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INTRODUCTION |
Astrocytes and microglia are glial cells that play a major role in
the inflammation observed in Alzheimer's disease
(AD)1 as well as many other
neurodegenerative diseases (1-5). Upon stimulation from various agents
or insults such as cytokines or trauma, these cells adopt a reactive
phenotype, a morphological hallmark in AD pathology, during which they
themselves may produce still more inflammatory cytokines and neurotoxic
free radicals. One potentially detrimental free radical is the highly
reactive nitrogen species peroxynitrite, which is a derivative of
nitric-oxide (NO) production (6). In the brain, astrocytes are able to
produce NO during inflammatory conditions by the enzyme inducible
nitric-oxide synthase (iNOS or NOS2) (7, 8).
Increased levels of iNOS production in astrocytes surrounding amyloid
plaques (9), and significant peroxynitrite damage to neurons have been
observed in AD brain (10). Peroxynitrite can also induce alterations in
synaptosomal membranes of neurons, and is thus a potent source for
oxidative stress in AD brain (11). Several lines of similar in
vitro evidence also show that 
-amyloid, the primary plaque
component in AD brain, can stimulate NO production from astrocytes
(12-15), and that activated astrocytes can produce NO levels that are
detrimental to neurons (12, 16).
While the 42-amino acid -amyloid peptide (A42) is capable of
activating astrocytes in vitro and in vivo,
resulting in the activation of NF
B and the induction of iNOS (17),
the molecular pathway(s) and participants governing astrocyte iNOS
induction by A42 have not yet been described. In addition to
activation of astrocytes, A42 can also activate microglia, leading
to the production of interleukin (IL)-1 (15, 18) and tumor necrosis factor (TNF) 
(12, 19, 20). Because IL-1 and TNF can themselves stimulate iNOS production in astrocytes (21, 22), A42
stimulation of iNOS in astrocytes could be a direct effect on the
astrocyte or an indirect result of A42 stimulation of IL-1 and
TNF induction in microglia.
IL-1 is a critical inflammatory cytokine in AD. Microglia localized
to amyloid plaques stain positively for IL-1, and increased numbers of
IL-1-expressing microglia are associated with AD progression (23).
IL-1 is also capable of stimulating astrocytes to produce additional
pro-inflammatory cytokines such as IL-6, another inflammation marker
associated with neurodegeneration (24-26).
A signal transduction pathway activated by IL-1 that ultimately
leads to NFB activation has been described (27-29) and involves the
TNF receptor-associated factor-6 (TRAF6). Similarly, TNF activates NFB via TRAF2 (28, 30). Both TRAF2 and TRAF6 participation can then lead to the activation of NFB-inducing kinase (NIK), the
common mediator of IL-1 and TNF activation of NFB (31), and
NIK can complex with and activate the IB kinase signalsome complex
(,-,-
) (32). Activated IB kinase signalsome complex phosphorylates IB, the inhibitor of the transcription factor NFB,
directing the inhibitor for proteasome-mediated degradation and
allowing NFB to translocate to the nucleus, where it binds to
specific promoter response element sequences and stimulates gene
transcription (for reviews, see Refs. 33 and 34). Activated NFB has
been observed in AD brain (35, 36), and we have shown previously that
NFB activation is necessary for A42-stimulated iNOS induction in
astrocytes (17).
The molecular events that coordinate the neurotoxic inflammatory
response in neurodegenerative diseases such as AD are crucial to
understand. To determine if IL-1 induction is required for the
A42 stimulation of iNOS in astrocytes, we used a series of approaches. We examined the temporal pattern of A42 stimulation of
IL-1 and iNOS production in mixed glial cultures, and whether new
protein synthesis was required for iNOS induction. The participation of
IL-1 signal transduction pathways in iNOS activity was blocked at
the ligand/receptor interface with the IL-1 receptor antagonist (IL-1ra). Finally, we inhibited either IL-1-mediated activation of
NFB by using the dominant-negative truncated form of TRAF6, or
TNF-mediated activation of NFB by using the dominant-negative truncated form of TRAF2, or both TNF- and IL-1-mediated
activation of NFB by the kinase-inactive form of NIK. We report here
that inhibition of IL-1 signal transduction (and, to a lesser
extent, TNF signal transduction) resulted in decreased
A42-stimulated nitrite production and iNOS promoter activity,
demonstrating that A42-stimulated iNOS induction is mediated at
least in part by inflammatory cytokine production.
