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12,1412,14-prostaglandin J2 in Activated Human Astrocytes, But Not in Human Brain Macrophages1
Laboratory of Molecular Medicine and Neuroscience, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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12,14-PGJ2 (15d-PGJ2)
is a ligand for the peroxisome proliferator-activated receptor
(PPAR)
. It has been proposed as an inhibitor of microglial
activation, based on the study of iNOS down-regulation in rodent
microglia. Because iNOS induction after cytokine activation remains
controversial in human microglia, we examined the effect of
15d-PGJ2 and other PPAR agonists on human microglia and
astrocytes, using COX-2 induction as an index of activation. We found
that PPAR
ligands (clofibrate and WY14643) enhanced IL-1
-induced
COX-2 expression in human astrocytes and microglia, while inhibiting
IL-1 plus IFN- induction of iNOS in astrocytes. This is the first
description of an inhibition of iNOS uncoupled from that of COX-2.
15d-PGJ2 suppressed COX-2 induction in human astrocytes. It
prevented NF-
B binding to the COX-2 promoter through a new pathway
that is the repression of NF-Bp50 induction by IL-1. In contrast,
15d-PGJ2 increased c-Jun and c-Fos DNA-binding activity in
astrocytes, which may result in the activation of other inflammatory
pathways. In human microglia, no effect of 15d-PGJ2 on
COX-2 and NF-Bp65/p50 induction was observed. However, the entry of
15d-PGJ2 occurred in microglia because STAT-1 and c-Jun
expression was modulated. Our data suggest the existence of novel
pathways mediated by 15d-PGJ2 in human astrocytes. They
also demonstrate that, unlike astrocytes and peripheral macrophages or
rodent brain macrophages, human microglia are not subject to the
anti-inflammatory effect of 15d-PGJ2 in terms of COX-2
inhibition. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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activates microglia and
astrocytes to secrete several potentially toxic products, including
lipid mediators and free radicals (3, 4, 5). Among these
mediators, PGs and NO are the products of the inducible isoforms of
cyclooxygenase
(COX-2)3 and NO
synthase (iNOS) enzymes, respectively (7). Overexpression
of these proinflammatory enzymes has emerged as an important
determinant of the cytotoxicity associated with inflammation in
pathologies such as Alzheimer’s disease, cerebral ischemia, multiple
sclerosis, and HIV-1 encephalitis (8, 9, 10, 11, 12). Important
differences exist in the molecular regulation of these enzymes between
rodent and human glial cells. For example, in rodents, microglia are
capable of high levels of NO production, particularly after LPS
stimulation in vitro (13). However, the ability of human
microglial cells to produce high levels of NO after LPS or
proinflammatory cytokine stimulation in vitro remains controversial
(14, 15, 16, 17, 18, 19). In contrast, human astrocytes can produce NO
after IL-1 and IFN- stimulation, but not after LPS treatment
(14, 15, 16, 17). Therefore, to study the therapeutic potential of
different anti-inflammatory drugs against human neuropathologies,
we should consider the relevance of results obtained in rodent systems,
compared with human glial cells.
