From the Department of Pediatrics, Medical University of South
Carolina, Charleston, South Carolina 29425
The present study underlines the importance of
reactive oxygen species in cytokine-mediated degradation of
sphingomyelin (SM) to ceramide. Treatment of rat primary astrocytes
with tumor necrosis factor-
(TNF-
) or interleukin-1
led to
marked alteration in cellular redox (decrease in intracellular GSH) and
rapid degradation of SM to ceramide. Interestingly, pretreatment of
astrocytes with N-acetylcysteine (NAC), an antioxidant and
efficient thiol source for glutathione, prevented cytokine-induced
decrease in GSH and degradation of sphingomyelin to ceramide, whereas
treatment of astrocytes with diamide, a thiol-depleting agent, alone
caused degradation of SM to ceramide. Moreover, potent activation of SM
hydrolysis and ceramide generation were observed by direct addition of
an oxidant like hydrogen peroxide or a prooxidant like aminotriazole.
Similar to NAC, pyrrolidinedithiocarbamate, another antioxidant, was
also found to be a potent inhibitor of cytokine-induced degradation of
SM to ceramide indicating that cytokine-induced hydrolysis of
sphingomyelin is redox-sensitive. Besides astrocytes, NAC also blocked
cytokine-mediated ceramide production in rat primary oligodendrocytes,
microglia, and C6 glial cells. Inhibition of TNF-
- and
diamide-mediated depletion of GSH, elevation of ceramide level, and DNA
fragmentation (apoptosis) in primary oligodendrocytes by NAC, and
observed depletion of GSH, elevation of ceramide level, and apoptosis
in banked human brains from patients with neuroinflammatory diseases
(e.g. X-adrenoleukodystrophy and multiple sclerosis)
suggest that the intracellular level of GSH may play a critical role in
the regulation of cytokine-induced generation of ceramide leading to
apoptosis of brain cells in these diseases.
 |
INTRODUCTION |
Sphingomyelin is preferentially concentrated in the outer leaflet
of the plasma membrane of most mammalian cells; it comprises sphingosine (a long chain sphingoid base backbone), a fatty acid, and a
phosphocholine head group. Ceramide is composed of a sphingoid base
with a fatty acid in amide linkage. Sphingomyelin was initially considered only a structural component of plasma membrane; however, several investigations established the involvement of sphingolipids and
its metabolites in the key events of signal transduction associated with cell regulation, cell differentiation, and apoptosis (1-3). The
sphingomyelin pathway-associated signal transduction pathway mediates
the action of several extracellular stimuli that lead to important
biochemical and cellular effects (4-8). This pathway is initiated by
the activation of two distinct forms of sphingomyelinase (SMase),1 a
membrane-associated neutral sphingomyelinase (9) and an acidic
sphingomyelinase (10), which reside in the caveola and the
endosomal-lysosomal compartment. Each type of SMase hydrolyzes the
phosphodiester bond of sphingomyelin to yield ceramide and phosphocholine. Proinflammatory cytokines (tumor necrosis factor-
, TNF-
; interleukin-1
, IL-1
; interferon-
, IFN-
) and
bacterial lipopolysaccharides have been shown as potent inducers of
SMases. One of the products, ceramide, has emerged as a second
messenger molecule that is considered to mimic most of the cellular
effects of cytokines and lipopolysaccharide in terminal
differentiation, apoptosis, and cell cycle arrest (4, 5).
Sphingomyelin turnover and ceramide generation in response to TNF-
and IL-1
occurs within minutes of stimulation; however, the sequence
of events linking receptor stimulation and SMase activation remains
largely unknown (7, 8, 11). In a number of cell systems, interaction of
TNF-
with its membrane receptors (p75 and p55) has been found to
activate phospholipase A2 and to induce release of
arachidonic acid from phosphatidylcholine and phosphatidylethanolamine
pools. This arachidonic acid has been shown as a mediator of
sphingomyelin hydrolysis in response to TNF-
(11). In addition,
proteases have also been implicated in the pathway leading from TNF-
to the activation of SMase (7, 12) recently. On the other hand, IL-1
and TNF-
are known to induce the production of reactive oxygen
species (ROS), a class of highly diffusible and ubiquitous molecules,
which have been suggested to act as second messengers (13-15). ROS
encompassing species such as superoxide, hydrogen peroxide, and
hydroxyl radicals are known to regulate critical steps in the signal
transduction cascade and many important cellular events including
protein phosphorylation, gene expression, transcription factor
activation, DNA synthesis, and cellular proliferation (16, 17). A
recent observation has shown that glutathione or similar molecules
inhibit the activity of magnesium-dependent neutral SMase
in vitro (18). However, surprisingly, the SH group of GSH
was not required as S-methyl GSH and GSSG inhibited neutral
SMase at lower concentrations than GSH (18). On the other hand,
N-acetylcysteine has also been found to inhibit the
synthesis of ceramide in cultured rat hepatocytes through the
inhibition of dihydroceramide desaturase (19).