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MATERIALS AND METHODS |
Cell Culture--
Rat primary cortical glial cultures were
prepared and maintained as described previously (16). Briefly, cells
were grown in MEM supplemented with 10% fetal calf serum (HyClone)
and 1% penicillin/streptomycin (Life Technologies, Inc.). At least
24 h prior to stimulation, the serum-containing medium was
removed, and cells were washed once with PBS and then incubated in
serum-free MEM containing N2 media supplements (Life Technologies,
Inc.). Experiments were conducted on no later than tertiary passages of
cells. From immunohistochemical analysis, the cell population was
determined to be approximately 90-95% astrocytes and 5-10% microglia. For cycloheximide and IL-1 receptor antagonist studies, inhibitors were added to medium immediately prior to stimulation.
Reagents--
Recombinant rat TNF, recombinant rat IL-1,
and recombinant rat IL-1 receptor antagonist were purchased from
R&D/Genzyme. Lipopolysaccharide (LPS), cycloheximide, and dibutyryl
cAMP (Bt2cAMP) were from Sigma and were diluted in MEM
(Sigma) prior to use.
-Amyloid (A42) Preparation--
The 42-amino acid peptide
form of -amyloid (A42) was obtained from Dr. C. Glabe (University
of California Irvine, CA) and was stored as lyophilized powder at
20 °C. Aggregates of A42 were prepared at room temperature as
described previously (14). A42 aggregated under these conditions is
a mixture of globular and fibrillar peptide which activates astrocytes
and is toxic to neurons (14, 37). The absence of any contaminating
endotoxin in the A42 preps was verified by direct measurement on
Pyroplate LAL assay microplates (Associates of Cape Cod, Inc.).
Plasmids--
The 7-kilobase pair human iNOS promoter-luciferase
reporter construct (iNOS-Luc) has been described previously (38). The NFB reporter construct pNFB-SEAP (CLONTECH)
contains four tandem NFB response element repeats upstream of the
thymidine kinase basal promoter from the herpes simplex virus, allowing
for the expression of the secreted alkaline phosphatase gene
(SEAP).
The TRAF expression constructs were made as follows; full-length murine
TRAF2 and murine TRAF6 cDNAs cloned into an SR (SV40-derived) promoter-driven vector (pME18S-FLAG) were kindly provided by Dr. J. Inoue (University of Tokyo, Tokyo, Japan) and have been
described previously (39). These cDNAs were used as templates for
subcloning by polymerase chain reaction into the CMV promoter-driven
vector pCMV2-FLAG (Sigma). The following primers were used: for
dominant-negative TRAF2 (containing the C-terminal TRAF2 region, amino
acids 87-501; termed dnTRAF2-C), 5'-AAG CTT GCG GCC GCG AAT AGT AGT
TCG GCC TTT CCA GAT AAC-3' and 5'-GGA TCC TCT AGA TGG CTA GAG TCC TGT TAG GTC CAC AAT AGC-3'; for dominant-negative TRAF6 (containing the
C-terminal TRAF6 region, amino acids 301-531; termed dnTRAF6-C), pME18S-FLAG-TRAF6 was used as a template with the following primers: 5'-AAG CTT GCG GCC GCG AAT CCA AAT TAT GAG GAA ACT ATC AAA CAG-3' and
5'-GGA TCC TCT AGA TGG CTA CAC CCC CGC ATC AGT ACT TCG-3'. All
polymerase chain reactions used the DNA polymerase PfuTurbo (Stratagene). Amplified products were digested with NotI and
XbaI and then subcloned into the
NotI/XbaI sites of pCMV2-FLAG.
