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Departments of
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Cell Biology and
Medicine, University of Alabama at Birmingham, Birmingham, AL 35294
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or IFN-
stimulation.
Primary astrocytes express high constitutive levels of Fas, which are
not further affected by cytokine treatment. In microglia, Fas
expression is regulated at the level of mRNA expression; TNF- and
IFN- induced Fas mRNA by 
20-fold. STAT-1 and NF-
B
activation are involved in IFN-- or TNF--mediated Fas
up-regulation in microglia, respectively. The cytokine TGF-ß inhibits
basal expression of Fas as well as cytokine-mediated Fas expression by
microglia. Upon incubation of microglial cells with FasL-expressing
cells, 20% of cells underwent Fas-mediated cell death, which
increased to 60% when cells were pretreated with either TNF-
or IFN-. TGF-ß treatment inhibited Fas-mediated cell death of
TNF-- or IFN--stimulated microglial cells. In contrast,
astrocytes are resistant to Fas-mediated cell death, however, ligation
of Fas induces expression of the chemokines macrophage inflammatory
protein-1ß (MIP-1ß), MIP-1, and MIP-2. These data demonstrate
that Fas transmits different signals in the two glial cell populations:
a cytotoxic signal in microglia and an inflammatory signal in the
astrocyte. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In experimental allergic encephalomyelitis (EAE), an autoimmune inflammatory disease of the CNS, the involvement of Fas/FasL was clearly demonstrated by observations that lpr or gld mice are relatively resistant to the clinical development of EAE (14, 15). These data suggested that the Fas/FasL pathway plays a significant role in the development of EAE, possibly by mediating apoptosis of potential target cells. However, in two separate studies, oligodendrocytes, the myelin-producing cells in the CNS that are considered a major target in the disease of EAE, were resistant to FasL-mediated apoptosis and did not undergo apoptosis during the pathogenesis of EAE (16, 17). To the contrary, infiltrating CD4+ T cells and parenchymal microglia were killed by apoptosis, suggesting that the effector cells, rather than the target cells, are eliminated by apoptosis in the EAE disease process (16). More recently, the involvement of Fas/FasL in EAE progression has been dissected using the adoptive transfer model of EAE (18, 19). According to these studies, Fas expressed in the recipient animal as well as FasL expressed on donor cells are both important for the development of EAE. These data suggest that Fas expression on CNS-resident glial cells contributes to EAE induction.
In an attempt to elucidate the involvement of Fas/FasL in EAE as well
as MS pathogenesis, Fas expression and function on glial cells has been
investigated in several in vitro studies. In a study using human
oligodendrocytes, Fas ligation with anti-Fas Ab induced rapid lysis
of oligodendrocytes, supporting the idea of Fas-dependent
oligodendrocyte elimination in MS (20). However, this
Fas-dependent cell lysis did not exhibit characteristics of typical
apoptosis. High constitutive expression of Fas on mouse astrocytes has
been reported as well, but astrocyte susceptibility to FasL-mediated
apoptosis is still controversial (21, 22). In a study
using astrocyte cultures from fetal CNS, although Fas was
constitutively expressed on astrocytes, Fas ligation failed to induce
astrocyte cell death (23). In another report, Fas was
constitutively expressed by human fetal astrocytes as well as adult
astrocytes and was up-regulated by several proinflammatory cytokines
such as IL-1, IL-6, IFN-, and TNF- (24). In
addition, the fetal astrocytes underwent apoptosis when treated with
agonistic anti-Fas Ab. It has been suggested that the
susceptibility of human astrocytes to Fas-mediated apoptosis is
dependent on cell passage and other microenvironmental factors; only
late passage astrocytes are sensitive to Fas-induced apoptosis and the
presence of IFN- enhances apoptosis (25). Apoptosis in
microglia has not been well studied compared with that in astrocytes.
In several studies using immunohistochemistry in EAE brain, microglial
cells were positively stained by the TUNEL assay more frequently than
astrocytes (16). Recently, Fas-mediated apoptosis was
reported in cultured microglia upon TNF- or IFN- stimulation
(26).