The peroxisome proliferator-activated receptors (PPARs) are
ligand-activated transcription factors belonging to the nuclear
receptor family. Three subtypes of PPARs have been described: PPAR,
, and (20). Several clinically important compounds,
such as the fibrate class of hypolipidemic drugs, the
thiazolidinediones, some nonsteroidal anti-inflammatory drugs, and
fatty acids, can bind to and activate these receptors
(21, 22, 23, 24). Recent studies suggest that some PPAR ligands
may be important anti-inflammatory agents, including several PGs,
of which
15-deoxy-12,14-PGJ2
(15d-PGJ2) is the most potent (21, 22). In stimulated murine and human peripheral blood
macrophages, 15d-PGJ2 binds to and activates
PPAR, resulting in various anti-inflammatory events such as
inhibition of iNOS, COX-2, gelatinase b expression, and inflammatory
cytokine production (21, 22, 23). More recently, this has been
extended to the study of the microglial activation in rodents. In
rodent microglial cells activated with LPS,
15d-PGJ2 has been proposed as a potent suppressor
of brain macrophage activation because it down-regulates iNOS
expression (25, 26). Because the regulation of iNOS
expression differs between human and rodent cells (27), we
examined the anti-inflammatory effects of
15d-PGJ2 and other PPAR agonists on primary
cultures of human microglia and astrocytes using COX-2 induction as an
index of cell activation. Our results demonstrated that
15d-PGJ2 inhibits COX-2 expression in activated
astrocytes by interfering with the NF-B pathway through a novel
mechanism, whereas human brain macrophages were resistant to the
inhibitory effect of this compound. These data suggest that different
pathways exist for the anti-inflammatory effects of
15d-PGJ2 in comparison with human astrocytes,
rodent brain macrophages or peripheral macrophages, and human brain
macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The preparation of cells from human fetal brain has previously been described (14, 28). Purified cultures of microglia and astrocytes were obtained from CNS cells 10–14 days after plating, as described (14). Briefly, microglial cells were released by circular shaking and selected by a 20-min adhesion cycle (>95% CD68/KiM-7-positive cells). After the release of microglial cells, the adherent cells remaining were trypsinized, plated, and passaged three to four times to obtain purified cultures of astrocytes (>95% glial fibrillary acidic protein-positive astrocytes). Cells were maintained in Eagle’s MEM supplemented with 10% FCS, 2 mM L-glutamine, and antibiotics. Cell viability was assessed using thiazolyl blue (MTT) assay, as described (29).
Cell stimulation and reagents
To stimulate cultures, cells were refed with serum-free medium
containing the tested inducer, inhibitor, or agonist. The following
reagents were purchased from the designated companies: IL-1 and
IFN- (Roche Molecular Biochemicals, Gaithersburg, MD); clofibrate
and ciglitazone (Biomol Research Laboratories, Plymouth Meeting, PA);
15d-PGJ2 (catalog no. 18570), WY14643,
carbaprostacyclin, oleic acid, 9-hydroxyoctadecadieroic acid,
and rabbit polyclonal anti-COX-2 and anti-iNOS Abs (1:1000),
mouse anti-COX-1 mAb (1:500), ovine COX-2, COX-1, and mouse iNOS
standards, and human COX-2 cDNA (Cayman Chemicals, Ann Arbor, MI);
mouse GADPH cDNA (Ambion, Austin, TX); actinomycin D, protease
inhibitors, and LPS from Escherichia coli serotype O26:B6
(Sigma-Aldrich, St. Louis, MO); and Abs to NF-Bp50, NF-Bp65,
STAT-1, c-Jun, c-Fos, PPAR, and PPAR (Santa Cruz Biotechnology,
Santa Cruz, CA). Concentrations of PPAR ligands were chosen from
previous published studies (30, 31, 32, 33).
Cell lysates and Western blotting
After the treatment of microglial cells and astrocytes, cells were lysed in Laemmli buffer containing 5% 2-ME and sonicated for 1 min. The protein concentration was measured by a noninterfering protein assay (Geno-Tech, St. Louis, MO). Cell lysate proteins (15–50 µg/lane) were loaded onto a denaturing SDS-polyacrylamide gel, electrophoresed, and transferred onto nitrocellulose membrane. The nitrocellulose membrane was then blocked in 5% nonfat dry milk in PBS containing 0.1% Tween 20. The membrane was incubated with the primary Ab 1 h at room temperature, followed by incubation with the secondary Ab conjugated to HRP. The blots were probed with the ECL Western blot detection system from Amersham Pharmacia Biotech (Piscataway, NJ).