The constructs pcDNA3-HA and pcDNA3-HA-NIK were kindly provided
by Dr. M. Levrero (Università degli Studi di Roma La Sapienza, Rome, Italy). The pcDNA3-HA contains an HA epitope for N-terminal tagging in the expression vector pcDNA3 (Invitrogen); the
pcDNA3-HA-NIK contains the full-length human NIK cDNA cloned
into pcDNA3-HA (40). Dominant-negative (kinase-inactive) NIK was
prepared by site-directed mutagenesis of pcDNA3-HA-NIK to change
amino acid residues Lys429-Lys430 to Ala-Ala
(QuikChange, Stratagene) using the following primers: 5'-GCT TCC AGT
GCG CTG TCG CCG CCG TGC GGC TGG AAG TAT TTC-3' and 5'-GGA ATA CTT CCA
GCC GCA CGG CGG CGA CAG CGC ACT GGA AGC-3'. The resultant construct,
termed dnNIK, was verified by DNA sequence across the mutated site and
by restriction analysis.
Plasmids containing cloned partial cDNA fragments of rat IL-1,
rat iNOS, and rat GAPDH have been described previously (14).
Transfections and Reporter Assays--
Plasmid DNA was prepared
by endotoxin-free Maxi-Preps (Qiagen) before transfections.
Transfection of astrocyte cultures has been described previously (17).
Briefly, for all transfections, the cationic lipid reagent Tfx-50
(Promega) at a 1:1 (charge:µg of DNA) ratio was used. For
transfections using the iNOS-Luc reporter construct, 4 × 105 cells/well in a six-well tissue culture plate were
transfected with 3.6 total µg of DNA/well (2.0 µg of expression
construct and 1.6 µg of reporter construct; in cases of increasing
amounts of expression construct, the total amount of DNA transfected
was kept constant by using the appropriate control cloning vector). For
all transfections using the pNFB-SEAP reporter construct, 5 × 104 cells/well in a 24-well tissue-culture plate were
transfected with a total of 1 µg of DNA/well (0.5 µg of expression
construct and 0.5 µg of reporter construct, total amount of DNA kept
constant as described above).
Luciferase assays were performed with the LucLite reagent (Packard) and
read on a LumiCount luminometer (Packard) as described previously (17).
Chemiluminescent SEAP assays were performed with the Phospha-Light
assay reagents (Tropix) by the manufacturer's protocol and read on the
same luminometer. Relative light units for both assays were calculated
as a percentage of appropriate controls. At least eight replicates were
conducted per experiment, and statistical significance
(p < 0.05) was determined by Student's t test.
Western Blotting--
Cell lysates for Western blotting were
prepared in SDS-containing sample buffer and separated on 10%
SDS-polyacrylamide gels. After transfer to polyvinylidene difluoride
membrane (Immobilon-P, Millipore), blots were probed with antibodies to
IL-1 (polyclonal goat anti-rat IL-1 diluted 1/2000; R&D/Genzyme),
the astrocyte-specific marker glial fibrillary acidic protein (GFAP,
monoclonal anti-pig GFAP diluted 1/109, Sigma), and iNOS
(monoclonal anti-mouse macNOS diluted 1/2000, Transduction
Laboratories). ECL detection of protein bands was performed using
LumiGLO reagent (New England Biolabs).
Northern Blotting--
Northerns blots were performed as
described previously (14). Briefly, total RNA was isolated from cells
using RNeasy spin columns (Qiagen). Equal amounts of total RNA (800 ng/sample) were slot blot-loaded onto Durulon-UV membrane (Stratagene)
and immediately UV cross-linked. Partial cDNA probes were
32P-labeled using the RediPrime labeling kit (Amersham
Pharmacia Biotech). Pre-hybridization and probe hybridization were
performed at 68 °C using QuikHyb solution (Stratagene) and by the
manufacturer's protocol. Washed blots were exposed to Storm
phosphorimaging plates (Molecular Dynamics) and densitometry was
measured using ImageQuant version 1.1 software (Molecular Dynamics). To
verify equal amounts of total RNA per slot, each blot was later
stripped and re-probed for rat GAPDH mRNA. IL-1 and iNOS
mRNA levels were normalized to the GAPDH mRNA levels, and
relative increases were calculated from five separate experiments.