In this study we have investigated the regulation and function of Fas
expression using mouse astrocytes and microglia. We examined the
influence of several pro- and anti-inflammatory cytokines (IFN-,
TNF-, TGF-ß) on Fas expression in glial cells as well as the
functional outcome of Fas ligation on these cells. Herein, we report
that Fas expression is differentially modulated by cytokines on
astrocytes and microglia, and Fas ligation transmits distinct signals
in these two glial cell types.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary glial cell cultures were established from neonatal CD1
as well as C57BL/6/lpr mouse cerebra as previously described
(27). Cells were cultured in DMEM, high glucose formula
supplemented with glucose to a final concentration of 6 g/L, 2 mM
glutamine, 0.1 mM nonessential amino acid mixture, 0.1% gentamicin,
and 10% FBS (HyClone, Logan, UT). After 2 wk in primary culture,
oligodendrocytes and microglia were separated from astrocytes by
mechanical dislodgment. Microglia-enriched cultures were obtained by
incubating the detached cells in six-well plates and changing the
medium after 1 h to remove nonadherent cells. Microglia were also
prepared from CD1 STAT-1-deficient mice (provided by Dr. David E.
Levy, New York University School of Medicine, New York, NY)
(28). Astrocyte cultures were routinely >97% positive
for glial fibrillary acidic protein, a specific marker for astrocytes,
and primary microglial cultures were >95% positive as assessed by
immunostaining for the Mac-1 surface Ag. The microglial cell line EOC13
was derived from C3H/HeJ CH-2k mice using a nonviral immortalization
procedure as previously described (29). This
CSF-1-dependent cell line is B7.1+,
Mac-1+, CD45+, and class I
MHC+ as well as phagocytic. The EOC13 cell line
was maintained in DMEM complete medium (2 mM glutamine, 10%
heat-inactivated FBS, and 20% LADMAC-conditioned medium, which
contains CSF-1) as previously described (27).
Reagents and plasmids
Recombinant murine IFN- was purchased from Genzyme (Boston,
MA), human TGF-ß1 was obtained from R&D Systems (Minneapolis, MN),
and rat TNF- was purchased from BioSource International (Camarillo,
CA). Hamster anti-mouse Fas Ab (Jo2), PE-conjugated hamster
anti-mouse Fas Ab (Jo2), hamster anti-mouse Fas ligand Ab
(MFL3), hamster anti-mouse ICAM-1 Ab (3E2), and hamster
anti-TNP IgG were purchased from PharMingen (San Diego, CA). The
NF-B inhibitor SN50, the p38 MAP kinase inhibitor SB202190, and
olomoucine were purchased from Calbiochem (San Diego, CA). The MAP/ERK
kinase (MEK) inhibitor U0126 was purchased from Promega (Madison, WI).
Murine Fas cDNA in the pcDNA 1.1 plasmid was generated by PCR as
previously described (30). The plasmid was cut with
XbaI, and the 500-bp fragment containing the C-terminal half
of mouse Fas cDNA was subcloned into pcDNA3 at an XbaI
restriction enzyme site. After linearization with HindIII,
this construct (pcDNA3-mouse Fas) was used for in vitro transcription
to generate a 420-bp antisense RNA probe. A pGEM-4Z vector containing a
fragment of mouse GAPDH cDNA (corresponding to bp 223–434) inserted at
the polylinker sites EcoRI/KpnI was linearized
with EcoRI. In vitro transcription of this plasmid with T7
RNA polymerase generated a 270-bp antisense RNA probe.