In the present study, we examined the possible involvement of ROS in
cytokine-mediated activation of sphingomyelin breakdown and ceramide
formation in rat primary glial cells. We show that intracellular GSH
plays a crucial role in the breakdown of SM to ceramide, in that low
GSH levels are required for ceramide generation and high GSH levels
inhibit production of ceramide. Inhibition of cytokine-mediated
breakdown of SM to ceramide by antioxidants like
N-acetylcysteine (NAC) and pyrrolidinedithiocarbamate (PDTC)
and induction of ceramide production by oxidants or pro-oxidants like
hydrogen peroxide, aminotriazole, diamide, and
L-buthione-(SR)-sulfoximine clearly delineate a
novel function of ROS and GSH in regulation of the first step of
sphingomyelin signal transduction pathway. Moreover, decreased levels
of GSH and increased levels of ceramide correlate with the DNA
fragmentation in rat primary oligodendrocytes as well as in the banked
human brains from patients with neuroinflammatory diseases
(e.g. multiple sclerosis and X-adrenoleukodystrophy).
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MATERIALS AND METHODS |
Reagents--
DMEM/F-12 and fetal bovine serum (FBS) were from
Life Technologies, Inc. Human IL1-
was from Genzyme. Mouse
recombinant TNF-
was obtained from Boehringer Mannheim, Germany.
Diamide, buthione-(SR)-sulfoximine, N-acetylcysteine, and pyrrolidinedithiocarbamate were from
Sigma.
Isolation and Maintenance of Rat Primary Microglia,
Oligodendrocytes, and Astrocytes--
Microglial cells were isolated
from mixed glial cultures according to the procedure of Guilian and
Baker (20). Briefly, after 7 days the mixed glial cultures were washed
3 times with DMEM/F-12 containing 10% FBS and subjected to a shake at
240 rpm for 4 h at 37 °C on a rotary shaker. The floating cells
were washed and seeded onto plastic tissue culture flasks and incubated
at 37 °C. After 30 min the non-attached cells (mostly
oligodendrocytes) were removed by repeated washes, and the attached
cells were used as microglia. These cells were seeded onto new plates
for further studies. Ninety to ninety-five percent of this preparation
was positive for nonspecific esterase, a marker for macrophages and microglia.
After 4 h shaking, the flasks were washed three times to remove
the floating cells. Medium with 10% FBS was added, and flasks were
subjected to another cycle of shaking for 24 h at 250 rpm. The
suspended cells were spun at 200 × g and incubated for
30 min in tissue culture dish. The non-attached or weakly attached cells (mostly oligodendrocytes) were removed and seeded onto
polylysine-coated dishes and cultured in medium containing 1% FBS.
Ninety-five to ninety-seven percent of these cells were positive for
galactocerebroside immunostaining.
Astrocytes were prepared from rat cerebral tissue as described by
McCarthy and DeVellis (21). After 10 days of culture astrocytes were
separated from microglia and oligodendrocytes by shaking for 24 h
in an orbital shaker at 240 rpm. To ensure the complete removal of all
oligodendrocytes and microglia, the shaking was repeated twice after a
gap of 1 or 2 days. Attached cells were trypsinized (1 mM
EDTA and 0.05% trypsin in 10 mM Tris-buffered saline, pH
7.4) and distributed into culture dishes. These cells when checked for
the astrocyte marker glial fibrillar acidic protein were found to be
95-100% positive. C6 glial cells obtained from ATCC were
also maintained in DMEM/F-12 containing 10% FBS as indicated above.
Brain Tissue--
Frozen and fixed X-adrenoleukodystrophy and
multiple sclerosis brain tissues were obtained from Brain and Tissue
Banks for Developmental Disorders, University of Maryland, Baltimore,
MD 21201. Two X-ALD brains were from 7- and 9-year-old males, and two
MS brains were from 30- and 33-year-old females. Control brain for
X-ALD studies was from an 8-year-old male (Control 1), and control
brain for MS studies was from a 30-year-old female (Control 2).
Lipid Extraction--
Approximately 1.0 × 106
cells were exposed to different cytokines in the presence or absence of
antioxidants for different periods, and lipids were extracted according
to the methods described by Welsh (22).
Quantification of Sphingomyelin by High Performance TLC and
Densitometry--
Sphingomyelin was separated from total lipid
extracts by high performance TLC (LHPK plates from Whatman) as
described by Ganser et al. (23) for phospholipids with the
following modification: the plate was overrun for 30 min during its
development and was dried overnight in vacuum desiccator. Sphingomyelin
was quantitated by densitometric scanning using Imaging Densitometer
(model GS-670; Bio-Rad), and software was provided with the instrument
by the manufacturer.