Nitrite Determination--
NO production was quantitated by
measuring the levels of nitrite in the conditioned media using the
Griess assay as described previously (16), except that nitrate in the
conditioned medium was first reduced to nitrite with nitrate reductase
and NADPH (Sigma) for 1 h at 37 °C before the assay.
Cytokine ELISAs--
For IL-1 measurements, 7.5 × 104 cells/well were plated in a 48-well tissue culture
plate. For TNF measurements, 5 × 104 cells/well
were plated in a 48-well tissue culture plate. After 36-48 h of
incubation in serum-free MEM containing N2 media supplement, cells
were stimulated with either control diluent or 10 µM
A42. Conditioned media from duplicate wells per sample were
collected at the indicated time points and then stored at 80 °C
until the ELISA. Immediately after the last time point was collected,
cytokine levels in the conditioned media were measured by ELISA (rat
IL-1 or rat TNF assay kit, R&D Systems) and performed by the
manufacturer's protocol.
Immunofluorescence--
Identification of cell types expressing
either IL-1 or iNOS was done by immunofluorescence as described
previously (41) using the same antibodies as for the Western blots.
Polyclonal goat anti-rat IL-1 antibody and monoclonal mouse
anti-macNOS were used at 1/200 dilution. Monoclonal mouse anti-pig GFAP
was used at 1/1000 dilution. Secondary fluorochrome-conjugated
antibodies donkey anti-goat IgG and donkey anti-mouse IgG (Jackson
ImmunoResearch) were used at 1/400 dilutions. Images were digitally
captured from a Zeiss Axiophot2 microscope using Spot camera
(Diagnostic Instruments) and Metamorph software.
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RESULTS |
A42-stimulated Cytokine Production Occurs before iNOS Production
in Vitro--
To determine the temporal sequence of events during
A42 activation of glial cultures, a time-course Western blot profile was used to demonstrate that IL-1 protein production (proIL-1 form associated with cellular content prior to secretion) occurs before
iNOS protein production (Fig. 1).
A42-stimulated IL-1 production was detected by 3 h, reached
a peak by 6 h, and continued to be present through 36 h after
stimulation. However, protein production of iNOS was not detected until
6 h after A42 stimulation, and iNOS levels steadily increased
through 36 h. In cells treated with diluent alone, there was a
delayed, minor IL-1 production detected at 6-12 h, but no iNOS
protein detected. GFAP levels did not increase at any time point
examined, consistent with previous findings in A42 stimulation of
astrocytes (14). To determine if A42 stimulates IL-1 (active
form) secretion from these cells, an IL-1 ELISA was performed on
conditioned medium (Fig. 2A). Similar to the Western blot time-course profile of A42-stimulated proIL-1 production, active IL-1 was secreted into the conditioned medium and detectable by 3 h, and active IL-1 levels
accumulated through 12 h.
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Fig. 1.
Time course of
A42-stimulated IL-1
and iNOS production. Rat cortical glial cultures were
stimulated with either control buffer or 10 µM A42
(except for 0-h time point, which represents untreated cells) and then
harvested at the time points indicated. Equal volumes of cell lysates
were separated by SDS-polyacrylamide gel electrophoresis and
immunoblotted with antibodies to IL-1 (the proIL-1 form is
detected in cell lysates), GFAP, or iNOS. Molecular weight standards
(× 103) are indicated on the right. Immunoblot
is representative of four experiments with similar results.
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Fig. 2.
Time course of
A42-stimulated cytokine production.
