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The Journal of Neuroscience, July 15, 2000, 20(14):5292-5299
Modulation of Interleukin-1 and Tumor Necrosis Factor  Signaling by P2 Purinergic Receptors in Human Fetal
Astrocytes
Judy S. H.
Liu1,
Gareth
R.
John1,
Andrew
Sikora2,
Sunhee C.
Lee1, and
Celia F.
Brosnan1, 3
Departments of 1 Pathology, 2 Microbiology
and Immunology, and 3 Neuroscience, Albert Einstein College
of Medicine, Bronx, New York 10461
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ABSTRACT |
In human astrocytes, interleukin-1 (IL-1) is a potent inducer
of genes associated with inflammation. In this study, we tested the
hypothesis that in primary cultures of human fetal astrocytes signaling
by the P2 purinergic nucleotide receptor pathway contributes to,
or modulates, cytokine-mediated signal transduction. Calcium imaging
studies indicated that most cells in culture responded to ATP, whereas
only a subpopulation responded to UTP. Pretreatment of astrocytes with
P2 receptor antagonists, including suramin and periodate oxidized ATP
(oATP), resulted in a significant downregulation of IL-1-stimulated
expression of nitric oxide, tumor necrosis factor (TNF ), and IL-6 at
both the protein and mRNA levels, without affecting cell viability. In
cells transiently transfected with reporter constructs, IL-1
demonstrated more potent activation of the transcription factors
nuclear factor - B (NF-B) and activator protein-1 (AP-1) than
TNF. However, pretreatment with oATP downregulated activation of
NF-B and AP-1 by IL-1 or TNF. Electromobility shift assays
using oligonucleotides containing specific NF-B binding sequences
confirmed that pretreatment with oATP or apyrase attenuated
cytokine-mediated induction of this transcription factor. From these
data, we conclude that P2 receptor-mediated signaling intersects with
that of IL-1 and TNF to regulate responses to cytokines in the
CNS. Because inflammation, trauma, and stress all lead to the release
of high levels of extracellular nucleotides, such as ATP and UTP,
signaling via P2 receptors may provide a mechanism whereby cells can
sense and respond to events occurring in the extracellular environment
and can fine tune the transcription of genes involved in the
inflammatory response.
Key words:
P2 receptors; IL-1; TNF; human fetal astrocytes; transcription factor NF-B; transcription factor AP-1
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INTRODUCTION |
Astrocytes maintain the homeostatic
environment of the CNS and play an important role in regulation
of immune responses by functioning as a source of proinflammatory
cytokines, chemokines, and immune effector molecules (Ransom and
Sontheimer, 1992 ; Norenberg, 1997). Astrocytic processes envelop
neurons and form a glial limitans at the subpial surface and around the
vasculature. Imaging studies have shown that these cells are
interconnected by at least two distinct pathways: an intercellular
pathway mediated by gap junctions and an extracellular pathway mediated
by P2 purinergic receptors that respond to nucleotides such as ATP
(Guthrie et al., 1999). Regulation of these properties of astrocytes in
various disease states may therefore represent an important component
of the maintenance of appropriate neuronal function.
The cytokine IL-1 acts on human astrocytes to induce expression of
multiple genes associated with inflammation, such as interleukin-6, tumor necrosis factor- (TNF), and the inducible form of nitric oxide synthase (NOS II) (Lee et al., 1993a,b), as well inducing a
reorganization of the cytoskeleton, resulting in a shape change (stellation) resembling a reactive gliosis (Liu et al., 1994). Recently, we showed that treatment of human astrocytes with IL-1 also profoundly affected astrocyte communication pathways, resulting in
an uncoupling of gap junctions and, conversely, an upregulation of P2
receptor-mediated signaling (John et al., 1999). Because events such as
mechanical stress, trauma, and inflammation all lead to the release of
high levels of extracellular nucleotides, the ability to detect changes
in the source and local concentration of these molecules may provide
the cell with important information about events occurring in the
extracellular environment. Most cell types express both P1 and P2 type
receptors, and signaling via these receptors modulates many biological
effects, including neurotransmission, inflammation, and immune-mediated
reactivity (Dubyak and el-Moatassim, 1993; DiVirgilio, 1995; Burnstock,
1997). Astrocytes express predominantly members of the metabotropic P2Y receptor family, which are seven transmembrane-spanning receptors coupled to G-proteins (King et al., 1996; Centemeri et al., 1997). P2Y1, a member of the P2 family of receptors, has
been shown recently to mediate calcium wave propagation in rat
astrocyte cultures derived from dorsal spinal cord (Fam et al.,
2000).
In this study, we tested the hypothesis that autocrine/paracrine
signaling via P2 receptors modulates the response of astrocytes to
IL-1. Binding of IL-1 drives dimerization of the IL-1 receptor type 1 with its accessory protein, followed by the recruitment and
phosphorylation of the receptor associated kinases (IRAK1 and IRAK2)
via MyD88. IRAK-1 subsequently interacts with TNF receptor-associated factor-6, followed by phosphorylation of nuclear factor-B
(NF-B)-inducing kinase, which is a mitogen-activated protein (MAP)
kinase kinase kinase that phosphorylates and activates the IB
kinases, resulting in the translocation of NF-B to the nucleus
(Mercurio and Manning, 1999). IL-1 also activates a distinct MAP
kinase cascade that results in DNA binding of activator protein-1
(AP-1) (Minden and Karin, 1997). TNF activates similar cascades, but
cell-type and species-specific differences exist in the relative
potency that these two cytokines display (Ghosh et al., 1998). We now
show that autocrine/paracrine signaling via P2 receptors significantly modulates both IL-1- and TNF-mediated activation of NF-B and AP-1 in human fetal astrocytes, supporting a regulatory role for these
receptors in inflammatory reactions in the human CNS.