Quantification of Ceramide Levels by Diacylglycerol Kinase
Assay--
Ceramide content was quantified essentially according to
Priess et al. (24) using diacylglycerol (DAG) kinase and
[
-32P]ATP. Briefly, dried lipids were solubilized in
20 µl of an octyl
-D-glucoside/cardiolipin solution
(7.5% octyl
-D-glucoside, 5 mM cardiolipin
in 1 mM DTPA) by sonication in a sonicator bath. The
reaction was then carried out in a final volume of 100 µl containing
the 20-µl sample solution, 50 mM imidazole HCl, pH 6.6, 50 mM NaCl, 12.5 mM MgCl2, 1 mM EGTA, 2 mM dithiothreitol, 6.6 µg of DAG
kinase, and 1 mM [
-32P]ATP (specific
activity of 1-5 × 105 cpm/nmol) for 30 min at room
temperature. The labeled ceramide-1-phosphate was resolved with a
solvent system consisting of methyl acetate:n-propyl alcohol:chloroform:methanol, 0.25% KCl in water:acetic acid
(100:100:100:40:36:2). A standard sample of ceramide was phosphorylated
under identical conditions and developed in parallel. Both standard and
samples had identical RF values (0.46).
Quantification of ceramide-1-phosphate was carried out by
autoradiography and densitometric scanning using Imaging Densitometer
(model GS-670; Bio-Rad). Values are expressed either as arbitrary units
(absorbance) or as percent change.
Measurement of GSH (Reduced Glutathione) and GSSG (Oxidized
Glutathione)--
Concentration of intracellular reduced GSH was
measured using a colorimetric assay kit for GSH from R & D Systems.
Briefly, 2 × 106 cells were homogenized in 500 µl
of ice-cold 5% metaphosphoric acid and centrifuged at 3000 × g for 10 min. Supernatants were used to assay GSH using
4-chloro-1-methyl-7-trifluromethyl-quinolinium methylsulfate and 30%
NaOH at 400 nm. Concentration of GSSG was determined according to the
method of Griffith (25) after derivatization with 2-vinylpyridine for
30 min at room temperature.
Detection of DNA Fragmentation--
Cells (1 × 106) were pelleted in an Eppendorf tube by centrifugation
at 1000 rpm for 5 min, washed with phosphate-buffered saline, pH 7.4, resuspended gently in 50 µl of a lysis buffer (200 mM
NaCl, 10 mM Tris-HCl, pH 8.0, 40 mM EDTA, pH
8.0, 0.5% SDS, 400 ng of RNase A/µl), and incubated at 37 °C for
1 h. The lysate received 200 µl of the digestion buffer (200 mM NaCl, 10 mM Tris-HCl, pH 8.0, 0.5% SDS, 125 ng of proteinase K/µl). The contents were mixed by inversion several
times and then incubated at 50 °C for 2 h. An equal volume of a
mixture of phenol, pH 8.0, chloroform, and isoamyl alcohol (25:24:1,
v/v) was added, gently mixed for 10 min, and stored at room temperature
for 2 min. The two phases were separated by centrifugation at 3000 rpm
for 10 min. The viscous aqueous phase was transferred to a fresh tube, and the phenol/chloroform extraction was repeated. The aqueous phase
was extracted with an equal volume of chloroform, and 1.0 M
MgCl2 was added to the aqueous phase to a final
concentration of 10 mM. The total DNA was precipitated by
the addition of 2 volumes of absolute ethanol with several inversions.
DNA was pelleted by centrifugation at 3000 rpm for 15 min, washed with
70% ethanol, and air-dried. The pellet was dissolved in 25 µl of 10 mM Tris-HCl containing 1.0 mM EDTA, pH 8.0, and
electrophoresed in 1.8% agarose gel at 4 °C. The gel was stained
with ethidium bromide, and DNA-intercalated ethidium fluorescence was
photographed on Polaroid film 665 (P/N) using an orange filter. To
study DNA fragmentation in banked human brain tissues, brain tissues
were gently homogenized in 0.85 M sucrose buffer, and
nuclei were purified according to the procedure described previously
(26). Total genomic DNA was isolated from the nuclei and
electrophoresed as described.
Fragment End Labeling of DNA on Paraffin-embedded Tissue Sections
of MS and X-ALD Brains--
Fragmented DNA was detected in
situ by the terminal deoxynucleotidyltransferase-mediated binding
of 3'-OH ends of DNA fragments generated in response to apoptotic
signals, using a commercially available kit (TdT FragELTM)
from Calbiochem. Briefly, paraffin-embedded tissue slides were deparaffinized, rehydrated in graded ethanol, treated with 20 µg/ml
proteinase K for 15 min at room temperature, and washed prior to
terminal deoxynucleotidyltransferase staining. After terminal
deoxynucleotidyltransferase staining, sections were lightly counterstained with methyl green.
 |
RESULTS |
NAC and PDTC Block TNF-
- and IL-1
-induced Degradation of
Sphingomyelin to Ceramide in Primary Rat Astrocytes--
Rat primary
astrocytes were cultured in serum-free media with TNF-
or IL-1
for different times to quantify the production of ceramide using
diacylglycerol (DAG) kinase. Since DAG kinase phosphorylates both DAG
and ceramide using [
-32P]ATP as substrate, both lipids
can be quantified in the same assay. It was found that in astrocytes,
the DAG content was much higher than the ceramide content (Fig.
