cytokine in
resistance to CD95-induced apoptosis was unknown. In this study, IL-8
was identified as an astrocyte survival factor. Further investigations
currently in progress should give insights into the second messengers
of the CD95 pathway that are affected by IL-8. In the brain, the IL-8
detected during viral and bacterial infections or in the presence of
malignancies 45 may thus be the product of CD95L-stimulated reactive
astrocytes. We propose that the regulation of IL-8 secretion via the
CD95 pathway participates in the pathogenesis of many brain diseases.
The dramatic consequences induced by CD95 ligation on astrocytes
need to be considered in the context of the biological source of CD95L
in normal and pathological conditions. Trafficking leukocytes include
CD95L-expressing cells that could mediate important interactions 50 ,
particularly in the context of inflammation and tumors. Furthermore,
CNS resident cells (neurons or glial cells) may potentially express
CD95L, although contradictory results have been reported 10, 51, 52, 53, 54 .
Nevertheless, CD95 and CD95L have recently been implicated in the
pathogenesis of experimental autoimmune encephalomyelitis 55, 56, 57 . In
addition to the cellular origin of CD95L, it is important to consider
factors present in the astrocyte microenvironment that may influence
the final consequences of CD95 ligation. IFN- was an interesting
candidate to test, since this cytokine is released from activated T
lymphocytes during CNS autoimmune and infectious diseases 58, 59, 60, 61, 62 .
Moreover, recent data suggest that neurons could be an additional
source of IFN- 63 . This cytokine is also implicated in cellular
differentiation during brain development 64 . We propose that IFN-
plays a pivotal role in the functions (inflammation and apoptosis)
mediated by CD95, since astrocytes cultured in the presence of IFN-
for 24 h are switched to respond to CD95 ligation by apoptosis.
This shows that microenvironmental factors produced by neurons and
infiltrating inflammatory cells can influence the central role of CD95
in astrocyte survival.
Overall, the present findings implicate that CD95 is an important
component in the complex control of CNS inflammation. The normal brain
is protected from overwhelming and deleterious immune responses by
several passive (e.g., physical isolation by blood-brain barrier) and
active (e.g., release of immunosuppressive factors) mechanisms.
However, after brain injury certain immune responses do proceed in the
CNS, but they must be tightly regulated. We propose that CD95 expressed
by astrocytes could perform this role during inflammatory responses
that are associated with reactive gliosis. Indeed, cultured astrocytes
share phenotypic and functional characteristics with reactive
astrocytes seen in various diseases (Alzheimer’s disease, MS, stroke,
HIV infection, and tumors). Both reactive and cultured astrocytes
express high levels of GFAP and show a high proliferation index at
initial stages 5, 12, 65 . Moreover, human (reported here
and in 54 or murine 55 astrocytes in culture as well as
reactive astrocytes 66 express CD95, whereas resting astrocytes,
either by immunochemistry 66, 67 or analyzed ex vivo (data not shown)
are negative. At early stages of culture, ligation of CD95 on
astrocytes leads to enhanced secretion of IL-8. Such a mechanism could
be responsible for perpetuating inflammatory responses in vivo. The
final outcome for cultured astrocytes is death by spontaneous apoptosis
(data not shown), and indeed, reactive astrocyte apoptosis has been
reported in many brain diseases 6, 7, 8, 9, 10 , colocalizing with CD95
staining in MS lesions 10 . Inflammatory cytokines such as IFN- may
influence the response of reactive astrocytes to CD95L, since IFN-
sensitizes astrocytes to CD95 triggering. Consistent with these
findings, it has recently been reported that intracerebral injection of
IFN- limits reactive astrocyte expansion following physical brain
injury 68 . Unraveling the complexities of the CD95 pathway in
astrocytes should shed light on the phenomenon of reactive gliosis and
clarify issues concerning the control of astrocyte number in
development.
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Acknowledgments |
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Footnotes |
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2 Current address: Etablissement de Transfusion Sanguine de Franche-Comté, Laboratoire de Thérapeutique Immuno-Moléculaire, 1 bld A. Fleming, F-25020 Besançon, France.
3 Address correspondence and reprint requests to Dr. Pierre-Yves Dietrich, Laboratory of Tumor Immunology, Division of Oncology, University Hospital, rue Micheli-du-Crest 24, CH-1211 Geneva 14, Switzerland. E-mail address:
4 Abbreviations used in this paper: CNS, central nervous system; MS, multiple sclerosis; GFAP, glial fibrillary acidic protein; PE, phycoerythrin; CD95L, CD95 (Fas/Apo-1) ligand; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; YVAD-cmk, acetyl-Tyr-Val-Ala-Asp-chloromethylketone; FAP-1, Fas-associated phosphatase-1; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; DEVD-pNA, succinyl-Asp-Glu-Val-Asp-p-nitroaniline; YVAD-pNA, acetyl-Tyr-Val-Ala-Asp-pNA; DEVD-CHO, succinyl-Asp-Glu-Val-Asp-aldehyde; YVAD-CHO, acetyl-Tyr-Val-Ala-Asp-aldehyde; CHx, cycloheximide; ICE, IL-1ß-converting enzyme.