Cultures were stimulated for up to 12 h with either control buffer
or 10 µM A42 (except for 0-h time point, which
represents untreated cells), and conditioned media were collected at
the indicated time-points. IL-1 (A) and TNF
(B) levels in the conditioned media were quantitated by
ELISA. Data shown represent the mean ± S.E. of three different
time-course experiments per ELISA, with each experiment conducted in
duplicate.
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Another cytokine rapidly induced in glia by A42 is TNF. Because
TNF is readily secreted from the cells, an ELISA was performed on
cell conditioned media to measure the time course of TNF production from A42-stimulated cells. As shown in Fig. 2B, there was
a rapid increase in cytokine production upon addition of A42, with
TNF protein detected as early as 1 h after stimulation and high
levels of TNF sustained through 12 h. These time courses
demonstrate that IL-1 and TNF responses are early, rapid
inflammatory responses to A42 with protein production occurring
within the first hr of exposure to A42, and support the possibility
that A42-stimulated iNOS production may be a response to an initial
IL-1 and/or TNF production.
Protein Synthesis Inhibitor Blocks A42-stimulated iNOS mRNA
Levels--
If cytokine production is necessary for A42-stimulated
iNOS expression, then we would expect that cells pre-treated with cycloheximide, a protein synthesis inhibitor, should fail to express iNOS when treated with A42. Northern blot analysis (Fig.
3) of cells stimulated with 10 µM A42 for 12 h in the presence of 1 µM cycloheximide demonstrates that IL-1 mRNA can
still be detected strongly, but iNOS mRNA levels are reduced. The
Northern blots were stripped and then re-probed with GAPDH for
normalization (data not shown). Quantitation of several experiments by
phosphorimaging densitometry showed that the A42-stimulated iNOS
mRNA levels were reduced in the presence of cycloheximide by
approximately 50% compared with the mRNA levels seen in the
A42-stimulated cells in the absence of cycloheximide (Fig. 3). In
cells treated with cycloheximide and stimulated with 1 mM
Bt2cAMP, which can directly induce iNOS expression, there
was no decrease in iNOS mRNA levels, but in fact a dramatic
increase in iNOS mRNA levels (data not shown). These data indicate
that the reduction of A42-stimulated iNOS mRNA in the presence
of cycloheximide is not due to cell toxicity, and suggest that new
protein synthesis is important for iNOS induction.
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Fig. 3.
Inhibition of
A42-stimulated iNOS mRNA levels by
cycloheximide. Cultures were stimulated for 12 h with either
control buffer (C) or 10 µM A42 in the
presence (+CHX) or absence (CHX) of 1 µM cycloheximide. IL-1 and iNOS mRNA levels were
quantitated by densitometry of Northern slot blots after normalization
to GAPDH mRNA levels. The IL-1 and iNOS mRNA levels are
expressed relative to the mRNA levels in the cultures treated with
control buffer in the absence of cycloheximide. Densitometry data are
the mean ± S.E. from five independent experiments. The IL-1
and iNOS slot blots shown are from one representative experiment.
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IL-1 Receptor Antagonist Decreases the Levels of
A42-stimulated iNOS and Nitrite Production--
As a more specific
means of inhibiting the effects of IL-1, we stimulated cells with
A42 for 18 h in the absence or presence of the IL-1 receptor
antagonist (IL-1ra). IL-1ra attenuated A42-stimulated levels of iNOS
protein as determined by Western blot analysis (Fig.
4A), but did not affect GFAP
protein levels. Similarly, we used increasing concentrations of IL-1ra
and measured the levels of nitrite, the stable metabolite of NO, that
accumulated in the conditioned medium. Cells pre-treated for
approximately 5 min with increasing concentrations of IL-1ra showed a
dose-dependent decrease in nitrite levels in both
A42-stimulated cells and IL-1-stimulated cells (Fig.