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MATERIALS AND METHODS |
Astrocyte cultures and cytokines. Enriched human
fetal brain astrocyte cultures were established from second trimester
abortuses as described previously (Lee et al., 1992). Culture purity
was determined by immunostaining for glial fibrillary acidic protein (astrocytes), microtubule-associated protein-2 (neurons), and CD68
(microglia). All tissue collection was approved by the Institutional Clinical Review Committee. Recombinant human IL-1 was a gift from
the Biological Response Modifiers Program at the National Cancer
Institute (Frederick, MD), and TNF and interferon- (IFN) were
from Genzyme (Cambridge, MA).
Treatment of cell cultures with antagonists of purinergic
receptors. Periodate oxidized ATP (oATP) (Sigma, St. Louis, MO), suramin (Calbiochem, San Diego, CA), and 8-(sulfophenyl) theophylline (SPX) (Sigma) were dissolved in DMEM and used at the
concentrations indicated. Cells were pretreated for 2 hr before
cytokine stimulation. Medium was changed in both oATP-treated and
control cultures immediately before addition of cytokines as indicated.
All other inhibitors were left in the medium during cytokine treatment
of cultures.
Calcium measurements. Astrocytes were plated on uncoated
glass-bottomed microwells (Mat-Tek, Ashland, MA) at a density of 2.5 × 105 cells/ml. At 7-30 d after
plating, confluent cultures were loaded with 10 µM Indo-1 AM (Molecular Probes, Eugene, OR) at
37°C for 45 min and rinsed in PBS, pH 7.4, and the ratio of
Indo-1 fluorescence intensity emitted at two wavelengths (390-440 and
>440 nm) was imaged using UV laser excitation at 351 nm on a Nikon
(Tokyo, Japan) RCM 8000 real-time confocal microscope. After
background and shading correction with UV large pinhole and Nikon 40×
water immersion objective (NA, 1.15; working distance, 0.2 mm), Indo-1 fluorescence ratio images were continuously acquired at 1 Hz before and
after application of a chemical stimulus. The efficacy of calcium
mobilization was calculated as the proportion of cells within the field
responding with an increase in intracellular calcium concentration.
Half-maximal calcium increases were obtained from sigmoidal curves
fitted to the ascending phase of each Indo-1 fluorescence ratio increase.
Detection of nitric oxide formation. After activation with
cytokines, the levels of nitrite in the cell supernatant were measured at the times indicated by the Griess reaction as described previously (Lee et al., 1993b).
Northern blot analysis. At indicated times, total RNA was
extracted by Trizol (Life Technologies, Gaithersburg, MD), and Northern blot analysis for type II NOS and IL-6 was performed as described previously (Lin et al., 1996, 1998). Human hepatocyte type II NOS cDNA
was kindly provided by Dr D. Geller (University of Pittsburgh, Pittsburgh, PA), and human IL-6 cDNA was provided by Dr. T. Hirano (Osaka University Medical School, Osaka, Japan).
Cytokine ELISA. Astrocytes were cultured at 2-4 × 104 cells per well in 96 well plates.
Culture medium was changed at 0 hr, astrocytes were stimulated in
triplicate by cytokines at doses indicated, supernatants were collected
at 16 hr, and levels of TNF and IL-6 in the supernatant was
determined by ELISA (R & D Systems, Minneapolis, MN; Immunotech,
Westbrook, ME).
Transient transfection of astrocytes and reporter gene
activity. Cultures of primary human astrocytes were transfected
using Lipofectamine (Life Technologies). Briefly, 1 µg of the
NF-B-luciferase reporter construct pIg -Luc (Fujita et al., 1993)
or AP-1 reporter construct 6AP-1-luc (gift from Roya Khosravi-Far,
Harvard Medical School, Boston, MA) and 10 µl of lipofectamine were
incubated for 30 min in 200 µl of serum- and antibiotic-free medium
to form DNA liposome complexes. Astrocytes in six well plates (80%
confluent) were incubated with the transfection mix for 5 hr and then
transferred to medium containing 5% FBS. Eighteen hours later, cells
were treated with the indicated agents for 6 hr and harvested with 300 µl of reporter lysis buffer (Promega, Madison, WI). Twenty microliters of lysate were added to 100 µl of luciferase substrate (Promega) for 7 sec, and relative light units (RLU) were determined (Lumat LB luminometer; Berthold, Bad Wildbad, Germany). In preliminary experiments, 0.3 µg of cytomegalovirus (CMV)--galactosidase
(Promega) was cotransfected with reporter constructs to control for
differences in transfection efficiency between wells; however, it was
found that cytokine simulation consistently upregulated CMV-driven
constructs and that transfection efficiency did not differ between
wells from the same cases plated at the same density. Percent reduction of IL-1-induced NF-B induction was determined after
subtraction of control values.