1). Stimulation of cells with TNF-
resulted in a time-dependent increase in the production of
ceramide (about 3-fold after 45 min). In contrast to induction of
ceramide production, the level of DAG, an activator of protein kinase C
and acidic sphingomyelinase, was unchanged at different time points of
stimulation (Fig. 1). Similar to TNF-
(Figs. 1 and
2), stimulation of astrocytes with
IL-1
for different times also induced a significant increase in the
ceramide content (Fig. 3). Almost
3-4-fold increase in ceramide production was found in astrocytes after
30 or 45 min of exposure with TNF-
or IL-1
. This increase in
ceramide was paralleled by TNF-
- and IL-1
-induced decrease in
sphingomyelin (Figs. 2 and 3). Sphingomyelin turnover of approximately
18-25% could be observed as early as 15 min following treatment of
astrocytes (Figs. 2 and 3), and maximal effects of up to 45-50% SM
hydrolysis were observed after 30-45 min of treatment with TNF-
or
IL-1
. These experiments suggest that both TNF-
and IL-1
can
modulate the degradation of sphingomyelin to produce ceramide, the
putative second messenger of the sphingomyelin signal transduction
pathway, in rat primary astrocytes within a short time. Interestingly,
we found that treatment of astrocytes with antioxidants like NAC (10 mM) 1 h before the addition of TNF-
or IL-1
potentially blocked the decrease in sphingomyelin as well as the
increase in ceramide (Figs. 2 and 3), whereas 10 mM acetate
had no effect on the degradation of SM to ceramide (data not shown).
Similar to NAC, another antioxidant PDTC also inhibited cytokine-mediated degradation of SM to ceramide (Figs. 2 and 3). These
experiments suggest that reactive oxygen species (ROS) are possibly
involved in cytokine-induced degradation of SM to ceramide.

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Fig. 1.
Effects of TNF- on DAG and ceramide
contents of rat primary astrocytes. Cells were exposed to TNF-
(50 ng/ml) for different time intervals. Lipids were extracted, and DAG
and ceramide contents were determined as described under "Materials
and Methods." Results are mean ± S.D. of three different
experiments.
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Fig. 2.
Inhibition of TNF- -induced degradation of
sphingomyelin to ceramide by NAC and PDTC in rat primary
astrocytes. Cells preincubated with either 10 mM NAC
or 100 µM PDTC for 1 h in serum-free DMEM/F-12
received TNF- (50 ng/ml). At different time intervals, cells were
washed with HBSS and scraped off. Lipids were extracted, and levels of
ceramide (A) (100% value is 4.51 ± 0.1 nmol/mg
protein) and sphingomyelin (B) (100% value is 25.39 ± 6.27 nmol/mg protein) were measured as described under "Materials and
Methods." Results are mean ± S.D. of three different
experiments.
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Fig. 3.
NAC and PDTC inhibit IL-1 -mediated
degradation of sphingomyelin to ceramide in rat primary
astrocytes. Cells preincubated with either 10 mM NAC
or 100 µM PDTC for 1 h in serum-free DMEM/F-12
received IL-1 (50 ng/ml). At different time intervals, cells were
washed with HBSS and scraped off. Lipids were extracted, and levels of
ceramide (A) (100% value is 4.51 ± 0.1 nmol/mg
protein) and sphingomyelin (B) (100% value is 25.39 ± 6.27 nmol/mg protein) were measured as described under "Materials and
Methods." Results are mean ± S.D. of three different
experiments.
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TNF-
and IL-1
Decrease Intracellular Level of Reduced
Glutathione (GSH) in Rat Primary Astrocytes and NAC Blocks This
Decrease--
Since the intracellular level of GSH is an important
regulator of the redox state of a cell, to understand the relationship between induction of ceramide production and intracellular level of GSH
in cytokine-stimulated astrocytes, cells were stimulated with TNF-
or IL-1
, and the level of GSH was measured at different times. Fig.
4 shows that stimulation of cells with
TNF-
or IL-1
resulted in an immediate decrease in intracellular
level of GSH with the maximal decrease (66-70% of control) found
within 15-30 min of initiation of stimulation, and with a further
increase in time of incubation, the level of GSH was found to be almost normal (88-95% of control at 90 min). These experiments suggest that
cytokine stimulation apparently induces rapid, short term production of
oxidants which transiently deplete GSH. However, the lack of decrease
of GSH (Fig. 4) and lack of hydrolysis of SM (Fig. 2 and 3) in the
presence of NAC in the cytokine-treated cells suggest that NAC
inhibited the cytokine-induced degradation of SM to ceramide by
maintaining the normal levels of GSH (Fig. 4).

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Fig. 4.
Depletion of intracellular GSH concentration
by TNF- and IL-1 in rat primary astrocytes is restored by
NAC. Cells preincubated with 10 mM NAC for 1 h
received either TNF- (50 ng/ml) or IL-1 (50 ng/ml). At different
time intervals, cells were scraped off, and GSH concentrations (100%
value is 182.5 ± 15.4 nmol/mg protein) were measured as described
under "Materials and Methods." Results are mean ± S.D. of
three different experiments.
