Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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We have
developed a cellular model in which cultured astrocytes and brain
capillary endothelial cells preconditioned with tumor necrosis
factor- (TNF-
) fail to upregulate intercellular adhesion
molecule-1 (ICAM-1) protein (80% inhibition) and mRNA (30%
inhibition) when challenged with TNF-
or exposed to hypoxia. Inasmuch as ceramide is known to mediate some of the effects of TNF-
, its levels were measured at various times after the TNF-
preconditioning. We present evidence for the first time that, in normal
brain cells, TNF-
pretreatment causes a biphasic increase of
ceramide levels: an early peak at 15-20 min, when ceramide levels
increased 1.9-fold in astrocytes and 2.7-fold in rat brain capillary
endothelial cells, and a delayed 2- to 3-fold ceramide increase that
occurs 18-24 h after addition of TNF-
. The following findings
indicate that the delayed ceramide accumulation results in cell
unresponsiveness to TNF-
: 1)
coincident timing of the ceramide peak and the tolerance period,
2) mimicking of preconditioning by
addition of exogenous ceramide, and
3) attenuation of preconditioning by
fumonisin B1, an inhibitor of
ceramide synthesis. In contrast to observations in transformed cell
lines, the delayed ceramide increase was transient and did not induce
apoptosis in brain cells.
ceramide; intercellular adhesion molecule-1; astrocytes; brain
endothelial cells; ischemic tolerance; tumor necrosis
factor-
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INTRODUCTION |
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VARIOUS SUBLETHAL STRESS conditions such as oxidative stress (43), radiation (48), or heat (36) induce an adaptive response that results in cell tolerance to a subsequent challenge that would otherwise be lethal. The best-studied stress adaptation phenomenon in mammals, ischemic preconditioning, is well documented in animal models of ischemic injury of brain and heart (for reviews see Refs. 6 and 51, respectively) and of other organs as well (21, 35).
Adaptation to one type of stress can be achieved even by preconditioning with a different type of stress. For example, heat stress preconditioning (23) or pretreatment with chemicals causing oxidative stress (55) has been found to confer protection against ischemia-reperfusion injury of the myocardium. Preconditioning exposure to global brain ischemia can protect against focal ischemia and vice versa (39). These observations suggest a common denominator underlying protective mechanisms.
One candidate mediator of the stress adaptation reaction is a
pleiotropic cytokine, tumor necrosis factor- (TNF-
). TNF-
is
known as a key contributor to cell dysfunction and death in many
pathological conditions including brain ischemia (8). In brain,
TNF-
is released early after trauma and ischemia-reperfusion injury (1) and triggers expression of adhesion molecules, activation of
inducible nitric oxide synthase, and release of cytokines, thus
contributing to leukocyte extravasation and free radical production,
key elements of the inflammatory reaction (18). Under certain
conditions, however, TNF-
has been shown to protect animals from
radiation (33), hyperoxia (47), endotoxic shock (25), oxidative stress
(50), and stroke (30). Recent studies in murine and rat models of brain
ischemia demonstrate that TNF-
can substitute for ischemic
preconditioning when applied intracisternally (32) or elicited
systemically via injection of lipopolysaccharide (42) 48-72 h
before ischemic insult. However, signal transduction pathways that
regulate TNF-
preconditioning have not been elucidated.
The goal of this work was to study molecular events triggered by
TNF- preconditioning of brain capillary endothelial cells and
astrocytes. One of the best-studied effects of TNF-
in
endothelial cells (41) and astrocytes (2) is upregulation of expression of adhesion molecules. Among the many cell adhesion ligands,
intercellular adhesion molecule-1 (ICAM-1) is an important mediator of
critical steps in leukocyte transmigration through the endothelium and accumulation at the site of injury. Animal models demonstrate a
substantial increase of ICAM-1 mRNA at the site of infarction as early
as 3 h after onset of permanent ischemia (46). Injection of
monoclonal antibodies directed against ICAM-1 has been shown to
decrease infarct size in models of transient focal ischemia (7). On the basis of these observations, ICAM-1 expression was chosen
as the biological readout of TNF-
-induced activation of cultured
astrocytes and capillary endothelium as we developed conditions in
which TNF-
pretreatment causes unresponsiveness to its own action in
these cells.
Ceramide, a product of sphingomyelin hydrolysis, has been implicated as
a second messenger in many of the multiple signaling pathways initiated
on TNF- binding to its p55 receptor (15). Although most of the
studies of ceramide focus on its role in TNF-
-triggered apoptosis
and cell cycle control (for recent reviews see Refs. 34 and 40), there
is also evidence that ceramide can cause cytoprotection (17). These
observations suggested that TNF-
-induced preconditioning of brain
cells could be mediated via ceramide. To test this hypothesis, we
measured intracellular ceramide levels after TNF-
pretreatment in
cultured astrocytes and rat brain capillary endothelial cells (RBEC).
We present evidence for the first time that in these differentiated
cells TNF-
causes a biphasic increase of ceramide levels similar to
that observed in TNF-
-sensitive tumor cell lines, but in contrast to
tumor cells, the delayed ceramide release in astrocytes and RBEC is transient and subsides after 4-6 h, causing no apoptosis but
resulting in cell unresponsiveness to subsequent activation by TNF-
.
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METHODS |
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Cell culture.
RBEC cultures were prepared from adult Wistar-Kyoto rat brains, as
previously described for similar human cultures (41), except fetal
bovine serum was substituted for human serum and heparin (90 µg/ml)
was added to the medium. The purity of the RBEC was >95%, as
determined by positive immunostaining for von Willebrand factor (factor
VIII) and angiotensin-converting enzyme, incorporation of acetylated
low-density lipoprotein, and negative staining for glial cells (glial
fibrillary acidic protein, galactocerebroside, ED-2), muscle cells
(-actin), and pericytes (tropomyosin). Four cell cultures at
passages 7-12 derived from four
different brains were used.