RNA isolation and Northern blots
Northern blots were performed on total cellular RNA isolated from cells using an RNA isolation kit from Qiagen (Chatsworth, CA). Ten micrograms of total RNA per lane were electrophoresed on a formaldehyde-containing 1.2% agarose gel and transferred to nylon-supported membranes. Hybridizations were conducted at 42°C with cDNA probes and labeled with [32P]CTP by random priming in Ultrahyb (Ambion). After hybridization, membranes were washed twice for 20 min in 2x SSC phosphate/EDTA and 0.1% SDS at room temperature, twice for 20 min in the same solution at 55°C, and twice for 20 min in 0.1x SSC phosphate/EDTA and 0.1% SDS at 55°C. To verify equivalency of RNA loading in the different lanes, the blots were rehybridized to determine the levels of GAPDH mRNA. For the quantification of COX-2 and GAPDH mRNA signals, densitometry of bands was performed using ImageQuant (Molecular Dynamics, Sunnyvale, CA), and relative COX-2 mRNA were determined as the ratio COX-2/GAPDH.
EMSA
Protein extraction from cells was conducted by a modification of
the Andrews and Faller procedure (34) in ice-cold buffer C
(20 mM Tris-HCl (pH 7.9), 1.5 mM MgCl2, 420 mM
NaCl, 0.2 mM EDTA, 1 mM dithiotreitol, 25% glycerol) containing
protease inhibitors, 1 mM PMSF, antipain, leupeptin, aprotinin,
pepstatin A, and chymostatin (10 µg/ml each). Protein concentration
was determined by the method of Bradford (Bio-Rad, Richmond, CA). AP-1
consensus and mutant oligonucleotides were from Santa Cruz
Biotechnology. Oligomers were designed and synthesized with their
complementary strands from the distal and proximal NF-B sites within
-455 to -428 and -232 to -205 from the transcriptional start site
(+1) on the human COX-2 promoter (35). The NF-B
sequences used were as follows: distal NF-B
(5'-CGGCGGCGGGAGAGGGGATTCCCTGCGCCC-3'); distal NF-B
mutant (5'-CGGCGGCGGGAGAGCTCATTCCCTGCGCCC-3'); proximal
NF-B (5'-AGACAGGAGAGTGGGGACTACCCCCTCTGC-3'); and
proximal NF-B mutant
(5'-AGACAGGAGAGTGGCCACTACCCCCTCTGC-3'). The
NF-B binding sequence is underlined. Sense and antisense oligomers
were annealed in 20 mM Tris-HCl (pH 7.6), 50 mM NaCl, 10 mM
MgCl2, and 1 mM dithiotreitol. The annealed
oligonucleotides were phosphorylated at the 5' end with
[-32P]ATP and T4 polynucleotide kinase. For
EMSA, the DNA-binding assay (20 µl) contained 10 mM Tris-HCl (pH
7.9), 50 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, 1 mM
DTT, 10% glycerol (v/v), 4 µg poly(dI-dC) (Amersham Pharmacia
Biotech), 5 µg nuclear extract, and 0.8 ng
32P-labeled DNA fragment. The reaction mixture
was incubated at room temperature for 30 min. In the supershift assays,
the Abs to NF-Bp50, NF-Bp65, c-Fos, or c-Jun (2 µg/reaction)
were incubated with the reaction mixture for 2 h at 4°C before
the addition of 32P-labeled DNA fragment. In cold
competition assays, 100-fold molar excess of cold wild-type or mutant
oligomers was used. The protein-DNA product was run onto a 4 or 6%
nondenaturing polyacrylamide gel. The gel was then dried and subjected
to autoradiography.
GST and GR assays
The rate of GST or glutathione reductase (GR) enzymatic
activities was assessed by measuring the rate of conjugation of
1-chloro-2,4-dinitrobenzene with reduced glutathione (GSH) or the rate
of NADPH oxidation in the presence of oxidized glutathione,
respectively. Whole cell extracts were prepared in cold buffer (50 mM
potassium phosphate (pH 7.5), 1 mM EDTA). Protein concentration was
determined, and GST and GR activities were measured at 25°C, using
enzymatic assay kits, according to the manufacturer’s instructions
(Cayman Chemicals). GST and GR sp. act. were expressed as nanomoles of
substrate converted per minute per milligram of cellular protein.