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Thiol-depleting Agents Induce the Production of Ceramide in Rat
Primary Astrocytes--
Since NAC, a thiol antioxidant, blocked
cytokine-mediated depletion of intracellular levels of GSH and
breakdown of SM to ceramide, we investigated the effect of
thiol-depleting agents (diamide and
buthione-(SR)-sulfoximine) on ceramide production. Diamide
reduces the intracellular level of GSH by its oxidation to GSSG,
whereas buthione-(SR)-sulfoximine does so by blocking the
synthesis of GSH (27, 28). Stimulating rat primary astrocytes with
diamide resulted in an immediate decrease in intracellular level of GSH
due to rapid consumption of intracellular GSH through its nonenzymatic
conversion to the oxidized dimer, GSSG (27), and marked induction of
ceramide production (about 7-fold after 30 min of stimulation) (Fig.
5) suggesting that intracellular level of
GSH is the important regulator of degradation of SM to ceramide.
Consistent with this hypothesis, treatment of cells with NAC blocked
diamide-mediated decrease in GSH level and induction of ceramide
production (Fig. 5). Similar to diamide,
buthione-(SR)-sulfoximine also decreased the level of GSH
and induced the production of ceramide (data not shown). In light of
the recent report that GSH and similar molecules inhibit the activity
of neutral SMase in vitro and GSSG has higher inhibitory
effect than GSH (18), we investigated the intracellular level of GSSG
in astrocytes treated with TNF-
and diamide. In contrast to the
decrease in intracellular level of GSH (Figs. 4 and 5), both TNF-
and diamide increased the intracellular level of GSSG (Fig.
6). Thus it appears that the low GSH
and/or high intracellular oxidant (ROS) levels induced by cytokines and
thiol-depleting agents facilitated the induction of ceramide
production, whereas the normal levels of GSH and/or low ROS induced or
maintained by the addition of NAC under these conditions blocked the
hydrolysis of sphingomyelin to ceramide. Taken together, these results
demonstrate that the intracellular levels of GSH and/or ROS regulate
the extent to which sphingomyelin is degraded to ceramide, and
ceramide-mediated signaling cascades are transduced.

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Fig. 5.
Effect of diamide, a thiol-depleting agent,
on the induction of ceramide production in rat primary astrocytes.
Cells preincubated with 10 mM NAC for 1 h received
diamide (0.5 mM). At different time intervals, cells were
washed with HBSS and scraped off. A, lipids were extracted,
and the level of ceramide (100% value is 4.51 ± 0.1 nmol/mg
protein) was measured as described under "Materials and Methods."
Results are mean ± S.D. of three different experiments.
B, at different time intervals, intracellular level of GSH
(100% value is 182.5 ± 15.4 nmol/mg protein) was measured as
described under "Materials and Methods." Results are mean ± S.D. of three different experiments.
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Fig. 6.
Effect of TNF- and diamide on
intracellular level of oxidized glutathione (GSSG) in rat primary
astrocytes. Cell were incubated with TNF- (50 ng/ml) and
diamide (0.5 mM), and at different time intervals, the
intracellular level of GSSG (100% value is 4.9 ± 0.52 nmol/mg
protein) was measured as described under "Materials and Methods."
Results are mean ± S.D. of three different experiments.
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Aminotriazole and Hydrogen Peroxide Induce the Production of
Ceramide in Rat Primary Astrocytes--
Inhibition of
cytokine-mediated induction of ceramide production by antioxidants and
induction of ceramide production by thiol-depleting agents alone
suggest the possible involvement of ROS in the induction of ceramide
production. Therefore, we examined the effect of exogenous addition of
an oxidant like H2O2 or endogenously produced
H2O2 by inhibition of catalase with
aminotriazole (ATZ), which inhibits endogenous catalase to increase the
level of H2O2, on the induction of ceramide
production. Fig. 7 depicts the time
course of ceramide production in rat primary astrocytes following the
addition of ATZ. Approximately 45 min following the addition of ATZ,
ceramide generation increased more than 5-fold over base line (Fig. 7). However, pretreatment of cells with NAC blocked the ATZ-mediated increase in ceramide production. Consistent with the increase in
ceramide production by ATZ, addition of exogenous
H2O2 to astrocytes also induced the production
of ceramide with the maximum increase of about 7-fold after 15 min.
These results clearly indicate that intracellular levels of ROS
regulate the production of ceramide.

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Fig. 7.
Effect of aminotriazole and
H2O2 on the induction of ceramide production in
rat primary astrocytes. Cells were incubated with 5 mM
aminotriazole (ATZ) (A) or 0.5 mM
H2O2 (B) in presence or absence of
10 mM NAC. At different time intervals, cells were washed
with HBSS and scraped off. Lipids were extracted, and the level of
ceramide (100% value is 4.51 ± 0.1 nmol/mg protein) was measured
as described under "Materials and Methods." Results are mean ± S.D. of three different experiments.
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Inhibition of Cytokine-mediated Production of Ceramide in Rat
Primary Microglia, Oligodendrocytes, and C6 Glial Cells by
NAC--
Since NAC inhibited the cytokine-mediated production of
ceramide in rat primary astrocytes, we examined the effect of NAC on
cytokine-mediated induction of ceramide production in rat primary oligodendrocytes, microglia and C6 glial cells. Fig.