Electromobility shift assay. Astrocytes in 100 mm dishes
were pretreated for 2 hr with 300 µM oATP, the
medium was replaced, and cells were treated with 10 ng/ml IL-1 for
30 min. Nuclear extracts were prepared on ice using a modified Dignam
method (Akama et al., 1998). All buffers were supplemented with 1 mM PMSF, 1 mM DTT, and a
cocktail of protease inhibitors (Boehringer Mannheim, Indianapolis,
IN). Cells (~1 × 107) were scraped
into 1.2 ml of 1 mM PMSF, calcium-magnesium free PBS, and pelleted in microcentrifuge tubes. Pellets were resuspended in
low-salt buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, and 10 mM KCl) and allowed to sit on ice for 10 min
before the addition of 75 µl of 10% Nonidet P-40. Samples were then
vortexed for 10 sec and spun at 13,000 × g for 30 sec.
The nuclear pellet was then resuspended in high-salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, and
0.2 mM EDTA) and allowed to rock gently at 4°C
for 30 min before centrifugation for 15 min (13,000 × g). Supernatants were collected, and protein content was
determined using the Bradford assay.
An electromobility shift assay (EMSA) was performed using the Gel Shift
Assay Core System (Promega) and -32P
labeled oligonucleotides containing the NF-B consensus binding sequence (5' AGT TGA GGG GAC TTT CCC AGG C-3'). Nuclear extracts (4 µg) were incubated in binding buffer [4% glycerol, 1 mM
MgCl2, 0.5 mM EDTA, 50 mM
NaCl, 10 mM Tris-HCl, and 50 µg/ml poly(dI-dC)] with
1.75 pmol of either specific or mutant oligonucleotides (Santa Cruz
Biotechnology, Santa Cruz, CA) or nonspecific competitor oligonucleotides for 15 min before addition of labeled probe. After
incubation at room temperature for 20 min, supershift antibodies (2 µg; Santa Cruz Biotechnology) were added for another 40 min. Samples
were separated on a 5.5% polyacrylamide, 5% glycerol gel in
Tris-glycine buffer.
Western blot analysis of NOS II protein expression. Cells
were harvested 48 hr after cytokine treatment, and NOS II protein expression was determined as described previously using an
affinity-purified rabbit polyclonal antibody against human macrophage
NOS II (1:1000; Transduction Laboratories, Lexington, KY).
Statistics. Statistical analysis was performed using
Student's t test or ANOVA. p < 0.05 was
considered significant.
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RESULTS |
Characterization of calcium response to nucleotide triphosphates in
cultures of primary human fetal astrocytes
Human fetal astrocyte cultures loaded with Indo-1 AM were
subjected to laser confocal microscopy to determine the response of
these cells to the nucleotide triphosphates UTP and ATP. The cells were
first challenged with UTP (100 µM). Approximately 30% responded with an increase in intracellular calcium concentration (Fig.
1, compare A, B).
The cells were then washed. When intracellular calcium had returned to
baseline levels, the same cells were challenged with ATP (100 µM); as demonstrated in C, all
astrocytes responded with a marked increase in intracellular calcium.
These results indicated that the population of cells within these
primary cultures was heterogeneous and that although, human fetal
astrocytes responded to ATP, a proportion also expressed a receptor
that responded to UTP. To determine whether the responses could be
blocked by P2 receptor antagonists, astrocyte cultures were pretreated
with oATP for 2 hr (300 µM)
(D-F). The chemical structure of oATP and its
affinity for a wide range of ATP binding proteins suggests that it
would interact with a number of P2 receptor subtypes. The cells were
then challenged with UTP (E) or ATP
(F), and their responses were compared with those of
control untreated cultures. The rise in intracellular calcium
stimulated by 100 µM UTP was completely blocked
by oATP, whereas the response to 100 µM ATP was
blocked in ~55% of cells by oATP.
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Figure 1.
Exogenously added nucleotides results in transient
elevations of intracellular calcium in human astrocytes that is blocked
by oATP. Astrocytes were loaded with 10 µM Indo-1 for 45 min and visualized by real-time confocal imaging microscopy. Increase
in intracellular calcium was visualized as a shift toward the red
spectrum. A-C show the same field without stimulation
(A) and after exposure to 100 µM
UTP (B) or 100 µM ATP
(C). D-F are also of the same
field in cultures pretreated with 300 µM oATP, showing
control no stimulation (D) and 100 µM UTP (E) or 100 µM
ATP (F). Note the similarity in basal
intracellular calcium levels in control (A) and
oATP-treated (D) cells. All panels
of nucleotide-treated cultures are shown at mean peak intracellular
calcium concentrations after treatments. The experiment shown is one of
three, each on cells derived from different brains. Scale bars, 25 µm.
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Blockade of P2 receptors on astrocytes downregulates
nitrite production
In previous studies, we showed that the expression of type II NOS
in human fetal astrocytes was critically dependent on activation with
IL-1, with IFN acting synergistically (Lee et al., 1993b; Liu et
al., 1996). To determine whether signaling via the P2 receptor pathway
modulated NOS II expression, astrocytes were pretreated with a panel of
P2 receptor antagonists for 2 hr and then stimulated with IL-1 (10 ng/ml) plus IFN (200 U/ml), and nitrite levels in the cell
supernatant were measured at 48 hr (Fig.