Comparisons of means were conducted by Student’s t test;
differences with a value of p 
0.05 were considered
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have demonstrated that both human microglial
cells and astrocytes activated by IL-1 are capable of enhanced
production of PGs, whereas only human astrocytes can be induced by
IL-1 in combination with IFN- to produce detectable levels of NO,
under our conditions of culture (14, 15, 16, 28, 36). To
investigate the effect of PPAR agonists on these inflammatory pathways
in human cells, we first monitored COX-2 and iNOS protein expression in
microglia and astrocytes after cytokine stimulation. As expected, COX-2
protein was induced by IL-1 in both microglia and astrocytes (Fig. 1
A). The induction was
detectable from 4 to 6 h and peaked at 12 h after stimulation
(data not shown). Under these culture conditions, IL-1 in
combination with IFN- (but not LPS) also induced iNOS protein
expression in human astrocytes, but not in human microglia (Fig. 1B).
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, clofibrate and WY14643, enhanced
IL-1-induced COX-2 expression in astrocytes and microglia (Fig. 2, A and B). Among
PPAR agonists tested, only 15d-PGJ2 displayed
a dose-dependent inhibitory effect on the induced COX-2 expression in
astrocytes, but not in human microglia (Fig. 2, A and
B). A specific and high-affinity agonist of PPAR,
ciglitazone, had no significant effect on COX-2 induction in
astrocytes, suggesting that the effect of
15d-PGJ2 was independent of PPAR activation.
The concentrations of 15d-PGJ2 that inhibited
COX-2 induction by IL-1 had no significant toxicity to astrocytes,
as judged by the MTT assay (data not shown). The only semisynthetic
agonist available for PPAR, carbaprostacyclin, had no effect on the
induced COX-2 expression in human astrocytes or microglia (Fig. 2, A and B). The various agonists tested had a
slight or no effect on the constitutive expression of COX-1 isoform
(Fig. 2, A and B) and did not affect COX-2
expression in unstimulated cells (data not shown).
15d-PGJ2 has been shown to inhibit
cytokine-induced iNOS expression in rodent microglial cells (25, 26). However, in human glial cells tested in this study, IL-1
plus IFN- induced iNOS expression only in astrocytes, but not in
microglia (Fig. 1
B). Similar to the observation in rodent
microglia, 15d-PGJ2, but not ciglitazone,
inhibited the induced iNOS expression in human astrocytes (Fig. 2C). PPAR agonists (clofibrate and WY14643), which
enhanced COX-2 expression in astrocytes and microglia, decreased
cytokine-induced iNOS expression in astrocytes (Fig. 2C).
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and . IL-1 slightly increased PPAR
expression in astrocytes and microglia (Fig. 3). In contrast, PPAR expression was
not affected by IL-1 treatment alone or in combination with
15d-PGJ2 or ciglitazone (Fig. 3).
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The anti-inflammatory effect of 15d-PGJ2
has been described on murine and human peripheral macrophages
(21, 22). We found that in glial cells, the inhibitory
effect of 15d-PGJ2 was not observed on COX-2
induction in human brain macrophages (microglia), but in astrocytes.
This observation prompted us to further examine the mechanism of the
differential effect of 15d-PGJ2 on the two cell
types. Northern blot analysis demonstrated a strong inhibition of
IL-1-induced COX-2 mRNA accumulation in human astrocytes in the
presence of 15d-PGJ2 (10-fold inhibition, Fig. 4). In human microglial cells,
15d-PGJ2 only partially inhibited COX-2 mRNA
induction by IL-1 (2-fold inhibition; Fig. 4), which would explain
the absence of a significant decrease in COX-2 protein level in these
cells (Fig. 2B). COX-2 message stability is an important
factor in regulation of its abundance due to the presence of large
number of instability motifs in its 3' untranslated region
(37). We observed no effect of
15d-PGJ2 on the stability of IL-1-induced
COX-2 mRNA in astrocytes and microglia (data not shown), indicating
that 15d-PGJ2 would mainly act as a
transcriptional inhibitor of COX-2 expression.