Analysis of Fas protein expression by immunofluorescence flow cytometry
Primary astrocytes were plated at 5 x
105 cells/well into six-well plates (Costar,
Cambridge, MA) and treated with IFN- or TNF- in the absence or
the presence of TGF-ß1 for various time periods. The cells were
trypsinized, washed with PBS, incubated with 20 µg/ml of
PE-conjugated anti-Fas Ab (Jo2) for 1 h at 4°C, washed, then
fixed in a final volume of 100 µl of 1% paraformaldehyde. The cells
were then analyzed on the FACScan (Becton Dickinson, Mountain View,
CA). Total fluorescence intensity was calculated as the mean
fluorescence intensity x percentage of positive cells. Negative
controls were incubated with isotype-matched Ab. For the analysis of
primary microglia or EOC13 cells, cells were treated as described
above, then scraped to detach from the well and incubated with 50 µl
of 2.4G2 supernatant (which contains rat anti-mouse FcR Ab)
supplemented with 10% normal mouse serum for 30 min at 4°C before
addition of PE-conjugated anti-Fas Ab.
RNA isolation and RNase protection assay (RPA)
Total cellular RNA was isolated from confluent monolayers of EOC13 cells or astrocytes that were incubated with cytokines or anti-Fas Ab, respectively, as previously described (31). Briefly, cells were washed once in PBS and lysed directly in the culture dish. RNA was extracted with guanidinium isothiocyanate and phenol, and precipitated with ethanol. Ten micrograms of total cellular RNA was analyzed by RPA using an RPA kit (Ambion, Austin, TX) as previously described (31). Total RNA was hybridized with both mouse Fas and GAPDH riboprobes (2.5 x 105 cpm) at 42°C overnight in 20 µl of 40 mM PIPES (pH 6.4), 80% deionized formamide, 400 mM NaOAc, and 1 mM EDTA. The hybridized mixture was treated with RNase A/T1 (1/100 dilution in 200 µl of RNase digestion buffer) at 37°C for 30 min, and then analyzed by 5% denaturing (8 M urea) PAGE. The protected fragments of the Fas and GAPDH riboprobes are 374 and 212 nucleotides in length, respectively. Quantification of protected RNA fragments was performed by scanning with the PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and values for Fas mRNA were normalized to GAPDH mRNA levels for each experimental condition. The linearized mouse chemokine multiprobe set mCK-5 (catalogue no. 45026P) was purchased from PharMingen. The mCK-5 was in vitro transcribed with T7 RNA polymerase to produce antisense chemokine RNA probes as previously described (32).
51Cr release assay
Fas-mediated microglial cell lysis was measured by the 51Cr release assay. FasL-expressing effector cells were generated by transfection of FasL cDNA-containing adenovirus vector into macrophages from Fas-deficient lpr mice as previously described (33). EOC13 cells were incubated with medium alone or different cytokines for 40 h and radiolabeled by incubation with 20 µCi of [51Cr]sodium chromate in 200 µl of DMEM containing 10% FBS at 37°C for 1 h. After washing the cells three times, 51Cr-labeled EOC cells (1 x 105) were incubated with an equivalent number of effector cells. The release of 51Cr in the supernatant was assessed 8 h later using a gamma counter. For each condition, FasL-negative macrophages were used as a negative control, 51Cr release was measured, and values were used as spontaneous release. Maximum 51Cr release was measured from each positive control in which cells are lysed in 1% Triton X-100. The percentage of specific cell death rate was calculated as (experimental 51Cr release - spontaneous release)/(maximum 51Cr release - spontaneous release).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We initially analyzed cell surface Fas expression on primary mouse
astrocytes and microglia by FACS analysis. Both cell types were
incubated in medium alone or with TNF- or IFN- for 40 h
before staining. On astrocytes, Fas is constitutively expressed and is
not significantly modulated by treatment with TNF- or IFN- (Fig. 1
). In primary mouse microglia, Fas is
expressed at a low level compared with that of astrocytes, and
expression is up-regulated upon TNF- or IFN- treatment (5-fold
induction; Fig. 1).
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and IFN-, we further investigated cytokine regulation of Fas
expression using the microglial cell line, EOC13. EOC13 is a
transformed microglial line that exhibits numerous characteristics of
primary microglia (27, 29). Surface Fas expression on
unstimulated or cytokine-stimulated EOC13 cells was regulated in a
comparable manner to that of primary microglia (Fig. 2A). Kinetic analysis
demonstrated that IFN- up-regulated Fas expression with slightly
different kinetics compared with those of TNF- (Fig. 2B).