8 shows that addition of TNF-
to
microglia (A), oligodendrocytes (B), or
C6 glial cells (C) induced the production of
ceramide. The increase in ceramide in these cells ranges from 2.5- to
4-fold with highest increase in glial cells and lowest in
oligodendrocytes. The ceramide levels peaked in glial cells at 30 min
following stimulation and 45 min of stimulation in oligodendrocytes and
C6 glial cells. These observations show that similar to
astrocytes, the SM cycle is also present in microglia, oligodendrocytes
and C6 glial cells. Consistent with the effect of NAC on
the production of ceramides in astrocytes, this antioxidant also
potently blocked the TNF-
-induced production of ceramide in
microglia, oligodendrocytes, and C6 glial cells indicating
that ROS are also involved in cytokine-mediated ceramide production in
these cells (Fig. 8).

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Fig. 8.
NAC inhibits TNF- -mediated ceramide
production in rat primary microglia (A), oligodendrocytes
(B), and C6 glial cells (C).
Cells preincubated with 10 mM NAC for 1 h in
serum-free DMEM/F-12 received TNF- (50 ng/ml). Cells were washed
with HBS and scrapped off at different intervals. Lipids were
extracted, and ceramide content (100% value for microglia,
oligodendrocytes, and C6 glial cells are 2.72 ± 0.53, 3.37 ± 0.32, 4.73 ± 0.21 nmol/mg protein, respectively) was
measured as described under "Materials and Methods." Results are
mean ± S.D. of three different experiments.
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NAC Inhibits TNF-
- and Diamide-mediated Apoptosis in Rat Primary
Oligodendrocytes by Increasing the Intracellular Level of GSH and
Decreasing the Production of Ceramide--
Since cytokine-mediated
ceramide production is implicated in apoptosis of different cells
including brain cells (29, 30), we investigated the effect of NAC on
TNF-
as well as diamide-mediated apoptosis in rat primary
oligodendrocytes as evidenced by electrophoretical detection of
hydrolyzed DNA fragments ("laddering"). To understand the role of
the intracellular level of GSH in inducing apoptosis, we treated
oligodendrocytes with TNF-
or with diamide, a thiol-depleting agent.
Both TNF-
and diamide decreased the intracellular level of GSH,
increased the level of ceramide, and induced internucleosomal DNA
fragmentation as evident from the typical ladder pattern (Fig. 9). Interestingly, blocking of the
diamide- and TNF-
-mediated decrease in intracellular levels of GSH
by pretreatment with NAC inhibited the induction of ceramide formation
and DNA fragmentation suggesting that intracellular levels of GSH may
regulate apoptosis in oligodendrocytes through ceramide formation.
To prove this hypothesis further, oligodendrocytes were treated with
C2-ceramide (a cell-permeable ceramide analog) in the
presence or absence of NAC. In contrast to the inhibitory effect of NAC
on TNF-
-mediated apoptosis, NAC had no effect on
C2-ceramide-mediated apoptosis in oligodendrocytes (Fig.
10).

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Fig. 9.
NAC blocks diamide- and TNF- -mediated DNA
fragmentation in rat primary oligodendrocytes by modulating the levels
of GSH and ceramide. Cells preincubated with 10 mM NAC
for 1 h received either diamide (0.5 mM) or TNF-
(50 ng/ml). After 12 h of incubation, cells were harvested and
washed with phosphate-buffered saline, and genomic DNA was extracted
and run on agarose gels (A) as mentioned under "Materials
and Methods." Ten micrograms of DNA was loaded in each lane. This is
the representative of three different experiments. Levels of ceramide
(B) (100% value is 3.37 ± 0.32 nmol/mg protein) and
GSH (C) were measured in homogenates as mentioned under
"Materials and Methods." Results are mean ± S.D. of three
different experiments.
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Fig. 10.
Effect of NAC on
C2-ceramide-mediated DNA fragmentation in rat primary
oligodendrocytes. Cells preincubated with 10 mM NAC
for 1 h received C2-ceramide. After 12 h of
incubation, cells were harvested and washed with phosphate-buffered
saline, and genomic DNA was extracted and run on agarose gels as
mentioned under "Materials and Methods." Ten micrograms of DNA was
loaded in each lane. This is the representative of three different
experiments.