Received for publication June 18, 1998. Accepted for publication November 2, 1998.
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 D. J. Brat, A. C. Bellail, and E. G. Van Meir The role of interleukin-8 and its receptors in gliomagenesis and tumoral angiogenesis Neuro-oncol, April 1, 2005; 7(2): 122 - 133. [Abstract] [PDF]
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 J. H. Song, C. X. Wang, D. K. Song, P. Wang, A. Shuaib, and C. Hao Interferon {gamma} Induces Neurite Outgrowth by Up-regulation of p35 Neuron-specific Cyclin-dependent Kinase 5 Activator via Activation of ERK1/2 Pathway J. Biol. Chem., April 1, 2005; 280(13): 12896 - 12901. [Abstract] [Full Text] [PDF]
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T. A. Neff, R.-F. Guo, S. B. Neff, J. V. Sarma, C. L. Speyer, H. Gao, K. D. Bernacki, M. Huber-Lang, S. McGuire, L. M. Hoesel, et al. Relationship of Acute Lung Inflammatory Injury to Fas/FasL System Am. J. Pathol., March 1, 2005; 166(3): 685 - 694. [Abstract] [Full Text] [PDF]
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N. Vij, L. Roberts, S. Joyce, and S. Chakravarti Lumican Regulates Corneal Inflammatory Responses by Modulating Fas-Fas Ligand Signaling Invest. Ophthalmol. Vis. Sci., January 1, 2005; 46(1): 88 - 95. [Abstract] [Full Text] [PDF]
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 D. R Balkundi, J. A Ziegler, J. F Watchko, C. Craven, and M. Trucco Regulation of FasL/Fas in Human Trophoblasts: Possible Implications for Chorioamnionitis Biol Reprod, August 1, 2003; 69(2): 718 - 724. [Abstract] [Full Text] [PDF]
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 J. F. Sanchez, L. F. Sniderhan, A. L. Williamson, S. Fan, S. Chakraborty-Sett, and S. B. Maggirwar Glycogen Synthase Kinase 3{beta}-Mediated Apoptosis of Primary Cortical Astrocytes Involves Inhibition of Nuclear Factor {kappa}B Signaling Mol. Cell. Biol., July 1, 2003; 23(13): 4649 - 4662. [Abstract] [Full Text] [PDF]
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D. R. Park, A. R. Thomsen, C. W. Frevert, U. Pham, S. J. Skerrett, P. A. Kiener, and W. C. Liles Fas (CD95) Induces Proinflammatory Cytokine Responses by Human Monocytes and Monocyte-Derived Macrophages J. Immunol., June 15, 2003; 170(12): 6209 - 6216. [Abstract] [Full Text] [PDF]
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 P.-Y. Dietrich, P. R. Walker, and P. Saas Death receptors on reactive astrocytes: A key role in the fine tuning of brain inflammation? Neurology, February 25, 2003; 60(4): 548 - 554. [Abstract] [Full Text] [PDF]
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 Y. Osawa, M. Nagaki, Y. Banno, D. A. Brenner, T. Asano, Y. Nozawa, H. Moriwaki, and S. Nakashima Tumor Necrosis Factor Alpha-Induced Interleukin-8 Production via NF-{kappa}B and Phosphatidylinositol 3-Kinase/Akt Pathways Inhibits Cell Apoptosis in Human Hepatocytes Infect. Immun., November 1, 2002; 70(11): 6294 - 6301. [Abstract] [Full Text] [PDF]
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J. Qiu, M. J. Whalen, P. Lowenstein, G. Fiskum, B. Fahy, R. Darwish, B. Aarabi, J. Yuan, and M. A. Moskowitz Upregulation of the Fas Receptor Death-Inducing Signaling Complex after Traumatic Brain Injury in Mice and Humans J. Neurosci., May 1, 2002; 22(9): 3504 - 3511. [Abstract] [Full Text] [PDF]
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C. Choi, O. Kutsch, J. Park, T. Zhou, D.-W. Seol, and E. N. Benveniste Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Induces Caspase-Dependent Interleukin-8 Expression and Apoptosis in Human Astroglioma Cells Mol. Cell. Biol., February 1, 2002; 22(3): 724 - 736. [Abstract] [Full Text] [PDF]
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 J. A. Drisko The Use of Antioxidants in Transmissible Spongiform Encephalopathies: A Case Report J. Am. Coll. Nutr., February 1, 2002; 21(1): 22 - 25. [Abstract] [Full Text] [PDF]