TNF- up-regulated Fas expression by 6–12 h after stimulation,
with a peak at 36 h, whereas IFN- most actively induced Fas
within a 24- to 36-h period, with expression still increasing at
40 h (Fig. 2B).
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, lanes 1 and 7),
but was induced by TNF- or IFN- in a time-dependent manner
(lanes 2–6 and 8–12). TNF- induced
Fas mRNA expression peaked at 6 h, while IFN--induced Fas mRNA
expression was optimal at 12 h of stimulation. These data
demonstrate that Fas is regulated by the cytokines TNF- and IFN-
at the mRNA level.
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and
NF-B
TNF-- or IFN--mediated Fas up-regulation has been reported
in several other cell types (34). However, the
transcriptional regulatory mechanisms and/or the transcription factors
involved in Fas expression have been only recently reported
(35). It is well known that the IFN- receptor transmits
signals through the JAK-STAT pathway to induce transcription of various
target genes (for review, see Ref. 36). Therefore, we
investigated the involvement of STAT-1 in IFN--mediated Fas
expression using microglia from STAT-1-deficient mice. As shown in
Fig. 4A, IFN--induced Fas
expression was severely impaired in STAT-1-deficient mouse
microglia, while TNF- mediated Fas up-regulation was intact,
demonstrating that STAT-1 is critical for IFN- mediated Fas
induction. TNF- binding to its receptor rapidly activates the
transcription factor NF-B in many cell types (for review, see Ref.
37). To further test the involvement of NF-B activation
in TNF--induced Fas expression, the activation of NF-B was
suppressed using the specific inhibitor, SN50. This oligopeptide
contains the nuclear localizing signal found in NF-B and
specifically blocks nuclear translocation of NF-B (38).
SN50 inhibited TNF--induced Fas mRNA expression by 60%, while
having no effect on IFN--mediated Fas expression (Fig. 4B). These data clearly demonstrate that NF-B activation
is required for TNF--mediated Fas expression in microglial
cells.
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Previous studies from our laboratory have shown that TGF-ß
exerts a strong anti-inflammatory effect on microglia by
antagonizing the effect of proinflammatory cytokines such as TNF-
and IFN-. Hence, we tested the effect of TGF-ß on cytokine-induced
Fas expression on EOC13 cells. FACS analysis demonstrated that TGF-ß
slightly inhibited the constitutive expression of Fas. In addition,
TGF-ß inhibited TNF--mediated Fas expression by 60% and
IFN--mediated Fas expression by 70% in EOC13 cells (Fig. 5). We also tested the inhibitory effect
of TGF-ß on Fas expression in primary mouse microglia and found
comparable inhibitory effects (data not shown). Regulation of Fas mRNA
expression was also tested upon TGF-ß stimulation (Fig. 6). TGF-ß alone had no effect on Fas
mRNA expression (lane 4), whereas it inhibited
TNF--induced Fas mRNA expression by 50% (compare lanes
2 and 5) and IFN--induced Fas expression by 45%
(compare lanes 3 and 6). These data demonstrate
that the inhibitory effect of TGF-ß on Fas expression is mediated at
the mRNA level.
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Fas transmits apoptotic signals into susceptible target cells
(39). We tested whether the Fas receptor on the surface of
microglia could induce cell death upon ligation with FasL.
FasL-expressing macrophage cells from the lpr mouse that
were transiently transfected with FasL cDNA were used. This cell line
expresses FasL on the cell surface and induces apoptosis in Fas-bearing
target cells (33). EOC13 cells were labeled with
51Cr and incubated with either
FasL+ lpr macrophages or
FasL-untransfected control cells, then 51Cr
released in the medium was measured. Without any stimulation, 20%
of EOC13 cells underwent Fas-mediated cell death upon Fas ligation.