2). Pretreatment of the cells with the
classical P2 receptor antagonist suramin or oATP resulted in a
significant dose-dependent downregulation of nitrite production, with
oATP demonstrating greater inhibition than suramin at the lowest doses
tested (75-300 µM). A similar effect was noted with
pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid (data not
shown). In contrast, the P1 receptor antagonist SPX had no effect,
indicating no role for P1 receptors that preferentially bind adenosine
or AMP but not ADP or ATP. None of the inhibitors when given alone
affected nitrite production, and none of the inhibitors was toxic for
the cells, as determined by lactate dehydrogenase release or trypan
blue exclusion (data not shown). These data suggested that ATP was
present in the culture medium, and this was confirmed using an assay
for the conversion of luciferin to luciferase, a reaction that requires
ATP. Medium from astrocyte cultures had 10,713 ± 1387 RLU (~ 0.4 µM) in contrast to unconditioned medium, 2217 ± 303 RLU (~0.05 µM). Because blockade of P2 receptors by
oATP has been shown to be irreversible (Murgia et al., 1993), it was
used for all subsequent experiments and washed out of the medium before
cytokine exposure.
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Figure 2.
Blockade of P2 receptors on astrocytes
downregulates nitrite production. Astrocytes were pretreated for 2 hr
with the inhibitors oATP, suramin, and SPX, at concentrations of 300, 150, and 75 µM. oATP was washed out before the addition
of IL-1 (10 ng/ml) plus IFN (200 U/ml). Nitrite was determined at
48 hr. The P2 inhibitors oATP and suramin demonstrated a dose-dependent
inhibition of nitrite production. In contrast, the P1 inhibitor SPX had
no effect. *p < 0.05 indicates statistically
significant reduction of nitrite compared with cytokine stimulation
without inhibitors. Data represent the mean and SD of astrocytes from
five different wells per data point and are representative of four
separate experiments on cells derived from different brains.
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Blockade of P2 receptors on astrocytes downregulates induction of
TNF and IL-6
To determine whether antagonism of P2 receptors affected the
expression of other genes activated in astrocytes by IL-1, the cells
were pretreated with oATP, washed, and activated with either IL-1
(10 ng/ml) alone or IL-1 plus IFN (200 U/ml). After incubation for 16 hr, the levels of TNF and IL-6 in the supernatant were determined by ELISA, and nitrite production was measured at 48 hr (Fig.
3). Consistent with previous data (Lee et
al., 1993b; Liu et al., 1996), IFN synergized with IL-1 in the
induction of NOS II and TNF in these cells, whereas the production
of IL-6 was not augmented by costimulation with IFN. Pretreatment
with oATP resulted in a dose-dependent inhibition of the production of
nitrite, TNF, and IL-6 in samples stimulated by either IL-1 alone
or IL-1 plus IFN.
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Figure 3.
Blockade of P2 receptors on astrocytes
downregulates induction of TNF, IL-6, and nitrite production.
Astrocytes were pretreated with varying doses of oATP (0, 300, or 30 µM) for 2 hr, washed, and exposed to IL-1(10 ng/ml) or
IL-1 plus IFN (200 U/ml) as indicated. Control cultures
(con) consisted of astrocytes treated with oATP alone
(300 µM) without cytokines. TNF
(B) and IL-6 (C) levels in
the supernatant were determined at 16 hr and nitrite
(A) at 48 hr. Pretreatment with oATP resulted in
a dose-dependent inhibition of nitrite, TNF, and IL-6 after
stimulation with IL-1 alone or in combination with IFN. Note also
that, whereas IFN potently synergized with IL-1 for nitrite and
TNF production, it had little effect on IL-6 expression.
*p < 0.05 indicates statistically significant
reduction compared with cytokine stimulation without inhibitors. Data
represent the mean and SD of results from three different wells and are
representative of cells derived from three different brains.
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Inhibition of astrocyte P2 receptors results in inhibition of NOS
II, TNF, and IL-6 mRNA accumulation
Cytokines regulate the production of nitrite by transcriptional
activation of the NOS II gene. To test whether pretreatment with oATP
downregulated IL-1-induced gene expression in these cultures, the
levels of mRNA for TNF, IL-6, and NOS II were determined by Northern
blotting. Astrocyte cultures were treated as described above, and RNA
was harvested at 6 hr for the determination of TNF mRNA and at 24 hr
for the determination of both IL-6 and NOS II mRNA, time points that we
have shown previously represent peak levels of gene expression after
activation with IL-1 (Liu et al., 1996). Pretreatment with oATP led
to a dose-dependent reduction in mRNA expression for NOS II and IL-6
(Fig. 4A) and TNF
(data not shown), in cultures activated with either IL-1 or IL-1
plus IFN. These data indicate that pretreatment with oATP blocked
IL-1-induced expression of these genes by inhibiting the
accumulation of specific mRNAs in the cell. Western blotting of
cellular homogenates from these cultures confirmed that pretreatment with oATP led to an inhibition of cytokine-induced expression of NOS II
protein (Fig. 4B). From these data, we conclude that antagonism of the P2 receptor downregulated the expression of multiple
genes induced in human fetal astrocytes by IL-1.