TNF- preconditioning and treatment.
Astrocytes and RBEC cultures were incubated for 4 h with 50 and 20 ng/ml rat recombinant TNF-
(Chemicon International, Temecula, CA),
respectively, in their culture media, washed, allowed to rest in
respective media without TNF-
for 20 h, and then activated again
with the same doses of TNF-
for 24 h.
Hypoxic treatment.
RBEC placed in modular incubator chambers (Billups Rothenberg, Del Mar,
CA) were first flushed with 5%
CO2-95%
N2 for 15 min, then sealed and
incubated at 37°C for 20 h and reoxygenated for 24 h.
O2 tension at the end of hypoxic
treatment was ~30 Torr (3%). For preconditioning, RBEC cultures were
incubated for 4 h with TNF- (20 ng/ml), and TNF-
was washed out
immediately before the beginning of hypoxic treatment.
Ceramide studies.
Astrocytes (100-mm culture dish; 2 × 106) or RBEC (60-mm
culture dish; 2 × 106)
were treated with TNF- for indicated periods of time. At the end of
each incubation, cells were washed twice with cold PBS, scraped off
into Eppendorf tubes, and microcentrifuged. Cell pellets were stored at
70°C. For lipid extraction, cells were transferred to 13 × 100-mm borosilicate screw-cap tubes, and lipids were extracted with 3 ml of 2:1 MeOH-CHCl3, as
described by Dbaibo et al. (9). The samples were centrifuged at 480 g for 10 min, the organic phase was
collected and evaporated under N2,
and the lipids were dissolved in 1 ml of
CHCl3. An aliquot of 100 µl was
set aside for lipid phosphate measurements.
Quantitation of cell apoptosis. Apoptosis was measured by means of a fluorescent, cell-permeable, DNA-binding dye, Hoechst-33342. Hoechst fluorescence on binding to DNA is inversely proportional to the degree of DNA degradation by DNase. On the basis of these characteristics of Hoechst-33342, a novel microtiter plate fluorescence assay for DNA fragmentation in the cells undergoing apoptosis has been developed and described elsewhere (16). In addition, we used the ethidium homodimer fluorescence exclusion test to confirm plasma membrane integrity and to identify necrotic cells, inasmuch as a prominent feature of apoptosis is cell shrinkage, without development of a leaky cellular membrane, whereas DNA fragmentation alone could result from nonapoptotic DNA damage. The third dye, calcein, was employed to estimate a possible cell loss resulting from the detachment of dead cells. Briefly, sister cultures of astrocytes or RBEC, plated in 96-well microtiter plates, were incubated with either 25 µM Hoechst-33342, 4 µM ethidium homodimer, or 1 µM calcein solutions in Hanks' balanced salt solution (all 3 dyes were from Molecular Probes, Eugene, OR), which were added at 100 µl/well for 45 min at 37°C. Cell fluorescence was measured using a CytoFluor 4000 fluorescent plate reader (PerSeptive Biosystems, Framingham, MA) at excitation/emission wavelengths of 360/460, 530/620, and 485/530 nm for Hoechst, ethidium homodimer, and calcein, respectively. Background fluorescence was measured on each plate and subtracted. The fluorescence signal measured on Hoechst- and ethidium iodide-labeled cultures was normalized to that of calcein-stained sister cultures. The percentage of apoptotic cells was calculated from Hoechst fluorescence by means of the following formula
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Flow fluorocytometric analysis.
Naive and TNF--pretreated cell cultures of RBEC and astrocytes were
activated with TNF-
for 24 h, trypsinized, and incubated with 10 µg/ml anti-rat ICAM-1 monoclonal antibody (Endogen, Woburn, MA) for 1 h at 4°C, washed, and stained with 4 µg/ml fluorescein-labeled fluorescein-conjugated goat anti-mouse IgG secondary antibody (Accurate
Chemicals and Scientific, Westbury, NY). Cells were fixed with 1%
paraformaldehyde and analyzed by means of a flow cytometer (FACScan,
Becton-Dickinson, San Jose, CA).
Fluorescent cell ELISA.
ICAM-1 expression was quantitated as previously described (4). Cells
were fixed with 1% paraformaldehyde, and then the following
incubations were performed at room temperature for 1 h each: first with
anti-rat ICAM-1 monoclonal antibody at 1 µg/ml, then with
biotinylated horse anti-mouse IgG, rat adsorbed (Vector, Burlingame,
CA), at 10 µg/ml, then with streptavidin -galactosidase (Molecular
Probes) at 10 µg/ml, and finally with
fluorescein-di-
-D-galactopyranoside (Molecular Probes). Fluorescence was measured in a CytoFluor 4000 fluorescent plate reader at an excitation/emission wavelength of
485/530 nm. Background fluorescence of the cells stained without anti-ICAM-1 antibody or with the isotype-matched control was measured and subtracted in each experiment. The results are presented as the
average of 10 wells.
RNA isolation and Northern blotting.
Total RNA was extracted with STAT-60 (Tel-Test "B," Friendswood,
TX) according to the manufacturer's instructions. RNA samples (15-µg
aliquots) were electrophoresed through a 1.2% (wt/vol) agarose gel
containing 5.4% (vol/vol) formaldehyde, transferred to a
nitrocellulose membrane (GIBCO), and baked in a vacuum oven for 2 h at
80°C. Prehybridization was performed at 42°C with Digene Fast
Pair Reagent. Hybridization was carried out at 42°C for 24 h by
adding to the prehybridization solution
[32P]DNA probes for
ICAM-1 (cDNA probe template for rat ICAM-1 mRNA was a generous gift
from Dr. Donald Anderson, Pharmacia). Cyclophilin probe was obtained
by random priming. The blots were then washed (0.1×
saline-sodium phosphate-EDTA-0.1% SDS at 45°C), dried, and quantitated using a PhosphorImager. Values for ICAM-1 mRNA were adjusted for background and normalized to cyclophilin mRNA. For nuclear
runoff analysis, cells were activated with TNF- for 2 h, then
transcription was stopped by addition of 5 µg/ml actinomycin D and
RNA was purified at different times.