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B to
the COX-2 promoter in human astrocytes, but not in microglial cells
The activation of the transcriptional factor NF-B is crucial to
the induction of many inflammatory response genes, including COX-2
(35, 38). The human COX-2 promoter contains two putative
binding sites for NF-B (35, 38). We investigated the
effect of 15d-PGJ2 on the binding activity of
NF-B to these sites, using EMSA. Low levels of constitutive
NF-B/DNA-binding activity were found in nuclear extracts from
unstimulated astrocytes and microglial cells (Fig. 5). The NF-B binding to the distal,
but not the proximal site in the COX-2 promoter was enhanced after
IL-1 treatment in both astrocytes and microglia and generated a fast
migrating specific gel-shifted complex (Fig. 5). A 100-fold molar
excess of cold wild-type NF-B oligomer, but not of the cold mutant
oligomer, completely abolished the formation of this complex,
demonstrating the specificity of the complex (Fig. 5). The treatment
with 15d-PGJ2 prevented the formation of the
NF-B/DNA complex in IL-1-stimulated astrocytes (Fig. 6, upper panel), whereas in
microglia only a slight decrease in this complex was observed (Fig. 6, lower panel). This complex was formed by both p50 and p65
NF-B subunits, because it was supershifted with either anti-p50
or anti-p65 Abs, resulting in high-m.w. NF-B bands migrating
near the origin (Fig. 6). These results suggest
15d-PGJ2 acts to inhibit NF-B binding in
astrocytes.
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ligands enhanced
IL-1-induced COX-2 expression in both astrocytes and microglia (Fig. 2C). Therefore, we investigated whether the effect of these
ligands could also involve the NF-B activity. To address this
question, we tested the effect of PPAR ligand, WY14643, on the
IL-1-induced NF-B activity in astrocytes and microglia. In
contrast to 15d-PGJ2, the treatment of cells with
WY14643 had no effect on the activation of NF-B binding in
IL-1-stimulated astrocytes and microglia (Fig. 7).
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Bp50 protein accumulation in
activated astrocytes, but not in microglia
The proteins that comprise NF-B are subject to complex modes of
regulation, including cytoplasmic compartmentalization and retention by
inhibitory proteins (I-B), phosphorylation, and various associations
of active subunits (39). Phosphorylation of I-B and the
consequent nuclear import of NF-B complex upon cellular stimulation
have been the most studied in the regulation of the NF-B activity.
We next investigated whether the differential effect of
15d-PGJ2 on NF-B transcriptional activity in
human astrocytes and microglia was due to a differential alteration of
its nuclear translocation. We monitored the levels of NF-Bp65 and
p50 expression by Western blotting in both total and nuclear extracts
from IL-1-stimulated astrocytes and microglia in presence or absence
of 15d-PGJ2. IL-1 induced NF-Bp50
expression in astrocytes and microglia, while the level of NF-Bp65
was not significantly modulated by IL-1.
15d-PGJ2 had no effect on the amount of
NF-Bp65 in total extracts from astrocytes or microglia, whereas in
nuclear extracts it slightly decreased NF-Bp65 in astrocytes, but
not in microglia (Fig. 8A). In
contrast, treatment with 15d-PGJ2 strongly
prevented the increase in NF-Bp50 protein induced by IL-1 in
total extracts from human astrocytes, but not from microglia (Fig. 8A). Consistently, the nuclear levels of NF-Bp50 were
also decreased in IL-1-activated astrocytes after treatment with
15d-PGJ2 (Fig. 8A). These results
demonstrate that 15d-PGJ2 modulates NF-B
transcriptional activity in human astrocytes at least through
repression of the NF-Bp50 induction by IL-1. In human microglia,
no significant effect of 15d-PGJ2 on
NF-Bp65/p50 could be evidenced.