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DNA Fragmentation in Banked Human Brains with X-ALD and MS--
In
the central nervous system, apoptosis may play an important
pathogenetic role in neurodegenerative diseases such as ischemic injury
and white matter diseases (31, 32). Both X-ALD and MS are demyelinating
diseases with the involvement of proinflammatory cytokines in the
manifestation of white matter inflammation. Several studies
demonstrating the induction of proinflammatory cytokines at the protein
or mRNA level in cerebrospinal fluid and brain tissue of MS
patients have established an association of proinflammatory cytokines
(TNF-
, IL-1
, IL-2, IL-6, and IFN-
) with the inflammatory loci
in MS (33-35). Recent documentation of the presence of TNF-
, IL-1
, and IFN-
in X-ALD brain has revealed the neuroinflammatory character of this disease (36, 37). Therefore, to understand the
underlying relationship among intracellular levels of GSH, levels of
ceramide, and DNA fragmentation in cytokine-inflamed central nervous
system of X-ALD and MS, we measured the levels of GSH and ceramide in
homogenates and also studied the DNA fragmentation in nuclei from
brains of patients with X-ALD and MS. Regions surrounding the plaques
in white matter were used for these studies. In contrast to white
matters of control brains, white matters of both X-ALD and MS brains
had several plaque regions. Control 1 is the age- and sex-matched
control for X-ALD and Control 2 is the age- and sex-matched control for
MS (Fig. 11). In both X-ALD and MS
brain homogenates, the level of GSH was lower (55-70% of control),
and the level of ceramide was higher (2-3-fold) compared with those found in control brains (Fig. 11). Consistent with a lower level of GSH
and a higher level of ceramide, genomic DNA isolated from nuclei of
X-ALD and MS brains when run on agarose gels formed the typical ladder
pattern, an indicator of apoptosis, which was absent in both of the
normal brains (Fig. 11). To confirm apoptosis in regions surrounding
the plaques of white matters of X-ALD and MS, paraffin-embedded tissue
sections of X-ALD and MS were stained with terminal
deoxynucleotidyltransferase-mediated fragment end labeling. Consistent
with increased DNA fragmentation in isolated nuclei of X-ALD and MS, we
observed increased terminal deoxynucleotidyltransferase staining on
brain sections of X-ALD and MS compared with those of controls (Fig.
12). These biochemical and
morphological observations indicate that intracellular level of GSH may
be an important factor in cytokine-mediated degradation of SM to
ceramide and apoptosis in inflammatory demyelinating diseases like
X-ALD and MS.

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Fig. 11.
Levels of GSH and ceramide and DNA
fragmentation in human brains with X-ALD and MS. Regions
surrounding plaques of brain white matter were used for DNA laddering
and to measure the levels of ceramide and GSH. Control 1 and control 2 are age- and sex-matched controls for X-ALD and MS, respectively. Since
there was no plaque in control brains, we used white matter of control
brain for this study. A, genomic DNA isolated from nuclei of
banked human brains was run on agarose gel and photographed as
mentioned under "Materials and Methods." Ten micrograms of DNA was
loaded in each lane. This is the representative of three different
experiments. B, same amount of brain material (based on
protein concentration) was used to measure the level of ceramide as
mentioned under "Materials and Methods." Results are mean ± S.D. of three different experiments. Concentrations of ceramide in
control-1 and control-2 were 46.6 ± 2.56 and 61.6 ± 6.69 nmol/mg protein, respectively. C, concentration of GSH was
measured in homogenates as mentioned under "Materials and Methods."
Results are mean ± S.D. of three different experiments.
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Fig. 12.
Fragment end labeling of DNA on tissue
sections of X-ALD and MS brains. Terminal
deoxynucleotidyltransferase-mediated end labeling of 3'-OH ends of DNA
fragments on paraffin-embedded tissue sections (A, control;
B, X-ALD; C, MS) was carried out using a
commercially available kit from Calbiochem. Arrows indicate
apoptotic bodies. Regions surrounding plaques were used for this
study.
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DISCUSSION |
Changes in the cellular redox state toward either prooxidant or
antioxidant conditions have profound effects on cellular functions. Several lines of evidence presented in this work suggest that the first
step of cytokine-induced sphingomyelin signal transduction pathway
(i.e. breakdown of sphingomyelin to ceramide and
phosphocholine) is redox-sensitive. First, cytokines like TNF-
and
IL-1
decreased intracellular GSH and induced the degradation of
sphingomyelin to ceramide in rat primary astrocytes, oligodendrocytes,
microglia, and rat C6 glial cells, and pretreatment of the
cells with antioxidants like NAC restored the levels of GSH and blocked
the degradation of sphingomyelin to ceramide. Second, depletion of
endogenous glutathione by diamide or buthione sulfoximine alone induces
the degradation of sphingomyelin to ceramide which is blocked by NAC. Third, the increase in intracellular H2O2 by
the addition of exogenous H2O2 or by the
inhibition of endogenous catalase by aminotriazole induced the
degradation of sphingomyelin to ceramide which is also blocked by NAC.
Fourth, besides NAC, PDTC, an antioxidant but not the precursor of GSH
(38), also inhibited the TNF-
- and IL-1
-induced hydrolysis of
sphingomyelin to ceramide.