Statistical analysis. Statistical analysis was carried out by two-factor ANOVA with replication and by paired t-test.
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RESULTS |
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TNF- pretreatment makes cells unresponsive to
subsequent activation by TNF-
and
hypoxia-reoxygenation.
FACS analysis showed that exposure of RBEC and astrocytes to TNF-
for 24 h caused a significant, twofold increase of surface ICAM-1
expression (Fig. 1). However,
when cells were pretreated with TNF-
for 4 h, washed, and 20 h later
challenged again with TNF-
, 83% and 92% inhibition of ICAM-1
induction was demonstrated in RBEC and astrocytes, respectively (Fig.
1, B and
D). Similar results were obtained
using ELISA (Table 1,
experiment A), demonstrating that
TNF-
pretreatment caused 81% and 74% inhibition of ICAM-1 induction in RBEC and astrocytes, respectively. The 4-h pretreatment of
astrocytes and RBEC with TNF-
did not cause upregulation of ICAM-1
expression (data not shown).
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TNF- pretreatment inhibits
TNF-
-induced ICAM-1 transcription.
The effect of TNF-
pretreatment was also studied at the level of
transcription. RBEC and astrocytes were pretreated with TNF-
for 4 h, and 20 h later they were treated again with TNF-
, as described
for the ICAM-1 protein quantitation. RNA was purified 2 h after the
second TNF-
addition, because preliminary experiments demonstrated
that maximum accumulation of ICAM-1 mRNA in naive and
TNF-
-pretreated RBEC and astrocytes occurred at this time. ICAM-1
mRNA levels in TNF-
-pretreated RBEC (Fig.
2, A and
C) and astrocytes (Fig. 2,
B and
D) were inhibited by 31.4% and
31.6%, respectively (the level of inhibition of the message was
significantly less than the inhibition of protein synthesis, suggesting
that, in addition to transcription, preconditioning affects
postranscriptional events). Comparison of the kinetics of ICAM-1 mRNA
degradation in control and preconditioned RBEC activated for 2 h with
TNF-
and then treated with the transcription inhibitor actinomycin D demonstrated that TNF-
preconditioning did not affect
ICAM-1 mRNA stability (Fig. 2E).
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Effect of exogenous ceramide on brain cell response to
TNF-.
Because ceramide has been implicated as a second messenger in many of
the TNF-
signaling pathways, we sought to investigate whether
inhibition of ICAM-1 expression in TNF-
-pretreated cells was
mediated by ceramide. Astrocyte and RBEC cultures were treated with the
cell-permeable ceramide analog
N-acetylsphingosine (C-2 ceramide).
Substitution of C-2 ceramide pretreatment (10 µM for 4 h, 20 h before
TNF-
activation) for TNF-
preconditioning neither produced
tolerance to subsequent TNF-
treatment nor upregulated ICAM-1
expression in control cultures of astrocytes and RBEC (data not shown).
When exogenous C-2 ceramide was added to RBEC 30 min before or even 60 min after TNF-
activation, however, an induction of ICAM-1 mRNA was
inhibited in RBEC (Fig. 3,
A and
C) and astrocytes (Fig. 3,
B and
D). The inhibitory effect of C-2
ceramide on ICAM-1 induction in RBEC and astrocytes has been confirmed
on the protein level as well (Table 1, experiment
C). The extent of inhibition of ICAM-1 achieved by
C-2 ceramide was comparable to that produced by TNF-
preconditioning, as documented by Northern blotting and cell ELISA.
Addition of C-2 ceramide 60 min after TNF-
inhibited transcription
of ICAM-1 mRNA by 39.5% and 49.3% in RBEC and astrocytes, respectively (Fig. 3, C and
D), and inhibited ICAM-1 protein
induction by 73.9% and 75.5% in RBEC and astrocytes, respectively
(Table 1, experiment C).
Biologically inactive C-2 dihydroceramide had no effect on ICAM-1
protein expression (tested in FACS assay and ELISA) or ICAM-1 mRNA
synthesis induced by TNF-
(data not shown). C-2 ceramide had a
dose-dependent inhibitory effect on ICAM-1 transcription, which reached
a maximum at 5-10 µM (Fig.
3E). As was noted with TNF-
preconditioning, C-2 ceramide had no effect on ICAM-1 mRNA stability
(Fig. 3F).
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TNF- induces biphasic ceramide release in primary
cultures of astrocytes and RBEC.
The observation that C-2 ceramide exerted its inhibitory effect only
when added at about the same time as TNF-
suggested that if ceramide
mediates the preconditioning effect of TNF-
, its levels should be
elevated near the time of the second TNF-
stimulation, i.e., ~24 h
after the start of the preconditioning treatment. Such late ceramide
release has been previously described in tumor cell lines (22, 43). To
test this possibility, ceramide concentrations were measured at
different times out to 30 h after TNF-
addition. In both types of
cells, two peaks of ceramide concentration were observed. The first
peak occurred between 15 and 20 min, reaching a level 2.7-fold over
baseline in the RBEC and 1.9-fold over baseline in the astrocytes (Fig.