This cell death rate increased to 60% when cells were stimulated
with either TNF- or IFN- (Fig. 7A), indicating that TNF-
and IFN- potentiate Fas-mediated cell death in EOC13 cells. This
cytokine-mediated potentiation of microglial cell death was almost
completely inhibited when cells were incubated with TGF-ß (Fig. 7A). In addition, incubation with TGF-ß alone inhibited
the basal level of cell death of EOC13 cells (Fig. 7A). The
specificity of cell death was demonstrated by addition of Abs specific
for Fas or FasL in the coculture. Abs against either Fas or FasL
completely blocked the potentiation of cell death by TNF-
stimulation (Fig. 7B). Likewise, the IFN--induced cell
death was completely blocked by Abs against Fas or FasL (data not
shown). However, neither anti-ICAM-1 nor isotype control Ab
(hamster IgG) inhibited Fas-induced cell death (Fig. 7B).
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Recently, several studies have reported that Fas can transmit
inflammatory signals depending on the cell type (25, 40).
Because primary mouse astrocytes were completely resistant to
Fas-mediated cell death in the 51Cr release assay
(data not shown), we examined whether Fas on astrocytes has an
alternative function, i.e., transduction of inflammatory signals.
Specifically, we examined whether Fas ligation on astrocytes affected
chemokine expression (Fig. 8). Astrocytes
incubated with anti-mouse Fas Ab (Jo2) for 1 h up-regulated
the mRNA expression of MIP-1ß by 3-fold, MIP-1 by 2-fold,
and MIP-2 by 2-fold (Fig. 8A, lane 2) compared
with that in the control sample (lane 1) in which
cells were incubated with isotype-matched Ab. The addition of
anti-hamster IgG Ab (as a secondary Ab) with anti-Fas Ab did
not further enhance anti-Fas Ab-induced chemokine expression
(lane 4), suggesting that anti-Fas Ab alone is
sufficient to transduce inflammatory signals. As well, the addition of
secondary Ab alone did not affect chemokine expression
(lane 3). Considering the abundant expression of the
proinflammatory cytokine TNF- in the CNS during EAE pathogenesis
(41), it is likely that Fas transmits signals in the
presence of TNF- during the disease process. To test possible
concerted effects of Fas-mediated signals and TNF-, we stimulated
cells with anti-Fas Ab in the absence or the presence of TNF-
(Fig. 8B). TNF- alone induced mRNA expression of MIP-1
and -ß by about 2-fold (lane 3). Furthermore, in
the presence of TNF-, cross-linking of Fas resulted in a synergistic
effect on MIP-1ß expression (7-fold induction) and an additive
effect on MIP-1 expression (4-fold induction; lane 4).
Possible additive/synergistic effects between Fas cross-linking and
TNF- on MIP-2 expression were not obvious, because TNF--induced
MIP-2 expression was so strong (Fig. 8B, lane 3).
To test the specificity of Fas signaling, we used primary astrocytes
isolated from lpr mice that are deficient in functional Fas
expression (Fig. 8C). Although TNF--mediated chemokine
induction was intact (lane 3),
anti-Fas Ab-induced chemokine expression was completely abolished
in lpr astrocytes (compare lanes 1 and
2). As well, addition of anti-Fas Ab plus TNF- showed
neither a synergistic nor an additive effect on chemokine expression in
lpr astrocytes compared with TNF- alone
(lanes 3 and 4). These data clearly
demonstrate that anti-Fas Ab generates an inflammatory signal
through binding to the Fas molecule on astrocytes. To test whether this
Fas-mediated chemokine induction is astrocyte-specific, we incubated
microglial cells with anti-mouse Fas Ab (Jo2) for 1 h (Fig. 8D). Unlike astrocytes, EOC13 cells constitutively express
MIP-1 and MIP-1ß mRNA at high levels, and Fas ligation did not
further enhance expression (compare lanes 1 and
2). Because microglia constitutively express low levels of
Fas, we stimulated EOC13 cells with TNF- for 40 h to enhance
Fas expression, then subjected these cells to Fas ligation. As shown in
Fig. 8D, TNF- stimulation enhances the expression of
MIP-1ß, MIP-1, and MIP-2 (compare lanes 1 and
3); however, Fas ligation did not affect expression of
MIP-1ß, MIP-1, or MIP-2 mRNA expression (compare lanes
3 and 4). These data show that the Fas-mediated
inflammatory signal occurs in astrocytes, but not in microglial cells.