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Figure 4.
Blockade of astrocyte P2 receptors results in
inhibition of NOS II, and IL-6 mRNA and NOS II protein expression.
A, RNA was extracted 24 hr after cytokine treatment from
the same cultures described in Figure 3 and subjected to Northern
analysis of NOS II, IL-6, and 18 S mRNA (A).
Densitometric ratios of NOS II and IL-6 to 18 S are given
below each lane. The results show that P2
blockade acts at the level of mRNA expression. Data shown are
representative of three separate experiments on cells derived from
three different brains. B, NOS II protein expression was
determined by Western blot analysis of homogenates. Astrocytes were
pretreated with 0, 30, or 300 µM oATP for 2 hr, washed,
and treated with IL-1 (10 ng/ml) plus IFN (200 U/ml) for 48 hr.
Cytokine stimulation resulted in strong induction of NOS II (130 kDa
band) that was markedly reduced in cells that had been pretreated with
300 µM oATP. Data shown are representative of four
independent experiments.
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IL-1 and TNF induction of NF-B transcriptional activation
is dose-dependent and downregulated by oATP
Because oATP pretreatment resulted in inhibition of three IL-1
inducible proteins NOS II, TNF, and IL-6, we hypothesized that
blockade of P2 receptors modulated the IL-1 signal transduction cascade. In most cell types, binding of IL-1 to the type I receptor activates the acute phase transcription factor NF-B, which is an
inducible enhancer of many inflammatory genes, including NOS II
(Marks-Konczalik et al., 1998; Taylor et al., 1998; Mercurio and
Manning, 1999). TNF is also a potent activator of NF-B, and the
signaling pathways activated by IL-1 and TNF are thought to
converge at the level of the NF-B inducing kinase (Ghosh et al.,
1998). However, in contrast to IL-1, TNF does not lead to the
activation of NOS II expression in human fetal astrocytes and only
minimally synergizes with IL-1 and IFN in NOS II expression (Liu
et al., 1996). To compare the level of activation of NF-B by IL-1
and TNF in human fetal astrocytes, we transiently transfected cells
with the NF-B-dependent reporter Ig-Luc, stimulated the cells
with three different doses of IL-1 and TNF, and measured luciferase activity after 6 hr. Both IL-1 and TNF treatment resulted in a dose-dependent induction of NF-B, but the fold induction over untreated control cultures induced by TNF was much
lower than that induced by IL-1 (Fig.
5A). Only the maximum dose
tested of TNF (100 ng/ml) resulted in activation that was significantly above baseline (p > 0.05).
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Figure 5.
IL-1 and TNF activation of NF-B is
dose-dependent and downregulated by P2 receptor blockade.
A, Astrocyte cultures were transiently transfected with
a reporter construct for NF-B activity. Cells were treated with
either IL-1 (0.1, 1, or 10 ng/ml) or TNF (1, 10, or 100 ng/ml),
and luciferase activity was measured at 5 hr (A).
Level of luciferase activity (expressed as RLU) in untreated cells is
represented by the transverse line, and SDs of these
background levels are demarcated by the dotted lines.
NF-B induction by both IL-1 and TNF was dose-dependent.
However, IL-1 treatment resulted in much higher levels of NF-B
activation. Data shown represent the mean and SD of three separate
measurements and are representative of three separate experiments
derived from cells from three different brains. B, Cells
were transfected as above and treated with both IL-1 (10 ng/ml) and
TNF (100 ng/ml) in the presence or absence of pretreatment with oATP
(300 µM). As before, IL-1 and TNF resulted in
induction of NF-B (with IL-1 induction greater than TNF
induction), and this was downregulated by pretreatment using oATP
blockade of P2 receptors, which had no effect on background levels of
NF-B activation. Data shown are the mean and SD of three separate
measurements and are representative of three separate experiments with
cells from three different brains.
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We then tested whether P2 receptor blockade affected IL-1- or
TNF-mediated induction of NF-B activity. Astrocytes were transfected with the NF-B reporter construct, as above, pretreated with oATP for 2 hr, washed, and then activated with either IL-1 (10 ng/ml) or TNF (100 ng/ml). The data showed that, whereas incubation
with oATP alone had no effect on baseline activity of NF-B, blockade
of P2 receptors significantly downregulated transcriptional activation
induced by IL-1 or TNF (Fig. 5B). Data for NF-B
activation with and without pretreatment with oATP from five different
brains are shown in Table 1.
IL-1 and TNF stimulate NF-B translocation and DNA binding,
which can be downregulated by P2 blockade and nucleotide diphosphate
and triphosphate depletion
Using an EMSA, we then examined the effect of P2 receptor blockade
on IL-1 (10 ng)- or TNF (100 ng)-induced nuclear translocation and binding to an NF-B consensus sequence. Astrocytes were
stimulated for 30 min with cytokines, and nuclear extracts were
prepared. EMSA revealed that, in samples from untreated cells, three
mobility shift complexes were detected (data for IL-1 are shown in
Fig. 6) that could be competed out with a
50-fold excess of cold oligonucleotide containing the NF-B binding
sequence (lanes NF). As an additional control for
specificity, the same extracts were incubated with a nonspecific cold
oligonucleotide of similar size and guanine and cytosine content
(lanes AP) or a mutant NF-B consensus sequence (see
below), neither of which affected cytokine-induced shift complexes.