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Cyclopentenone PGs are actively transported into cells via a
membrane transporter (40). The absence of effect of
15d-PGJ2 on the induction of COX-2 and
NF-B-binding activity in human brain macrophages could be due to a
less efficient transport of the PG, as compared with astrocytes. In
peripheral macrophages, 15d-PGJ2 has been shown
to interfere with other transcription factors such as AP-1 family
(c-Fos, c-Jun) and STAT-1 that are also important in various
proinflammatory pathways (21). To investigate whether
15d-PGJ2 could affect inflammatory pathways other
than NF-B in human astrocytes and microglia, we studied the
expression of c-Fos, c-Jun, and STAT-1 in activated astrocytes and
microglia after treatment with 15d-PGJ2.
IL-1-induced STAT-1 protein accumulation was reduced in both
astrocytes and microglial cells, whereas c-Jun protein accumulation was
enhanced in both cells after treatment with
15d-PGJ2 (Fig. 8B). In contrast,
15d-PGJ2 induced c-Fos protein accumulation only
in astrocytes, but not in microglia (Fig. 8B). To verify
that the increase in c-Fos and c-Jun corresponded to the active form of
these proteins, EMSA experiments using an AP-1 consensus
oligonucleotide were conducted (Fig. 8C). The data
demonstrated that 15d-PGJ2 enhanced the binding
activity of AP-1 complex in nuclear extracts from both astrocytes and
microglia. Consistent with the Western blotting results, supershift
experiments in presence of anti-c-Fos and anti-c-Jun Abs
demonstrated that the AP-1 complex formed in astrocytes comprised
c-Fos/c-Jun heterodimers after treatment with
15d-PGJ2, whereas it contained mainly c-Jun
homodimers in microglia (Fig. 8C). These data indicate that
1) the entry of 15d-PGJ2 also occurs in human
brain macrophages; and 2) 15d-PGJ2 does not act
only as an inhibitor of inflammatory response through NF-B
inhibition in human astrocytes, but can also activate other
inflammatory pathways through the increase in c-Fos and c-Jun
expression in astrocytes and microglia.
GST and GR enzymatic activities in human astrocytes and microglia
Our data presented an important difference in the ability of
15d-PGJ2 to inhibit NF-B-binding activity in
astrocytes as compared with human microglial cells. Cyclopentenone PGs
are highly reactive with nucleophilic agents such as the thiol groups
of nuclear proteins (40). In contrast, the transported PG
in the cytosol may also conjugate with intracellular GSH, inhibiting
the binding of the PG to the nuclear proteins (41). This
event can be modulated by two enzymatic activities: 1) the cytoplasmic
GST that is responsible for the metabolic elimination of cyclopentenone
PGs by conjugation to GSH (41), and 2) the GR that
catalyzes the reduction of oxidized glutathione to GSH and therefore
maintains adequate levels of reduced cellular GSH. Thus, we compared
GST and GR activities in human astrocytes and microglial cells. No
significant difference in the GST sp. act. was observed between
untreated or IL-1-treated astrocytes or microglial cells (data not
shown). However, a 35% decrease in the GR sp. act. was detected after
IL-1 treatment only in human astrocytes, but not in microglia
(448 ± 13 vs 293 ± 50 nmol/min/mg protein, untreated vs
IL-1-treated astrocytes, n = 3, p <
0.05). This suggests that intracellular GSH levels in astrocytes might
decrease after IL-1 treatment, and thereby could increase the
ability of the transported 15d-PGJ2 to interfere
with NF-B pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced COX-2 mRNA accumulation. In human brain macrophages,
the inhibition level of IL-1-induced COX-2 mRNA was much lower than
in astrocytes and was not sufficient to inhibit COX-2 protein
accumulation. 15d-PGJ2 is a ligand for PPAR,
but both PPAR-dependent and independent mechanisms have been
suggested for its anti-inflammatory effects among different cell
types studied (21, 22, 23, 25, 26). Although human astrocytes
expressed detectable levels of PPAR, our results suggest that the
effect of 15d-PGJ2 on COX-2 induction in
astrocytes is independent of PPAR activation, because 1) the
inhibition of COX-2 did not occur in human microglia that also express
PPAR; 2) a selective synthetic PPAR agonist, ciglitazone, was not
able to inhibit COX-2 in astrocytes; and 3) the inhibition of COX-2 in
astrocytes was observed at higher 15d-PGJ2
concentrations than those required for the PPAR activation.