4A). The
second peak occurred much later, between 18 and 24 h after addition of TNF-
, and then quickly subsided (Fig. 4,
B and
C). The exact time of this second
ceramide peak varied from culture to culture, but at least a twofold
increase in ceramide levels was observed in all experiments. In three
of five experiments in RBEC and in four of seven experiments in
astrocytes, TNF-
was washed out after 4 h of treatment, as for
TNF-
preconditioning, but its withdrawal at this time had no effect
on the delayed generation of ceramide. The average time of the peak in
RBEC calculated on the basis of ceramide measurements in five different
batches of RBEC derived from four animals was ~21 h
(P < 0.025 vs. baseline values). A
representative experiment is also shown for comparison (Fig.
4B). In astrocyte cultures, delayed
ceramide accumulation peaked between 21 h
(P < 0.02 vs. baseline levels) and
24 h (P < 0.002 vs. baseline levels
and P < 0.005 vs. 27-h levels; Fig. 4C).
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Time of cell unresponsiveness to TNF- correlates
with the time of the ceramide peak.
Because C-2 ceramide exerted the inhibitory effect only if added
shortly before or after TNF-
addition and because the increase of
endogenous ceramide levels was transient, we hypothesized that if
ceramide is a mediator of tolerance, TNF-
-preconditioned cells should remain unresponsive to the second TNF-
addition only during the short time when ceramide levels are elevated. Extending or shortening the time between the two TNF-
stimuli should result in
the loss of preconditioning effect. To test this prediction, astrocytes
or RBEC preconditioned for 4 h with TNF-
were given the second
TNF-
stimulus 18, 21, 24, or 27 h later, and ICAM-1 mRNA (Fig.
5, A and
B) or surface expression of ICAM-1
protein (Fig. 5C) was measured. In
RBEC (Fig. 5A) and astrocytes (Fig. 5B) the degree of inhibition of
ICAM-1 mRNA synthesis was dependent on the time between preconditioning
and the subsequent TNF-
challenge. When cells were activated with
TNF-
at 21 h after addition of the preconditioning TNF-
stimulus,
maximum inhibition of ICAM-1 mRNA synthesis was observed in RBEC
(n = 4, P < 0.04) and astrocytes (n = 3, P < 0.05). This temporal optimum (21 h after the first TNF-
addition) for cell unresponsiveness to
TNF-
activation corresponds to the time when ceramide release caused
by TNF-
reaches peak levels.
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TNF--induced delayed ceramide accumulation does not
trigger apoptosis in cultured brain cells.
Studies in tumor cell lines have linked stress- or cytokine-induced
prolonged and persistent elevation in ceramide levels to signaling for
apoptosis (19). Delayed ceramide release in nontumor cells has not been
previously reported. Thus our observation of TNF-
-triggered delayed
accumulation of ceramide in astrocytes and RBEC prompted us to
investigate whether increased ceramide levels result in cell death. A
single TNF-
treatment of astrocyte and RBEC cultures resulted in no
significant apoptosis even after 48 h of incubation (Table
2, Figs.
6E and
7E).
Similarly, two TNF-
treatments, as was done in the above
preconditioning studies (the first for 4 h and the second 20 h later
for 24 h) caused no significant apoptosis in astrocytes or RBEC (Table
2). The effect of exogenous C-2 ceramide strongly depended on its dose. When treated with 10 µM C-2 ceramide, only 7% and 12% of astrocytes and RBEC, respectively, became apoptotic (Table 2, Figs.
6C and 7C). In contrast, when treated with
50 µM C-2 ceramide, 70% of astrocytes and 56% of RBEC underwent
apoptosis (Table 2). Morphological changes characteristic of apoptosis
similar to that observed in cultures treated with methyl iodide, an
alkylating agent known to cause cell apoptosis (Figs.
6B and
7B), appeared as early as 24 h after
addition of 50 µM C-2 ceramide (Figs.
6D and
7D). After 48 h of incubation with
50 µM C-2 ceramide, practically all RBEC were dead; significant cell
loss occurred, which interfered with the assays. To mimic the apoptotic
effect of high doses of C-2 ceramide, the ceramidase inhibitor
N-oleoylethanolamine (NOE; Alexis
Biomedicals, San Diego, CA), which has been shown to increase ceramide
formation in tumor cells (48), was added to the cells to potentiate
TNF-
-induced production of endogenous ceramide. As shown above,
TNF-
treatment alone caused no apoptosis in astrocyte or RBEC
cultures, even if the treatment was prolonged for 48 h; however, at 48 h of TNF-
-NOE treatment, morphological changes characteristic of
apoptosis could be seen in both cultures (Figs. 6F and
7F). Treatment of RBEC with NOE
alone had no significant effect on Hoechst fluorescence and cell
morphology (data not shown).
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DISCUSSION |
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The data presented here demonstrate that TNF-, a pleiotropic
cytokine implicated as a major pathogenic factor in oxidative stress
and in inflammatory diseases, can induce tolerance to its own action if
applied for a short time to precondition cells. Two
observations suggest a general role for TNF-
as an inducer of cell
tolerance to stress: 1) TNF-
induced unresponsiveness in two types of brain cells, astrocytes and
RBEC, with the same pretreatment schedule (4 h of pretreatment 24 h
before the challenge), and 2)
pretreatment with TNF-
rendered cells unresponsive not only against
subsequent TNF-
challenge but also against subsequent hypoxia.
Although in vitro models of brain tolerance to hypoxia and
ischemia have tended to emphasize the effect of preconditioning on neuronal survival, our data show that other brain cells such as
endothelial cells and astrocytes also participate. This work addresses
signaling steps in endothelial cells and astrocytes that support
adaptation to TNF- and hypoxia via a TNF-
-ceramide-dependent mechanism.
Induction of ICAM-1 protein and mRNA has been measured as a biological
readout of TNF--triggered cell activation. The effect of TNF-
on
astrocytes and endothelial cells is well studied (2, 42) and is often
used as a paradigm for the proinflammatory action of TNF-
and other
types of stress such as oxidative stress or hypoxia (20). We found that
both types of TNF-
-preconditioned cells failed to upregulate surface
expression of ICAM-1 in response to TNF-
or hypoxia and that this
correlated with lower levels of ICAM-1 mRNA. The absence of any effect
on ICAM-1 mRNA stability indicates that TNF-
preconditioning
affected ICAM-1 transcription (and possibly that of other genes).