4B). Complete inhibition of IL-1-stimulated nitrite production was not observed as the concentration of receptor antagonist necessary for such an effect was not reached (IL-1ra concentrations were initially optimized for inhibition of A42-stimulated nitrite levels, which require less receptor antagonist than direct IL-1 administration). IL-1ra did not inhibit the levels of nitrite produced
by 1 mM Bt2cAMP-stimulated astrocytes,
demonstrating the specificity of the receptor antagonist as cAMP can
stimulate iNOS transcription via molecular mechanisms different from
IL-1 (42, 43).
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Fig. 4.
Inhibition of
A42-stimulated iNOS and nitrite production by
IL-1 receptor antagonist. A, Western blot analysis of
cultures stimulated for 18 h with either 10 µM
A42 or 10 µM A42 plus 50 ng/ml recombinant murine
IL-1 receptor antagonist (IL-1ra). IL-1ra attenuates A42-stimulated
iNOS protein levels, but does not affect GFAP protein levels. The blot
shown is a representative of 4 independent experiments. B,
cultures were stimulated for 30 h with 5 µM A42,
100 ng/ml recombinant rat IL-1, or 1 mM
Bt2cAMP in the absence (0) or presence of increasing
concentrations (3, 10, 30 ng/ml) of IL-1ra. Nitrite levels in the
conditioned medium were quantitated by Griess assay, and expressed as
percentage of control, where control is the maximum amount of
stimulated nitrite production in the absence of receptor antagonist.
Values represent mean ± S.E. of eight experiments.
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Dominant-negative TRAF Proteins Can Block NFB Activation in
Astrocytes--
In many cell types, the IL-1 signaling pathway
stimulates the activation of NFB via TRAF6, and the TNF signaling
pathway stimulates NFB activation via TRAF2. Dominant-negative TRAF6 (N-terminal truncation, leaving only the amino acids from 301 to 531;
termed dnTRAF6-C) has been reported to be able to inhibit NFB
activation by IL-1; similarly, dominant-negative TRAF2 (amino acids
87-501; termed dnTRAF2-C) can inhibit NFB activation by TNF
(27). We tested the effects of these dominant-negative constructs on
A42 stimulation of astrocytes. First, we overexpressed dnTRAF6-C and
dnTRAF2-C in the cells and verified that each construct can inhibit
NFB activity, as measured by a co-transfected NFB SEAP reporter
construct. As expected, dnTRAF2-C was able to inhibit TNF but not
IL-1 activation of the NFB reporter in astrocytes (Fig.
5A), and conversely, dnTRAF6-C
was able to inhibit IL-1 but not TNF activation of the NFB
reporter (Fig. 5B). Interestingly, it has recently been
reported that dnTRAF6-C but not dnTRAF2-C can inhibit LPS-stimulated
NFB activity in an endothelial cell line and a monocyte cell line
(44), suggesting the importance of TRAF6 in LPS stimulation of
NFB.
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Fig. 5.
Demonstration of selective functionality for
dominant-negative TRAFs in NFB
activation. Cultures were co-transfected with the reporter
construct pNFB-SEAP and increasing amounts (0.05, 0.15, or 0.5 µg)
of dnTRAF2-C or dnTRAF6-C (in all transfections, the total amount of
DNA transfected per well was kept constant with the addition of
pCMV2-FLAG, the control vector). After 48 h, cells were stimulated
for 12 h with either 20 ng/ml rat TNF (A) or 50 ng/ml rat IL-1 (B), and then conditioned medium was
collected. NFB activity was determined by secreted alkaline
phosphatase (SEAP) assays on equal amounts of conditioned medium.
Activity is expressed as percentage of control, where control is the
maximum amount of stimulated SEAP activity in the absence of
dominant-negative construct. Data represent mean ± S.E. of eight
independent experiments.