Finally, we investigated the possible signal transduction pathways
involved in this Fas-mediated inflammatory signal using pharmacological
inhibitors (Fig. 8E). Among several kinase inhibitors that
we tested, U0126, a specific inhibitor of MEK (42), most
potently inhibited anti-Fas Ab-induced chemokine mRNA expression; a
near-complete inhibitory effect was observed at 10 µM U0126
(lanes 3–5). Olomoucine, a cyclin-dependent kinase
inhibitor as well as a weak inhibitor of extracellular signal-related
kinase-1 (ERK-1), and SB202190, a specific p38 MAP kinase inhibitor,
showed only moderate inhibitory effects at concentrations of 50 µM
(lanes 6–11). MEK is a serine/threonine kinase that
phosphorylates the MAP kinases ERK-1 and -2 (43). Taken
together, these data suggest that cross-linking of Fas on astrocytes
signals through the MAPK pathway, probably by ERK activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
IFN-. Fas is also highly expressed on human fetal astrocytes and is
not modulated by IFN- stimulation (23). However, our
data differ from the report by Choi et al. (24) in which
they could up-regulate Fas expression on human fetal astrocytes by
TNF- stimulation and less so by IL-1ß or IL-6. We did not detect
any increase in Fas expression on murine astrocytes by TNF-; this
suggests a species-specific differential sensitivity of astrocytes to
TNF- stimulation of Fas expression.
In distinction to astrocytes, microglia express low basal levels of
Fas, which are further up-regulated by TNF- or IFN-. The RPA data
demonstrate that Fas expression is regulated by cytokines at the mRNA
level. Interestingly, we observed that cytokine induction of Fas mRNA
expression is higher than that of Fas protein expression, suggesting
the existence of possible regulatory mechanisms at the
post-transcriptional and/or translational level. Studies using
STAT-1-deficient microglia and the specific NF-B inhibitor SN50
prove that STAT-1 and NF-B activation are required for IFN--
and TNF--mediated Fas induction in microglia, respectively. In HeLa
cells, CCAAT/enhancer-binding protein ß (C/EBPß) is responsible for
influenza virus-infection mediated Fas up-regulation (44).
In addition, NF-B and C/EBP consensus elements are suggested to be
involved in rat Fas expression (45). Recently, STAT-1
involvement in IFN--induced Fas expression in the human colon
carcinoma cell line HT29 was reported (35). To date, the
transcriptional regulatory mechanism of Fas expression by TNF- has
not been reported. Within a 1-kb region of the mouse Fas promoter, five
NF-B consensus elements have been found (46). Further
investigation of which NF-B element(s) is critical for TNF-
induction of Fas and which isoforms of NF-B are induced to bind to
the elements is warranted. IFN- activation of STAT-1 leads to
STAT-1 binding to IFN- activation sequence (GAS) elements in the
promoter regions of IFN- inducible genes (for review, see Ref.
36). Interestingly, a GAS element is not present in the
1-kb promoter region of the mouse Fas gene (46).
Considering the relatively late induction of Fas mRNA by IFN-, it is
possible that additional transcription factors are induced/activated by
STAT-1 to ultimately transcribe Fas. As well, a GAS element may be
present in other regions of the Fas promoter.
In our study of the function of Fas expression on microglial cells, we
observed that incubation of microglia with FasL-bearing cells induced
cell death. This Fas-mediated microglial cell death was further
increased by TNF- or IFN- stimulation, consistent with a previous
report (26). Interestingly, microglial cell death was not
observed when cells were incubated with anti-Fas Ab alone or with
anti-hamster IgG Ab as a secondary Ab (data not shown). These
results suggest that engagement of Fas by membrane-bound FasL is
necessary for subsequent cell death. Although the
51Cr release assay used in our study does not
differentiate cell death through apoptosis vs necrosis, in conjunction
with data from previous reports (16, 26), our data argue
that activated microglia undergo Fas-mediated apoptosis by FasL-bearing
cells.