IL-1 treatment resulted in the enhancement of the shift complex,
labeled A. To identify the subunits of the IL-1-induced shift complex, the nuclear extracts were incubated with antibodies to
specific subunits of NF-B family proteins (Fig. 6, right
panel). Only antibodies to p65 and p50 resulted in the
appearance of a supershift in the IL-1-induced complex. This result
indicated that the NF-B detected was the conventional p65/p50
heterodimer most commonly induced by inflammatory stimuli. TNF
treatment of astrocytes resulted in a much lower level of NF-B DNA
binding compared with IL-1, although again supershift assays
indicated induction of p65/p50 heterodimer (data not shown). When
nuclear extracts of cells that had been pretreated with oATP before
IL-1 stimulation were subjected to EMSA, less NF-B DNA binding
was observed compared with cultures treated with IL-1 alone. Thus, blockade of P2 receptor signaling downregulated IL-1 induction of an
NF-B mobility shift complex in human astrocytes.
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Figure 6.
IL-1 induction of NF-B binding is
downregulated by blockade of P2 receptor signaling. Astrocyte cultures
were treated with IL-1 in the presence or absence of pretreatment
with oATP. Nuclei were harvested at 30 min and subjected to EMSA with a
radiolabeled oligonucleotide probe containing the NF-B binding site
along with specific (lane NF) and nonspecific
(lane AP) competitor oligonucleotides. The
left shows control samples, IL-1-treated samples, and
IL-1-treated samples after oATP pretreatment. Three shift complexes
were observed (A-C) in control cells. Complex A
was strongly induced by IL-1 and downregulated by oATP pretreatment.
The right panel shows the results of EMSA on
IL-1-treated astrocytes incubated with specific antibodies for
NF-B family members. Only incubation with antibodies to p50 and p65
resulted in the formation of a supershift complex. The experiment shown
is representative of four separate experiments using astrocytes derived
from four different brains.
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The relatively short incubation period with cytokines needed to
demonstrate translocation to the nucleus of NF-B allowed us to test
the effect of depletion of P2 receptor ligands from the medium using
apyrase, an enzyme that degrades nucleotide triphosphates and
diphosphates to nucleosides or nucleotide monophosphates. Astrocyte
cultures were treated for 30 min with apyrase before IL-1
stimulation (10 ng/ml) for 30 min and nuclear extracts were prepared as
above. EMSA showed that treatment with apyrase resulted in a
downregulation of IL-1-mediated binding to the NF-B consensus sequence, which could be competed out with 50-fold excess of cold oligonucleotide, but not one containing a point mutation that abrogates
NF-B binding (Fig. 7). A similar
result was obtained when cells were pretreated with suramin (data not
shown). Thus, either blockade of P2 receptors or depletion from the
culture medium of nucleotide triphosphates and diphosphates that
function as ligands for these receptors resulted in the inhibition of
IL-1-stimulated NF-B nuclear translocation and DNA binding,
implicating P2 receptor signaling in the activation of NF-B by
IL-1.
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Figure 7.
IL-1 induction of NF-B binding is
downregulated by breakdown of extracellular nucleotides by apyrase.
Astrocyte cultures were treated with IL-1 in the presence or absence
of apyrase (lane apy; 20 U/ml) pretreatment for 30 min
or oATP (lane oATP) pretreatment as described
previously. Nuclei were harvested at 30 min and subjected to EMSA with
a radiolabeled oligonucleotide probe containing the NF-B binding
site along with wild-type (lane NF) and mutant
(lane mut) competitor oligonucleotides. Control samples,
IL-1-treated samples, and IL-1-treated samples after apyrase
pretreatment or oATP are shown. Three shift complexes were observed
(A-C) in control cells. Complex A was strongly
induced by IL-1 and downregulated by oATP pretreatment, as well as
by apyrase. Quantitative analysis of these data is shown
below each lane and represents the
densitometric ratios for complex A compared with the value for the
complex A in the control sample (left lane). Data shown
are representative of two separate experiments with cells from two
different brains.
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IL-1 and TNF activation of AP-1 are also downregulated by
P2 blockade
In addition to activation of NF-B, cytokine stimulation also
leads to activation of AP-1, which together with NF-B regulates the
inducible expression of many genes involved in the inflammatory response, including NOS II (May and Ghosh, 1998). To determine whether
the modulatory effect of P2 receptor antagonism was specific for
activation of NF-B, we also transiently transfected primary astrocytes with an AP-1 reporter construct. In human fetal astrocytes, stimulation with either IL-1 or TNF induced a dose-dependent activation of AP-1 (Fig.
8A). IL-1 again
provided a stronger stimulus than TNF for AP-1 activation, although
the fold induction of AP-1 was not as potent as that obtained for
NF-B, which we ascribe to the higher background activity of AP-1 in
these cells. Pretreatment with oATP again led to an inhibition of
cytokine-induced activation (Fig. 8B). A similar
effect was observed after pretreatment with suramin (data not shown).
Because blockade of P2 receptors downregulates both NF-B and AP-1
activation in response to IL-1 or TNF, this would imply that
signals from P2 receptor activation interact with common or homologous
elements of both IL-1- and TNF-mediated signal transduction.