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Dominant-negative TRAF6 Inhibits iNOS Promoter Activation by
A42--
While the participation of TRAF2 and TRAF6 in NFB
activation by specific cytokines is now well identified, the
involvement of TRAF2 and TRAF6 in stimulation of gene expression is
less well studied. Only recently has TRAF6 been implicated in IL-8
promoter activity in macrophages (45). To address the possibility that iNOS promoter activation by A42 is mediated by TNF or IL-1, we
co-transfected cells with the iNOS promoter-luciferase reporter construct and increasing amounts of dnTRAF2-C or dnTRAF6-C. As shown in
Fig. 6, increasing amounts of dnTRAF6-C
reduced A42-stimulated iNOS promoter activity by approximately 80%,
and increasing amounts of dnTRAF2-C reduced A42-stimulated iNOS
promoter activity by approximately 60%, suggesting that IL-1, and
to some extent TNF, are critical mediators of A42-stimulated iNOS
activation. In contrast, dnTRAF2-C and dnTRAF6-C decreased
Bt2cAMP-stimulated iNOS promoter activity by only ~25%
(Fig. 6), suggesting only a minor role for IL-1 and TNF in
Bt2cAMP-stimulated iNOS activity.
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Fig. 6.
Dominant-negative TRAFs and iNOS promoter
activation. Cultures were co-transfected with the iNOS-Luc
reporter construct and increasing amounts (0.6 or 2.0 µg) of
dnTRAF2-C or dnTRAF6-C (total amount of DNA transfected equalized with
control vector as in Fig. 5). After 48 h, cells were stimulated
with either 10 µM A42 (A) or 1 mM Bt2cAMP (B) for 12 h, and
then the luciferase activity in cell lysates was determined. Data are
expressed as percentage of control, where control is the maximum amount
of stimulated luciferase activity in the absence of dominant-negative
construct. Data represent mean ± S.E. of 10 independent
experiments.
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Because NIK is involved in mediating both IL-1 and TNF signaling
pathways, an over-expressed kinase-inactive NIK (dominant-negative NIK,
termed dnNIK) should inhibit both IL-1- and TNF-stimulated NFB
activation (46). When cells were co-transfected with the iNOS promoter
reporter construct and increasing amounts of dnNIK, A42-stimulated
iNOS activity was inhibited by approximately 80%, whereas the
Bt2cAMP-stimulated iNOS promoter activity was not significantly reduced (Fig. 7). The dnNIK
inhibition curve for A42-stimulated iNOS promoter activity is
similar to the dnTRAF6-C inhibition curve of the iNOS promoter
activity, suggesting that TRAF6 (and thus IL-1) is the major
mediator of A42-stimulated iNOS production in these rat glial cells,
and that TRAF2 (and thus TNF) is a secondary mediator of
A42-stimulated iNOS production.
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Fig. 7.
Dominant-negative NIK and iNOS promoter
activation. Cultures were co-transfected with the iNOS-Luc
reporter construct construct and increasing amounts (0.6 or 2.0 µg)
of dnNIK (in all transfections, the total amount of DNA transfected per
well was kept constant with the addition of pcDNA3-HA, the control
vector). After 48 h, cells were stimulated with either 10 µM A42 or 1 mM Bt2cAMP for
12 h, and then luciferase activity in cell lysates was determined.
Data are expressed as percentage of control, where control is the
maximum amount of stimulated luciferase activity in the absence of
dnNIK. Data represent mean ± S.E. of eight independent
experiments.
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IL-1 Localizes to Microglia and iNOS Localizes to Astrocytes in
A42-stimulated Glial Culture--
To identify the cell type(s)
responsible for IL-1 and iNOS production in the rat glial cultures,
cells were stimulated with A42 for 12 or 36 h and then IL-1
and iNOS localization monitored by FITC immunofluorescence. Double
labeling for GFAP with Texas Red immunofluorescence was done to allow
for astrocyte identification. As shown in Fig.
8A, IL-1 localizes to
non-astrocytic cells (no overlapping FITC/Texas Red fluorescence). The
IL-1-positive cells were primarily microglia, as determined by OX42
staining (data not shown). In contrast, as shown in Fig. 8B,
iNOS was detected in astrocytes (overlapping FITC/Texas Red fluorescent
cells). These data suggest that A42 stimulates IL-1 production by
activated microglia, which in turn can participate in the activation of astrocytes to produce iNOS.