We subsequently investigated the effect of
15d-PGJ2 on NF-B-binding activity in human
astrocytes and microglia. We found that IL-1 induced the NF-B
binding to the distal, but not the proximal, site in the COX-2 promoter
in both human astrocytes and microglia. Interestingly, in human
vascular endothelial cells, hypoxia has been shown to induce COX-2
expression via an enhanced NF-B binding to the proximal site of the
promoter but not the distal site (35). This suggests that
the differential use of the NF-B binding sites in human COX-2
promoter depends on the inducer and/or tissue-specific factors.
Therefore, it is conceivable that other factor(s) might be involved in
the NF-B-binding activity to the human COX-2 promoter.
15d-PGJ2 suppressed the NF-B binding to the
distal site in COX-2 promoter specifically in astrocytes. In human
microglia, 15d-PGJ2 had almost no effect on
NF-B-binding activity, which was consistent with the low level of
inhibition of COX-2 mRNA accumulation and the absence of effect on
COX-2 protein accumulation. In a murine macrophage cell line,
15d-PGJ2 was shown to directly prevent the
NF-B-binding activity through multiple mechanisms, including
modification of I-B kinase activity, which reduces the NF-Bp65
nuclear translocation, and by direct modification of DNA binding domain
of NF-Bp65 (43). Interestingly,
15d-PGJ2 could affect NF-B-binding activity
via either mechanism depending on the cell type (43).
Consistent with the absence of effect of 15d-PGJ2
on COX-2 induction and NF-B/DNA-binding activity in human brain
macrophages, we found no changes in the total or nuclear accumulation
of both NF-B subunits in these cells after treatment with
15d-PGJ2. In human astrocytes, although
15d-PGJ2 slightly decreased the nuclear amount of
NF-Bp65, it mainly acted by reducing the IL-1 induction of
NF-Bp50 subunit. This suggests a new mode of action of
15d-PGJ2 on NF-B activation pathway in human
astrocytes different from that described in murine peripheral
macrophages (43). In contrast, we found that the
expression and the DNA-binding activity of AP-1 proteins (c-Fos and
c-Jun) were induced in human astrocytes and microglia in presence of
15d-PGJ2. These data demonstrate that
15d-PGJ2 can differentially affect
transcriptional factors among different cell types because it has been
shown to down-regulate AP-1-binding activity in peripheral macrophages
(21). Regulatory sequences corresponding to the activating
transcription factor (ATF)/CRE site have been reported in the
5'-flanking region of the human COX-2 gene (for a review, see Ref.
44). This site is typically activated by hetero- and
homodimers of the c-Fos, c-Jun, ATF, and CREB. The ATF/CRE site has
been shown to be critical for the COX-2 gene induction in several cell
types, such as fibroblasts or epithelial cells (44).
However, the relative importance of one transcription factor in the
induction of the COX-2 gene over the others seems to be
dependent on the stimulus and the cell type (44). In the
case of human astrocytes, the increase in AP-1-binding activity by
15d-PGJ2 could suggest an antagonistic role of
these factors in COX-2 expression. In human microglia,
15d-PGJ2 also increases IL-1-induced
AP-1-binding activity with no effect on COX-2 expression, perhaps due
to the maintained activation of NF-B. Our data also suggest that,
besides the negative feedback on COX-2 expression,
15d-PGJ2 could lead to the activation of other
inflammatory response genes involving AP-1 protein activation.