Furthermore, we have found that TGF-ß exerts a strong antiapoptotic
effect by inhibiting surface Fas expression on microglia. TGF-ß has
differential inhibitory mechanisms on the apoptotic process depending
on the cell type under study. TGF-ß protects dendritic cell
precursors from apoptosis by reducing Fas expression (47).
In contrast, TGF-ß decreases apoptosis of human T cells by inhibiting
FasL expression, with no apparent effect on Fas expression
(48). These findings in conjunction with the results from
this study indicate that TGF-ß uses different pathways to inhibit the
apoptotic process. It has been reported that TNF- or IFN-
potentiate microglial cell death partly by down-regulating
antiapoptotic molecules, such as Bcl-2 and Bcl-xL
(26). In this regard, we tested the effect of TGF-ß on
mRNA expression of Bcl family members, including antiapoptotic members
(Bcl-w, Bcl-xL, and Bcl-2) as well as
proapoptotic members (Bak, Bax, and Bad). In these experiments TGF-ß
slightly down-regulated mRNA expression of all of the above-mentioned
Bcl family members (data not shown), indicating that TGF-ß-mediated
inhibition of microglial cell death does not involve down-regulation of
proapoptotic molecules or up-regulation of antiapoptotic molecules.
Rather, TGF-ß inhibits microglial cell death by inhibiting surface
Fas expression.
TGF-ß is a strong immunosuppressive cytokine, and its expression is
elevated in various diseases, including MS (49). In the
CNS, astrocytes, microglia, and oligodendrocytes can be activated to
express TGF-ß (50, 51, 52). In EAE, TGF-ß expression is
prominent during the recovery phase (53) and has been
proposed to contribute to disease recovery (54, 55). The
immunosuppressive function of TGF-ß on microglia has been reported in
our laboratory as well as others. TGF-ß inhibits IFN--induced
class II MHC and CD40 expression by microglia (27, 56, 57). As well, TGF-ß inhibits TNF-, IL-1, IL-6, and IL-12
production by microglia (56, 58). These results in
conjunction with our findings that TGF-ß inhibits cell death of
microglia suggest that TGF-ß expression at the recovery phase of EAE
can protect microglia from cell death as well as inhibit their ability
to function as an APC within the CNS. Given that microglia can exert
protective effects in the CNS, such as neurotropin production
(59, 60) and regeneration of neurons (61),
preservation of these cells may be viewed as beneficial at certain
stages of disease.
Compared with microglia, the function of Fas molecules on astrocytes has been controversial. In one report mouse astrocytes were shown to be susceptible to Fas-mediated apoptosis in vitro (17). Apoptotic astrocytes have been detected in the white matter of Theiler’s virus-infected mice as well (62). However, no apoptotic astrocytes have been detected in brains from EAE-induced mice (16). In our study we could not detect astrocyte cell death based on the 51Cr release assay (data not shown). It is not clear why astrocytes, which express high constitutive levels of Fas, are resistant to Fas-mediated cell death. Recently, it was proposed that susceptibility to Fas-mediated apoptosis of human astrocytes varies depending on the cell passage state and also on microenvironmental factors, such as the presence of cytokines (25). In that study the resistance of early passage fetal astrocytes to Fas-mediated apoptosis correlated with the expression of Fas-associated phosphatase-1 (FAP-1), a tyrosine phosphatase interacting with the C-terminal region of Fas (63). FAP-1 inhibits Fas-mediated apoptosis upon overexpression and is highly expressed in tissues and cell lines that are resistant to Fas-mediated apoptosis (64). Interestingly, we detected mRNA expression of FAP, a mouse homologue of human FAP-1, in mouse astrocytes but not in microglia (data not shown), which may explain the relative resistance of astrocytes to Fas-mediated cell death compared with microglia.