However, inhibition of ICAM-1 protein was more pronounced than
inhibition of transcription (~75% vs. 40%), which suggests that
mechanisms controlling ICAM-1 translation or its transport to the
surface could be also affected.
The sphingomyelin cycle has been demonstrated to participate in signal
transduction pathways initiated by TNF- and other types of stress
(9, 24, 19, 15, 34, 40). We have investigated whether ceramide serves
as a second messenger in the preconditioning of cells by TNF-
. When
added in place of TNF-
for 4 h, the cell-permeable ceramide analog
N-acetylceramide (C-2 ceramide) failed
to precondition astrocytes and RBEC; they remained fully responsive to
the TNF-
activation 24 h later. However, if the ceramide was added 1 h before or even 1 h after TNF-
, it did have an inhibitory effect on
ICAM-1 mRNA and protein levels. This finding suggested that ceramide
could participate in preconditioning if it were released sufficiently
late in the course of TNF-
pretreatment that it would be elevated at
the time of the second TNF-
stimulus. This hypothesis prompted us to
systematically measure ceramide levels in astrocyte and RBEC cultures
over a long period of time (30 h) after TNF-
treatment. We have
demonstrated two peaks of ceramide response to TNF-
: an early
1.9-fold (astrocytes) and 2.7-fold (RBEC) ceramide increase at
15-20 min and a late 2.5- to 3-fold increase at 18-24 h after TNF-
addition. Our observed early ceramide kinetics in RBEC are consistent with published data on ceramide release in human umbilical vein endothelial cells (peaks at 30 min) (29) and bovine aortic endothelial cells (peaks at 30 min) (45), whereas the ceramide response
of primary astrocytes to TNF-
has not been previously investigated.
Our findings of high ceramide levels later in the course of TNF-
treatment are novel, inasmuch as no delayed ceramide formation has been
reported in normal cells.
Several lines of evidence suggest that the late ceramide release
observed in astrocytes and RBEC cultures is responsible for induction
of unresponsiveness in TNF--preconditioned cells.
1) The degree of attenuation of
ICAM-1 mRNA caused by C-2 ceramide in both types of cells was
comparable to that of TNF-
pretreatment (30-40% reduction of
the normal TNF-
-induced increase).
2) C-2 ceramide exposure and TNF-
preconditioning affected ICAM-1 transcription levels and had no effect
on ICAM-1 mRNA stability. 3) TNF-
preconditioning resulted in inhibition of the ICAM-1 response to the
subsequent TNF-
activation only when the time of the second TNF-
addition coincided with the peak of late ceramide release.
4) Fumonisin B1, a natural inhibitor of
ceramide synthase, an enzyme that converts sphinganine to
dihydroceramide, attenuated TNF-
-induced ceramide release in
astrocytes and the effect of TNF-
preconditioning; astrocytes
pretreated with TNF-
in the presence of fumonisin B1 upregulated surface expression
of ICAM-1 in response to TNF-
as well as naive cells. Similarly,
fumonisin B1 attenuated
endothelial cell apoptosis resulting from high ceramide levels
triggered by TNF-
plus cycloheximide (51).
On the basis of multiple observations in tumor cell lines, one would
expect that the high levels of ceramide observed in our cultures of
brain cells should lead to apoptosis. However, our experiments
demonstrate that treatment of both cell types with TNF- continuously
for 48 h or with the schedule used to study tolerance (4 h for the 1st
treatment, 20 h of rest, 24 h for the 2nd treatment) results in no
significant apoptosis. In addition, when the time interval between the
two TNF-
treatments was increased to 27 h, no adaptation was
observed, which would not be the case if cell unresponsiveness were an
artifact caused by apoptosis. Similarly, exogenous ceramide also failed
to induce apoptosis at doses that inhibited TNF-
-induced ICAM-1
expression in brain cells. Our observations are consistent with the
recent data of Casaccia-Bonnefil and co-workers (5) that showed that
addition of 10 µM C-2 to neuronal and astrocyte cultures had no
effect on their survival and with earlier studies (37) demonstrating resistance of cultured endothelial cells to the cytotoxic effects of
TNF-
.
Comparison of the kinetics of delayed ceramide formation in tumor cell
lines with that observed in our model reveals major differences. For
example, in T cell lymphoma Jurkat cells, activation of a specific
death receptor, Fas, with anti-Fas antibody resulted in a gradual
increase of ceramide levels with no tendency to subside over 10 h of
observation (43). TNF- treatment of L929 cells caused similar
continuous accumulation of ceramide over 12 h (22), and in B cell
lymphoma WEHI 231 cells, high ceramide levels triggered by the anti-IgM
antibody persisted for 48 h (48). In contrast, we report here that in
normal cultured brain cells the ceramide increase was transient,
achieving highest levels 18-24 h after addition of TNF-
and
then quickly subsiding to the baseline levels. These observations
suggest that ceramide levels in differentiated cells are under tighter
metabolic control than in TNF-
-sensitive tumor cell lines. Indeed,
it has been shown that inhibition of protein synthesis or transcription
is required for TNF-
to produce a cytotoxic response in endothelial
cells (37, 51). In our model, TNF-
caused rapid apoptosis of
astrocytes and RBEC only if added together with the ceramidase
inhibitor NOE, which has been shown to perturb ceramide metabolism
(48). Thus constitutive deregulation of ceramide metabolism in
transformed cells might be responsible for the disproportionate
ceramide response to stress, resulting in persistently high ceramide
levels that trigger cell death. This explanation fits our data and that
of D'Souza and co-workers (11), who demonstrated differential
sensitivity of normal and malignant astrocytes to TNF-
.