The unresponsiveness of human brain macrophages to the
anti-inflammatory effect of 15d-PGJ2 in terms
of COX-2 and NF-B inhibition is surprising and is apparently
specific to this macrophage population, because inflammatory pathways
have been shown to be down-regulated by this compound in human
peripheral blood macrophages and peritoneal macrophages (21, 22). We observed that 15d-PGJ2 could
affect the expression of other transcription factors, such as c-Jun and
STAT-1, in human microglia. This indicates that the transport of
15d-PGJ2 occurs in microglia. The effect of
15d-PGJ2 on AP-1 and STAT-1 factors might likely
occur via PPAR activation, as suggested by other studies
(21). Reactive oxygen species are believed to be important
in the activation of NF-B by a mechanism that is not completely
understood (45). Among free radicals, superoxide anion
production is induced upon inflammatory activation in microglia
(14). However, it is not clear whether this production of
superoxide anions could be in part responsible for antagonizing the
repression of NF-B activation by 15d-PGJ2 in
these cells. Another biological factor that interferes with
15d-PGJ2 is the glutathione (GSH) that can
conjugate with the cyclopentenone ring of the PG (40, 41).
High levels of GSH could result in the quenching of
15d-PGJ2, thereby inhibiting its binding to
nuclear proteins (41, 43). Thus, a difference in the
intracellular levels of GSH could also explain the difference in the
effect of 15d-PGJ2 observed between human
microglia and astrocytes. In support of this hypothesis, we observed
decreased activity of GR in IL-1-stimulated astrocytes, which could
lead to a decrease in the intracellular levels of GSH in these
cells.
Under pathological conditions, COX-2 and iNOS are up-regulated within a
single cell type or tissue (7). Most of the
anti-inflammatory drugs, such as dexamethasone, down-regulate both
enzymes. The general inhibition of inflammatory product secretion by
such drugs may explain the inconsistency of their effects on the
neurological lesions in different in vivo experimental systems for
neuroinflammatory disorders (46, 47, 48). In addition to
15d-PGJ2, we also tested the effect of synthetic
activators of PPAR, clofibrate and WY14643, on astrocyte and
microglial activation. We demonstrated that PPAR agonists increased
COX-2 expression in human astrocytes and microglia stimulated with
IL-1, while they inhibited iNOS induction by IL-1 plus IFN- in
human astrocytes. This finding provides, for the first time, the
possibility of an inhibition of iNOS uncoupled from that of COX-2 in
human astrocytes. A PPAR-dependent effect of clofibrate and WY14643
in activated glial cells would be suggested by our following
observations: 1) both agonists exerted similar effects; 2) they exerted
their effect only on IL-1-activated glial cells, but not on
unstimulated cells, and IL-1 stimulation of glial cells enhanced the
amount of PPAR protein; and 3) the effect of WY14643 did not occur
through modulation of IL-1-induced NF-B activity.
In conclusion, 15d-PGJ2 and PPAR agonists
exert differential effects on the inflammatory response among human
astrocytes and microglial cells, in comparison with peripheral
macrophages. Further studies on the molecular modes of action of these
molecules will reinforce our understanding of the mechanisms that
regulate inflammation in brain and should provide new insights in the
treatment of neuroinflammatory diseases in humans.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nazila Janabi at the current address: UMR960 Institut National de la Recherche Agronomique, Laboratoire de Microbiologie Moleculaire, Ecole Nationale Veterinaire de Toulouse 23 Chemin des Capelles, 31076 Toulouse Cedex, France. E-mail address: n.janabi{at}envt.fr
3 Abbreviations used in this paper: COX, cyclooxygenase; 15d-PGJ2, 15-deoxy-12,14-PGJ2; ATF, activating transcription factor; GR, glutathione reductase; GSH, reduced glutathione; iNOS, inducible NO synthase; PPAR, peroxisome proliferator-activated receptor.
Received for publication August 21, 2001. Accepted for publication March 6, 2002.
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