View larger version (21K):
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|
Figure 8.
IL-1 and TNF activation of AP-1 is
dose-dependent and downregulated by P2 receptor blockade.
A, Astrocyte cultures were transiently transfected with
a reporter construct for AP-1 activity. Cells were treated with either
IL-1 (0.1, 1, or 10 ng/ml) or TNF (1, 10, or 100 ng/ml), and
luciferase activity was measured in RLU after 4-5 hr
(A). Levels of luciferase activity (expressed as
RLU) in untreated cells is represented by the transverse
line, and SDs of these background levels are demarcated by the
dotted lines. AP-1 induction by both IL-1 and TNF
was dose-dependent. However, IL-1 treatment resulted in higher
levels of AP-1 activation. B, cells were transfected as
above and treated with either IL-1 (10 ng/ml) or TNF (100 ng/ml)
in the presence or absence of pretreatment with oATP as described
above. As before, IL-1 and TNF resulted in induction of AP-1 (with
IL-1 greater than TNF) that was downregulated by pretreatment
with oATP. Blockade of P2 receptors with oATP had no effect on
background levels of AP-1 activation. Data shown are representative of
three separate experiments on cells from three different brains.
|
|
| |
DISCUSSION |
In the normal CNS, the cytokine IL-1 is detected at very low
levels and is restricted to specific neuronal elements (Breder et al.,
1988), but levels increase dramatically in a number of different
inflammatory and degenerative conditions, such as multiple sclerosis
and Alzheimer's disease, with expression localized to activated
microglia and macrophages. Experiments both in vivo and
in vitro have strongly implicated a role for this cytokine in disease pathogenesis in many of these disorders (Rothwell, 1999).
The neurodegenerative activities of IL-1 are thought to be
attributable to the induction of inflammatory mediators such as
the cytokines TNF and IL-6, as well as toxic factors such as the
free radical gas nitric oxide. In cells of human origin, we and others
have proposed that astrocytes are an important source of these factors
after activation with IL-1 (Liu et al., 1996; Chao et al., 1997). In
this study, we now show that the stimulatory effect of IL-1 for
astrocytes can be significantly downregulated by blockade of P2
receptors. This inhibition occurred at the level of mRNA accumulation,
suggesting that signaling via P2 receptors regulates elements of the
IL-1 signaling cascade, and this was confirmed using specific gene
reporter constructs and EMSA for the transcription factors NF-B and
AP-1. Activation by TNF was also found to be downregulated by
blockade of P2Y receptors, although this cytokine mediated only a
low-level activation of NF-B and AP-1. Because astrocytes use P2
receptor signaling as part of an astrocyte-to-astrocyte communication
system (Guthrie et al., 1999), these data provide strong support for
the hypothesis that autocrine/paracrine signaling via these receptors
regulates astrocyte activation and function in response to changes in
the extracellular environment.
That physiologically relevant levels of extracellular ATP are involved
in P2 receptor regulation of astrocytic function is supported by the
observation that this effect was obtained in the absence of added
ligand. In addition, apyrase, an enzyme that catalyzes hydrolysis of
extracellular nucleotides, also downregulated IL-1-mediated
activation of NF-B. In the CNS, ATP has been identified as a
neurotransmitter and would be expected to exist only transiently in the
extracellular space because of the activity of ubiquitous ectoATPases
(Wang and Guidotti, 1998). However, high levels of extracellular
nucleotides are released from sources such as platelets, activated
leukocytes, and damaged or dying cells in a number of injurious
conditions. Extracellular ATP has been implicated in the induction of a
reactive astrogliosis, because in cultured rat astrocytes ATP increases
DNA synthesis and cell proliferation (Neary et al., 1998, 1999),
induces the release of arachidonic acid metabolites (Bruner and Murphy,
1993; Stella et al., 1997; Brambilla et al., 1999), and induces
cytoskeletal changes resulting in process extension (Neary et al.,
1994). In most of these studies, activation of members of the P2Y
family of receptors has been implicated, and our data are in
agreement with this.
Similarly, in the immune system, ATP and its metabolites also modulate
the response of cells to inflammatory signals through interactions with
P2 receptors. In human macrophages, ATP has been shown to provide a
second stimulus required for the processing and secretion of
lipopolysaccharide (LPS)-induced IL-1 (Griffiths et al., 1995;
Ferrari et al., 1997a). In these studies, the activation of the
P2X7 receptor has been implicated, which results
in the formation of a large transmembrane pore that allows the
bidirectional passage of molecules up to 900 Da (DiVirgilio, 1995).
Activation of this receptor with high levels of ATP (3-15
mM) has also been implicated in the induction of NF-B
and subsequent cytotoxicity involving both apoptotic and necrotic
mechanisms (Surprenant et al., 1996; Ferrari et al., 1997b; von
Albertini et al., 1998). We have no evidence from Lucifer yellow
permeability assays on ATP-treated cells that this receptor is active
in our system (data not shown). In both human and murine macrophages,
ATP has also been found to modulate the generation of reactive oxygen
intermediates (Schmid-Antomarchi et al., 1997; Sikora et al., 1999) and
to regulate LPS-induced NOS II and TNF expression both in
vivo and in vitro (Tonetti et al., 1995; Denlinger et
al., 1996; Greenberg et al., 1997; Hu et al., 1998; Sikora et al.,
1999). In most of these studies, the action of extracellular
nucleotides has been found to be stimulatory and to be dependent on
specific agonists. For example, in murine J774 cells, costimulation of
cells with LPS plus UTP potently enhanced NOS II expression through a
mechanism that involved the activation of a
Ca2+/calmodulin-dependent protein kinase,
whereas ATP only modestly increased NOS II expression (Chen et al.,
1998).