We hypothesize that ceramide in normal cells does not serve as a
messenger for apoptosis but, rather, represents one of the feedback
mechanisms inhibiting cell reaction to TNF- and perhaps other types
of stress. In support of this hypothesis, the ceramide has been shown
to inhibit the respiratory burst and cell spreading in human
neutrophils if added simultaneously with TNF-
(12); ceramide also
mediated TNF-
-induced inhibition of GLUT-4 in adipocytes (28) and of
sodium-myo-inositol cotransporter in
endothelial cells (53).
How ceramide causes cell unresponsiveness to TNF- has yet to be
elucidated. The data presented here indicate that ceramide can act at
the level of gene transcription, suggesting that ceramide may act by
inhibiting a common transcription factor. For example, a nuclear
factor-
B (NF-
B) site has been identified in the ICAM-1 promoter
(26), and ceramide has been shown to inhibit NF-
B activation
triggered by phorbol 12-myristate 13-acetate (14). In a recent study,
inhibition of NF-
B by ceramide was associated with excessive
production of p50 homodimers (3). Interestingly, the same mechanism was
reported for lipopolysaccharide-induced tolerance in a monocytic cell
line (55). The transient nature of the TNF-
-induced
accumulation of ceramide, which parallels the short duration of cell
desensitization, argues that ceramide-induced inhibition of ICAM-1
expression depends on a fast-acting mechanism such as phosphorylation
or dephosphorylation. Recently identified ceramide-activated kinases,
protein kinase C
(13), a proline-directed serine-threonine kinase
(27), and a serine-threonine phosphatase (10) could play a role.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Richard Kolesnick (Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center) for expert advice on ceramide assay, Joliet Bembry for preparation of rat brain endothelial cell cultures, and Nancy Merkel for technical assistance in ceramide measurements.
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FOOTNOTES |
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U. Schweizer was supported by Siemens Med GT, Erlangen and Hans-Krüger-Stiftung, Berlin, Germany.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: I. Ginis, Stroke Branch, NINDS, Bldg. 36, Rm. 4A03, National Institutes of Health, Bethesda, MD 20892-4128 (E-mail: ginis{at}codon.nih.gov).
Received 11 December 1998; accepted in final form 12 February 1999.
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REFERENCES |
---|
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---|
1.
Arvin, B.,
L. F. Neville,
F. C. Barone,
and
G. Z. Feuerstein.
The role of inflammation and cytokines in brain injury.
Neurosci. Biobehav. Rev.
20:
445-452,
1996[Medline].
2.
Ballestas, M. E.,
and
E. N. Benveniste.
Interleukin-1- and tumor necrosis factor-
-mediated regulation of ICAM-1 gene expression in astrocytes requires protein kinase C activity.
Glia
14:
267-278,
1995[Medline].
3.
Boland, M. P.,
and
L. A. O'Neill.
Ceramide activates NF-B by inducing the processing of p105.
J. Biol. Chem.
273:
15494-15500,
1998
4.
Carlson, S. L.,
D. J. Beiting,
C. A. Kiani,
K. M. Abell,
and
J. P. McGillis.
Catecholamines decrease lymphocyte adhesion to cytokine-activated endothelial cells.
Brain Behav. Immun.
10:
55-67,
1996[Medline].
5.
Casaccia-Bonnefil, P.,
L. Aibel,
and
M. V. Chao.
Central glial and neuronal populations display differential sensitivity to ceramide-dependent cell death.
J. Neurosci. Res.
43:
382-389,
1996[Medline].
6.
Chen, J.,
and
R. Simon.
Ischemic tolerance in brain.
Neurology
48:
306-311,
1997[Medline].
7.
Chopp, M.,
Y. Li,
N. Jiang,
R. L. Zhang,
and
J. Prostak.
Antibodies against adhesion molecules reduce apoptosis after transient middle cerebral artery occlusion in rat brain.
J. Cereb. Blood Flow Metab.
16:
578-584,
1996[Medline].
8.
Dawson, D. A.,
D. Martin,
and
J. M. Hallenbeck.
Inhibition of tumor necrosis factor- reduces focal cerebral ischemic injury in the spontaneously hypertensive rat.
Neurosci. Lett.
218:
41-44,
1996[Medline].
9.
Dbaibo, G. S.,
D. K. Perry,
C. J. Gamard,
R. Platt,
G. G. Poirier,
L. M. Obeid,
and
Y. A. Hannun.
Cytokine response modifier A (CrmA) inhibits ceramide formation in response to tumor necrosis factor (TNF)-: CrmA and Bcl-2 target distinct components in the apoptotic pathway.
J. Exp. Med.
185:
481-490,
1997
10.
Dobrowsky, R. T.,
C. Kamibayashi,
M. C. Mumby,
and
Y. A. Hannun.
Ceramide activates heterotrimeric protein phosphatase 2A.
J. Biol. Chem.
268:
15523-15530,
1993
11.
D'Souza, S.,
K. Alinauskas,
E. McCrea,
C. Goodyer,
and
J. P. Antel.
Differential susceptibility of human CNS-derived cell populations to TNF-dependent and independent immune-mediated injury.
J. Neurosci.
15:
7293-7300,
1995[Abstract].
12.
Fuortes, M.,
W. Jin,
and
C. Nathan.
Ceramide selectively inhibits early events in the response of human neutrophils to tumor necrosis factor.
J. Leukoc. Biol.
59:
451-460,
1996[Abstract].
13.
Galve-Roperh, I.,
A. Haro,
and
I. Diaz-Laviada.
Ceramide-induced translocation of protein kinase C in primary cultures of astrocytes.
FEBS Lett.