Based on our in vitro data as well as the in vivo data reported by
Bonetti et al. (16), we argue that mouse astrocytes do not
undergo Fas-mediated apoptosis during EAE pathogenesis. To the
contrary, we propose that Fas expressed on astrocytes can facilitate
inflammatory responses by inducing the expression of chemokines such as
MIP-1, MIP-1ß, and MIP-2. MIP-1 and -ß are members of the C-C
chemokine family that function as chemoattractants primarily for
monocytes and T lymphocytes (for review, see Ref. 65).
MIP-2 is a functional mouse homologue of human IL-8, a potent
chemoattractant for neutrophils (for review, see Ref. 65).
The importance of chemokine expression in EAE induction has been
previously reported (for review, see Ref. 65). In the
mouse model of EAE, production of MIP-1 in the CNS correlated with
development of severe clinical disease, and administration of
anti-MIP-1 Ab inhibited EAE induction (66).
Similarly, MIP-1ß expression was detected in rat brain upon EAE
induction (67). It has been noted that CNS infiltration of
activated immune cells is ameliorated in EAE-induced lpr
mice (14). Considering the critical role of chemokines in
recruitment of activated immune cells to the CNS, it is quite possible
that the lack of Fas-mediated induction of chemokines by astrocytes is
partly responsible for the resistance of lpr mice to EAE
induction. Furthermore, we found that Fas-mediated signaling exerts a
synergistic effect with TNF- for MIP-1ß mRNA expression. This
suggests that during EAE pathogenesis, when TNF- expression in the
CNS is prominent, signaling through Fas on astrocytes can enhance
MIP-1ß expression. In our study we could not detect MIP-1ß
expression at the protein level due to the lack of a sensitive mouse
MIP-1ß detection system. However, in another study we have found that
Ab cross-linking of Fas on human astrocytes can induce inflammatory
chemokine and cytokine expression at both the mRNA and protein levels
(C. Choi, X. Xu, J. W. Oh, S. J. Lee, and E. N.
Benveniste, manuscript in preparation). To date, CNS-infiltrating
activated T cells, microglia, and astrocytes have been reported to
express FasL (16, 68, 69). However, it is not clear which
cell type(s) contributes to the ongoing inflammatory response by
engaging Fas on astrocytes in vivo. A preliminary analysis of the
signaling cascades activated by Fas ligation on astrocytes suggests
that the MAPK pathway is used, based on the fact that U0126, a specific
inhibitor of MEK, abrogates Fas-induced chemokine expression. It has
been reported that Daxx is involved in Fas-induced c-Jun N-terminal
kinase activation (70). Activation of MAP kinase kinase
kinase-1 by caspase-3 upon Fas ligation was documented
(71). However, Fas-induced MEK activation has not been
reported. In this regard, it will be interesting to test whether Daxx
and MAP kinase kinase kinase-1 are involved in Fas-mediated MEK
activation in astrocytes. Future studies on the signaling pathways
activated through Fas on astrocytes may enable us to interrupt
Fas-facilitated inflammatory responses in the CNS.
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Acknowledgments |
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deficient mice, and Dr. William Walker (St. Jude Children’s Research
Hospital, Memphis, TN) for the EOC13 cell line. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Etty N. Benveniste, Department of Cell Biology, 350 MCLM, University of Alabama at Birmingham, Birmingham, AL 35294-0005. E-mail address:
3 Abbreviations used in this paper: FasL, Fas ligand; DISC, death-inducing signaling complex; EAE, experimental allergic encephalomyelitis; MIP, macrophage inflammatory protein; MS, multiple sclerosis; MAPK, mitogen-activated protein kinase; RPA, RNase protection assay; ERK, extracellular signal-related kinase; MEK, MAP/ERK kinase; FAP-1, Fas-associated phosphatase-1.
Received for publication May 3, 1999. Accepted for publication November 22, 1999.
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