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A. M. Hohlbaum, M. S. Gregory, S.-T. Ju, and A. Marshak-Rothstein Fas Ligand Engagement of Resident Peritoneal Macrophages In Vivo Induces Apoptosis and the Production of Neutrophil Chemotactic Factors J. Immunol., December 1, 2001; 167(11): 6217 - 6224. [Abstract] [Full Text] [PDF]
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C. D. Riffkin, A. Z. Gray, C. J. Hawkins, C.W. Chow, and D. M. Ashley Ex vivo pediatric brain tumors express Fas (CD95) and FasL (CD95L) and are resistant to apoptosis induction Neuro-oncol, October 1, 2001; 3(4): 229 - 240. [Abstract] [PDF]
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C. Choi, X. Xu, J.-W. Oh, S. J. Lee, G. Y. Gillespie, H. Park, H. Jo, and E. N. Benveniste Fas-induced Expression of Chemokines in Human Glioma Cells: Involvement of Extracellular Signal-regulated Kinase 1/2 and p38 Mitogen-activated Protein Kinase Cancer Res., April 1, 2001; 61(7): 3084 - 3091. [Abstract] [Full Text]
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C. Comi, M. Leone, S. Bonissoni, S. DeFranco, F. Bottarel, C. Mezzatesta, A. Chiocchetti, F. Perla, F. Monaco, and U. Dianzani Defective T cell Fas function in patients with multiple sclerosis Neurology, October 10, 2000; 55(7): 921 - 927. [Abstract] [Full Text] [PDF]
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 O. Kutsch, J.-W. Oh, A. Nath, and E. N. Benveniste Induction of the Chemokines Interleukin-8 and IP-10 by Human Immunodeficiency Virus Type 1 Tat in Astrocytes J. Virol., October 1, 2000; 74(19): 9214 - 9221. [Abstract] [Full Text]
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P. R. Walker, T. Calzascia, V. Schnuriger, N. Scamuffa, P. Saas, N. de Tribolet, and P.-Y. Dietrich The Brain Parenchyma Is Permissive for Full Antitumor CTL Effector Function, Even in the Absence of CD4 T Cells J. Immunol., September 15, 2000; 165(6): 3128 - 3135. [Abstract] [Full Text] [PDF]
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 S. Aquaro, S. Panti, M. C. Caroleo, E. Balestra, A. Cenci, F. Forbici, G. Ippolito, A. Mastino, R. Testi, V. Mollace, et al. Primary macrophages infected by human immunodeficiency virus trigger CD95-mediated apoptosis of uninfected astrocytes J. Leukoc. Biol., September 1, 2000; 68(3): 429 - 435. [Abstract] [Full Text] [PDF]
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M. D. S. Jean, C. Debbasch, M. Rahmani, F. Brignole, G. Feldmann, J.-M. Warnet, and C. Baudouin Fas- and Interferon {gamma}-Induced Apoptosis in Chang Conjunctival Cells: Further Investigations Invest. Ophthalmol. Vis. Sci., August 1, 2000; 41(9): 2531 - 2543. [Abstract] [Full Text]
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S. J. Lee, T. Zhou, C. Choi, Z. Wang, and E. N. Benveniste Differential Regulation and Function of Fas Expression on Glial Cells J. Immunol., February 1, 2000; 164(3): 1277 - 1285. [Abstract] [Full Text] [PDF]
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S. Faouzi, B. E. Burckhardt, J. C. Hanson, C. B. Campe, L. W. Schrum, R. A. Rippe, and J. J. Maher Anti-Fas Induces Hepatic Chemokines and Promotes Inflammation by an NF-kappa B-independent, Caspase-3-dependent Pathway J. Biol. Chem., December 21, 2001; 276(52): 49077 - 49082. [Abstract] [Full Text] [PDF]
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 C. Limatola, M. T. Ciotti, D. Mercanti, F. Vacca, D. Ragozzino, A. Giovannelli, A. Santoni, F. Eusebi, and R. Miledi The chemokine growth-related gene product beta protects rat cerebellar granule cells from apoptotic cell death through alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors PNAS, May 23, 2000; 97(11): 6197 - 6201. [Abstract] [Full Text] [PDF]
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T. Sugawara, M. Fujimura, Y. Morita-Fujimura, M. Kawase, and P. H. Chan Mitochondrial Release of Cytochrome c Corresponds to the Selective Vulnerability of Hippocampal CA1 Neurons in Rats after Transient Global Cerebral Ischemia J. Neurosci., November 15, 1999; 19(22): RC39 - RC39. [Abstract] [Full Text] [PDF]
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