415:
271-274,
1997[Medline].
14.
Gamard, C. J.,
G. S. Dbaibo,
B. Liu,
L. M. Obeid,
and
Y. A. Hannun.
Selective involvement of ceramide in cytokine-induced apoptosis. Ceramide inhibits phorbol ester activation of nuclear factor-B.
J. Biol. Chem.
272:
16474-16481,
1997
15.
Ghosh, S.,
J. C. Strum,
and
R. M. Bell.
Lipid biochemistry: functions of glycerolipids and sphingolipids in cellular signaling.
FASEB J.
11:
45-50,
1997
16.
Ginis, I.,
and
D. V. Faller.
Protection from apoptosis in human neutrophils is determined by the surface of adhesion.
Am. J. Physiol.
272 (Cell Physiol. 41):
C295-C309,
1997
17.
Goodman, Y.,
and
M. P. Mattson.
Ceramide protects hippocampal neurons against excitotoxic and oxidative insults and amyloid -peptide toxicity.
J. Neurochem.
66:
869-872,
1996[Medline].
18.
Hallenbeck, J. M.
Inflammatory reactions at the blood-endothelial interface in acute stroke.
Adv. Neurol.
71:
281-300,
1996[Medline].
19.
Hannun, Y. A.
Functions of ceramide in coordinating cellular responses to stress.
Science
274:
1855-1859,
1996
20.
Hess, D. C.,
W. Zhao,
J. Carroll,
M. McEachin,
and
K. Buchanan.
Increased expression of ICAM-1 during reoxygenation in brain endothelial cells.
Stroke
25:
1463-1468,
1994[Abstract].
21.
Islam, C. F.,
R. T. Mathie,
M. D. Dinneen,
E. A. Kiely,
A. M. Peters,
and
P. A. Grace.
Ischaemia-reperfusion injury in the rat kidney: the effect of preconditioning.
Br. J. Urol.
79:
842-847,
1997[Medline].
22.
Jayadev, S.,
H. L. Hayter,
N. Andrieu,
C. J. Gamard,
B. Liu,
R. Balu,
M. Hayakawa,
F. Ito,
and
Y. A. Hannun.
Phospholipase A2 is necessary for tumor necrosis factor--induced ceramide generation in L929 cells.
J. Biol. Chem.
272:
17196-17203,
1997
23.
Joyeux, M.,
D. Godin-Ribuot,
and
C. Ribuot.
Resistance to myocardial infarction induced by heat stress and the effect of ATP-sensitive potassium channel blockade in the rat isolated heart.
Br. J. Pharmacol.
123:
1085-1088,
1998[Abstract].
24.
Kolesnick, R.,
and
D. W. Golde.
The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling.
Cell
77:
325-328,
1994[Medline].
25.
Lamping, N.,
R. Dettmer,
N. W. J. Schroder,
D. Pfeil,
W. Hallatschek,
R. Burger,
and
R. R. Schumann.
LPS-binding protein protects mice from septic shock caused by LPS or gram-negative bacteria.
J. Clin. Invest.
101:
2065-2071,
1998
26.
Ledebur, H. C.,
and
T. P. Parks.
Transcriptional regulation of the intercellular adhesion molecule-1 gene by inflammatory cytokines in human endothelial cells. Essential roles of a variant NF-B site and p65 homodimers.
J. Biol. Chem.
270:
933-943,
1995
27.
Liu, J.,
S. Mathias,
Z. Yang,
and
R. N. Kolesnick.
Renaturation and tumor necrosis factor- stimulation of a 97-kDa ceramide-activated protein kinase.
J. Biol. Chem.
269:
3047-3052,
1994
28.
Long, S. D.,
and
P. H. Pekala.
Lipid mediators of insulin resistance: ceramide signalling down-regulates GLUT4 gene transcription in 3T3-L1 adipocytes.
Biochem. J.
319:
179-184,
1996[Medline].
29.
Masamune, A.,
Y. Igarashi,
and
S. Hakomori.
Regulatory role of ceramide in interleukin (IL)-1-induced E-selectin expression in human umbilical vein endothelial cells. Ceramide enhances IL-1
action but is not sufficient for E-selectin expression.
J. Biol. Chem.
271:
9368-9375,
1996
30.
Mattson, M. P.
Neuroprotective signal transduction: relevance to stroke.
Neurosci. Biobehav. Rev.
21:
193-206,
1997[Medline].
31.
Merrill, A. H., Jr.,
E. Wang,
R. E. Mullins,
W. C. Jamison,
S. Nimkar,
and
D. C. Liotta.
Quantitation of free sphingosine in liver by high-performance liquid chromatography.
Anal. Biochem.
171:
373-381,
1988[Medline].
32.
Nawashiro, H.,
K. Tasaki,
C. A. Ruetzler,
and
J. M. Hallenbeck.
TNF- pretreatment induces protective effects against focal cerebral ischemia in mice.
J. Cereb. Blood Flow Metab.
17:
483-490,
1997[Medline].
33.
Neta, R. Modulation with cytokines of radiation injury:
suggested mechanisms of action. Environ. Health
Perspect. 105, Suppl.
6: 1463-1465, 1997.
34.
Pena, L. A.,
Z. Fuks,
and
R. Kolesnick.
Stress-induced apoptosis and the sphingomyelin pathway.
Biochem. Pharmacol.
53:
615-621,
1997[Medline].
35.
Peralta, C.,
G. Hotter,
D. Closa,
E. Gelpi,
O. Bulbena,
and
J. Rosello-Catafau.
Protective effect of preconditioning on the injury associated to hepatic ischemia-reperfusion in the rat: role of nitric oxide and adenosine.
Hepatology
25:
934-937,
1997[Medline].
36.
Piper, P. W.
Molecular events associated with acquisition of heat tolerance by the yeast Saccharomyces cerevisiae.