In contrast to the primarily proinflammatory activities of
nucleotides on immune function, breakdown products of extracellular nucleotides, most notably adenosine, have been shown to have
immunomodulatory properties. Some purinergic receptors specific for
adenosine have been shown to differentially inhibit macrophage
expression of the proinflammatory cytokines TNF but have variable
effects on IL-6 (Haskó et al., 1996; Sajjadi et al., 1996). In
multiple sclerosis patients, in contrast to normal controls,
dysregulation of adenosine has been demonstrated and correlated with
increased levels of TNF (Mayne et al., 1999). In addition, adenosine
receptor A1 levels were also decreased in multiple sclerosis patients. These studies would suggest that ATP and adenosine have opposing effects on cytokine-stimulated gene expression.
An additional observation made in the course of these studies was that,
in human fetal astrocytes, TNF mediated only a low-level activation
of NF-B and AP-1 compared with IL-1. This is in marked contrast
to what has been shown in the human monocytic cell lines U937 in which
TNF has been found to provide a more potent stimulus for
proinflammatory gene expression (Nasuhara et al., 1999). Intracellular signaling mediated by IL-1 and TNF is thought to converge at the
level of activation of the MAP kinase kinase kinases involved in the
activation of NF-B and AP-1. Both of these transcription factors
have been shown to be required for induction of the human NOS II gene,
and multiple NF-B binding sites have been detected in the human NOS
II promoter (Marks-Konczalik et al., 1998; Taylor et al., 1998). In our
studies, activation of NF-B was reduced but not completely
downregulated by oATP-mediated P2 receptor blockade, whereas NOS II
expression was completely abrogated by the same concentration of oATP.
These data support the concept that threshold levels of NF-B must be
achieved for successful induction of transcription of the human NOS II
gene and that, in cells activated with TNF or with IL-1 plus
oATP, this threshold level of NF-B was not reached (Ghosh et al.,
1998). Alternatively, NOS II induction may depend on the interaction of
multiple transcription factors, including AP-1, that may be
differentially affected by purinergic receptor blockade
(Marks-Konczalik et al., 1998).
The observation that P2Y receptor blockade affected both IL-1 and
TNF activation of NF-B suggests that P2Y signaling interacts with
components of the signal transduction cascade common to both cytokines.
In this regard, it is of interest to note that both Neary et al. (1999)
and Chen et al. (1998) have observed that signaling via ATP also
interacts with the extracellular signal-regulated protein kinases ERK1
and ERK2. Together, these data suggest that P2Y receptor signaling
intersects with common or related components of this family of
serine/threonine protein kinases (Eder, 1997).
In summary, our results indicate that cross-talk between a P2 receptor
signaling pathway and components of the cytokine-activated serine/threonine kinase pathways critically regulates inflammatory gene
expression in human fetal astrocytes. Because inflammatory events in
the CNS have the potential to cause irreversible damage, mechanisms
must exist whereby the cell can appropriately target responses to areas
of immune attack, infection, or trauma but spare adjacent uninvolved
tissues. The different specificities for endogenous agonists displayed
by P2 receptors suggests that the composition and concentration of
nucleotide triphosphates, diphosphates, and monophosphates, as well as
nucleosides, in the extracellular space provides the cell with
important information concerning changes in the extracellular
environment. Furthermore, changes in the expression of these receptors
may alter sensitivity to these events. In this regard, it is of
interest to note that IL-1 also modifies the functional population
of P2Y receptor on both human and rat astrocytes, resulting in the
transient increased expression of a response to UTP, most probably
mediated by a P2Y2 receptor (Stella et al.,
1997; John et al., 1999). From these data, we propose that
autocrine/paracrine activation of P2 receptors permits different cell
types to communicate with each other, and with the extracellular
environment, through the release and sensing of nucleotides such as ATP
and to use this information to fine-tune the response to the extent and
nature of the injury.
| |
FOOTNOTES |
Received Sept. 22, 1999; revised April 18, 2000; accepted April 26, 2000.
This work was supported by National Multiple Sclerosis Society
Grant RG2771 and United States Public Health Service Grants NS11920,
MH55477, and T32GMO7288. We thank Dr. Karen Weidenheim, Director of the
Human Fetal Tissue Repository, for tissue collection. We also thank
David Spray and Eliana Scemes for help with the calcium imaging, and
Meng-Liang Zhao and Wa Shen for culture preparation.
Correspondence should be addressed to Dr. Celia F. Brosnan, Department
of Pathology, Albert Einstein College of Medicine, 1300 Morris Park
Avenue, Bronx, NY 10461. E-mail: brosnan{at}aecom.yu.edu.
| |
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G. R. John, J. E. Simpson, M. N. Woodroofe, S. C. Lee, and C. F. Brosnan
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TOP
ABSTRACT
INTRODUCTION
Compound via MeSH