The signaling events in cytokine-mediated activation of sphingomyelin
degradation to ceramide are poorly understood. Since the discovery of
the sphingomyelin cycle, several inducers have been shown to be coupled
to sphingomyelin-ceramide signaling events, including
1
,25-dihydroxyvitamin D3, radiation, antibody
cross-linking, TNF-
, IFN-
, IL-1
, nerve growth factor, and
brefeldin A (1-5, 39). In the case of TNF-
, the pathway is
initiated by the action of TNF-
on its 55-kDa receptor, leading to
phospholipase A2 activation, generation of arachidonic
acid, and subsequent activation of sphingomyelinase (11). Over the
years a number of sphingomyelinase activities have been observed in the
cell. The major activities are the acid sphingomyelinase present in
lysosomes, an enzyme with deficient activity in Niemann-Pick disease
(10), and plasma membrane-associated magnesium-dependent neutral pH optimal sphingomyelinase
(9). In addition, a cytosolic magnesium-independent (40) and
zinc-dependent acidic (41) sphingomyelinase have also been
reported. The lysosomal acidic sphingomyelinase is believed to be
responsible for degradation of sphingomyelin associated with
turnover of membrane. The membrane-associated neutral sphingomyelinase
is known to be activated in serum deprivation, TNF-
, and
Fas-associated growth suppression and apoptosis (42, 43). Although the
studies reported here do not identify the sphingomyelinase that is
redox-sensitive, it is likely that the observed redox-sensitive
hydrolysis of sphingomyelin in cytokine-induced production of ceramide
is mediated by the plasma membrane-associated neutral sphingomyelinase.
A recent observation has shown that glutathione or similar molecules
inhibit the neutral magnesium-dependent neutral
sphingomyelinase (18). However, surprisingly, the SH group of GSH was
not required because S-methyl GSH and GSSG inhibited sphingomyelinase at lower concentrations than GSH. Our studies show
that cytokine-mediated degradation of SM to ceramide in astrocytes, oligodendrocytes, microglia, and C6 cells is a
redox-sensitive process that may have a role in neuroinflammatory
disease process.
Several studies support a role for hydrolysis of sphingomyelin as a
stress-activated signaling mechanism in which ceramide plays a role in
growth suppression and apoptosis in various cell types including glial
and neuronal cells (29, 30). Ceramide activates the proteases of the
interleukin-converting enzyme (ICE) family (especially
prICE/YAMA/CPP32), the protease responsible for cleavage of
poly(A)DP-ribose polymerase (44), and that the activation of prICE by
ceramide and induction of apoptosis are inhibited by overexpression of
Bcl-2 (45). Addition of exogenous ceramides or sphingomyelinase to
cells induces stress-activated protein kinase-dependent
transcriptional activity through the activation of c-jun (46), and a
dominant negative mutant of SEK1, the protein kinase responsible for
phosphorylation and activation of stress-activated protein kinase,
interferes with ceramide-induced apoptosis (47). These observations
also suggest that both Bcl-2 and stress-activated protein kinase
function downstream of ceramide in the apoptotic pathway. Our studies
showing DNA fragmentation and increase in ceramide and decrease in GSH
in primary oligodendrocytes and banked human brains with X-ALD and MS
clearly indicate that intracellular redox (level of GSH) is an
important regulator of apoptosis via controlling the generation of
ceramide. Our conclusion is based on the following observations. First,
treatment of oligodendrocytes with TNF-
decreased intracellular
level of GSH, increased degradation of SM to ceramide, and induced DNA
fragmentation; however, pretreatment of oligodendrocytes with NAC
blocked the TNF-
-mediated decrease in GSH level, increase in
ceramide level, and increase in DNA fragmentation. In contrast, NAC had
no effect on ceramide-mediated DNA fragmentation. Second, treatment of
oligodendrocytes only with diamide, a thiol-depleting agent, decreased
intracellular level of GSH, increased level of ceramide, and induced
DNA fragmentations which are prevented by pretreatment of NAC, a
thiol-replenishing agent. Third, we observed increased fragmentation of
DNA in the white matter region surrounding plaques from patients with
X-ALD and MS where the levels of GSH and ceramide were lower and
higher, respectively, compared with those found in white matters of
control human brains. These observations clearly suggest that
maintenance of the thiol/oxidant balance is crucial for protection
against cytokine-mediated ceramide production and thereby against
ceramide-induced cytotoxicity.
Recent observations demonstrated that ceramide potentiates the
cytokine-mediated induction of inducible nitric oxide synthase in
astrocytes and C6 glial cells (48). Although ceramide by itself did not induce the expression of inducible nitric oxide synthase
and production of NO, it markedly stimulated the cytokine-induced expression of inducible nitric oxide synthase and production of NO
suggesting that sphingomyelin-derived ceramide generation may be an
important factor in cytokine-mediated cytotoxicity in neurons and
oligodendrocytes in neuroinflammatory diseases. The NAC, which has been
used to block the cytokine-induced ceramide production in this study
and to inhibit cytokine-mediated induction of inducible nitric oxide
synthase in a previous study (49), is a nontoxic pharmaceutical drug
that enters the cell readily and serves both as a scavenger of ROS and
a precursor of GSH, the major intracellular thiol (50). Therefore, the
use of reductants such as NAC or other thiol compounds may be
beneficial in restoring cellular redox and in inhibition of
cytokine-mediated induction of inducible nitric oxide synthase and
breakdown of sphingomyelin thus reducing NO-mediated cytotoxicity as
well as ceramide-mediated apoptosis in neuroinflammatory diseases.