FEMS Microbiol. Rev.
11:
339-355,
1993[Medline].
37.
Pohlman, T. H.,
and
J. M. Harlan.
Human endothelial cell response to lipopolysaccharide, interleukin-1, and tumor necrosis factor is regulated by protein synthesis.
Cell Immunol.
119:
41-52,
1989[Medline].
38.
Santana, P.,
L. A. Pena,
A. Haimovitz-Friedman,
S. Martin,
D. Green,
M. McLoughlin,
C. Cordon-Cardo,
E. H. Schuchman,
Z. Fuks,
and
R. Kolesnick.
Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis.
Cell
86:
189-199,
1996[Medline].
39.
Simon, R. P.,
M. Niiro,
and
R. Gwinn.
Prior ischemic stress protects against experimental stroke.
Neurosci. Lett.
163:
135-137,
1993[Medline].
40.
Smyth, M. J.,
L. M. Obeid,
and
Y. A. Hannun.
Ceramide: a novel lipid mediator of apoptosis.
Adv. Pharmacol.
41:
133-154,
1997[Medline].
41.
Spatz, M.,
N. Kawai,
N. Merkel,
J. Bembry,
and
R. M. McCarron.
Functional properties of cultured endothelial cells derived from large microvessels of human brain.
Am. J. Physiol.
272 (Cell Physiol. 41):
C231-C239,
1997
42.
Tasaki, K.,
C. A. Ruetzler,
T. Ohtsuki,
D. Martin,
H. Nawashiro,
and
J. M. Hallenbeck.
Lipopolysaccharide pre-treatment induces resistance against subsequent focal cerebral ischemic damage in spontaneously hypertensive rats.
Brain Res.
748:
267-270,
1997[Medline].
43.
Tepper, A. D.,
J. G. Cock,
E. de Vries,
J. Borst,
and
W. J. van Blitterswijk.
CD95/Fas-induced ceramide formation proceeds with slow kinetics and is not blocked by caspase-3/CPP32 inhibition.
J. Biol. Chem.
272:
24308-24312,
1997
44.
Trosko, J. E. Hierarchical and cybernetic nature
of biologic systems and their relevance to homeostatic adaptation to
low-level exposures to oxidative stress-inducing agents.
Environ. Health Perspect. 106, Suppl. 1: 331-339, 1998.
45.
Verheij, M.,
R. Bose,
X. H. Lin,
B. Yao,
W. D. Jarvis,
S. Grant,
M. J. Birrer,
E. Szabo,
L. I. Zon,
J. M. Kyriakis,
A. Haimovitz-Friedman,
Z. Fuks,
and
R. N. Kolesnick.
Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis.
Nature
380:
75-79,
1996[Medline].
46.
Wang, X.,
A. L. Siren,
Y. Liu,
T. L. Yue,
F. C. Barone,
and
G. Z. Feuerstein.
Upregulation of intercellular adhesion molecule 1 (ICAM-1) on brain microvascular endothelial cells in rat ischemic cortex.
Brain Res. Mol. Brain Res.
26:
61-68,
1994[Medline].
47.
White, C. W.,
and
P. Ghezzi.
Protection against pulmonary oxygen toxicity by interleukin-1 and tumor necrosis factor: role of antioxidant enzymes and effect of cyclooxygenase inhibitors.
Biotherapy
1:
361-367,
1989[Medline].
48.
Wiesner, D. A.,
J. P. Kilkus,
A. R. Gottschalk,
J. Quintans,
and
G. Dawson.
Anti-immunoglobulin-induced apoptosis in WEHI 231 cells involves the slow formation of ceramide from sphingomyelin and is blocked by bcl-XL.
J. Biol. Chem.
272:
9868-9876,
1997
49.
Wolff, S. The adaptive response in radiobiology: evolving
insights and implications. Environ. Health
Perspect. 106, Suppl.
1: 277-283, 1998.
50.
Wong, G. H.,
R. L. Kaspar,
and
G. Vehar.
Tumor necrosis factor and lymphotoxin: protection against oxidative stress through induction of MnSOD.
EXS
77:
321-333,
1996[Medline].
51.
Xu, J.,
C. H. Yeh,
S. Chen,
L. He,
S. L. Sensi,
L. M. Canzoniero,
D. W. Choi,
and
C. Y. Hsu.
Involvement of de novo ceramide biosynthesis in tumor necrosis factor-/cycloheximide-induced cerebral endothelial cell death.
J. Biol. Chem.
273:
16521-16526,
1998
52.
Yellon, D. M.,
G. F. Baxter,
D. Garcia-Dorado,
G. Heusch,
and
M. S. Sumeray.
Ischaemic preconditioning: present position and future directions.
Cardiovasc. Res.
37:
21-33,
1998[Medline].
53.
Yorek, M. A.,
J. A. Dunlap,
M. J. Thomas,
P. R. Cammarata,
C. Zhou,
and
W. L. Lowe, Jr.
Effect of TNF- on SMIT mRNA levels and myo-inositol accumulation in cultured endothelial cells.
Am. J. Physiol.
274 (Cell Physiol. 43):
C58-C71,
1998
54.
Zhou, X.,
X. Zhai,
and
M. Ashraf.
Direct evidence that initial oxidative stress triggered by preconditioning contributes to second window of protection by endogenous antioxidant enzyme in myocytes.
Circulation
93:
1177-1184,
1996
55.
Ziegler-Heitbrock, H. W.,
A. Wedel,
W. Schraut,
M. Strobel,
P. Wendelgass,
T. Sternsdorf,
P. A. Bauerle,
J. G. Haas,
and
G. Riethmuller.
Tolerance to lipopolysaccharide involves mobilization of nuclear factor-B with predominance of p50 homodimers.
J. Biol. Chem.
269:
17001-17004,
1994