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Fig. 8.
Immunofluorescent identification of
IL-1- and iNOS-positive glial cells.
Mixed glial cell cultures containing both microglia and astrocytes were
stimulated with 10 µM A42 for either 12 h (for
IL-1, panel A) or 36 h (for iNOS,
panel B), and then processed for
immunofluorescent microscopy. Texas red-conjugated secondary antibody
identifies GFAP-positive astrocytes. FITC-conjugated secondary antibody
identifies IL-1-positive cells, which do not overlap with
GFAP-positive astrocytes (separate green and red
cells) (A), and iNOS-positive cells, which
overlap with GFAP astrocytes (superimposition of green and
red signal resulting in yellow color)
(B).
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DISCUSSION |
We demonstrate here that in rat glial cultures, the amyloid plaque
component A42 stimulates IL-1 and TNF cytokine production in
microglia prior to astrocyte iNOS production, and that the A42-stimulated iNOS promoter activity and NO production occur in an
IL-1-dependent (and to a lesser extent,
TNF-dependent) manner via a signal transduction pathway
that involves TRAF6 and NIK. We not only therefore define a clear
linkage among A42, inflammatory cytokines, and putative
peroxynitrite neurotoxicity, but we also demonstrate for the first time
that TRAF6, TRAF2, and NIK can mediate an inflammatory response that is
relevant to AD. Because there is an imperative need to understand
inflammation in AD, identifying each component such as these in a
signal transduction pathway that ultimately leads to deleterious
effects in the central nervous system such as peroxynitrite-mediated
damage to neurons is important from a therapeutic standpoint.
The pro-inflammatory cytokine IL-1 is a critical neurotoxic component
to such an inflammatory pathway, and is found in activated microglia
localized to amyloid plaques in AD (47). Evidence that IL-1 can
mediate neuronal injury has been shown recently in transgenic mouse
models of IL-1-converting enzyme (ICE). Significantly less ischemic
brain injury occurs in ICE-knockout mice (48) as well as in
dominant-negative ICE transgenic mice (49). Similarly, adenoviral-directed cerebral expression of IL-1ra in mouse brain also resulted in reduced ischemia-induced brain injury (50). A major
consequence of IL-1 production in the brain is the stimulation of
astrocytic iNOS activity. IL-1 stimulation of iNOS in rat, mouse,
and human astrocytes as well as in other cell types that can
participate in inflammation, such as endothelial cells, hepatocytes, and macrophages, has been well characterized (for reviews, see Refs. 51
and 52), and such IL-1-stimulated iNOS activity has been suggested
to be detrimental in several neurodegenerative disorders including AIDS
dementia complex, the murine multiple sclerosis model of experimental
autoimmune encephalitis, and AD (53). This detrimental effect of iNOS
activity is supported by evidence from knockout mice studies, which
demonstrate that under ischemic conditions, there is a delayed
reduction of brain injury and neurological deficits in iNOS-null mice
(54). Previously, we showed (17) that A42, the primary amyloid
plaque component in the brain, can activate glia in vitro in
a dose- and time-dependent manner, as determined by
morphological response and IL-1 expression, and that A42
stimulates iNOS activity in astrocytes via the transcription factor
NFB. Whether this astrocyte iNOS activity is a direct result of
A42 stimulation of astrocytes or an indirect result, requiring the
participation of other glial factors or inflammation mediators such as
IL-1 had not been defined previously. While studies consistent with
ours (12, 13, 18) have shown that different forms and preparations of
A42 can stimulate glial cells to produce cytokines or NO, there have
been no previous reports to demonstrate and define the linkage between
A42-stimulated microglial IL-1 (or TNF) and astrocytic
iNOS.
Our results demonstrating that A42-stimulated astrocyte iNOS
mRNA production requires new protein synthesis is consistent with
previous studies using cycloheximide to inhibit LPS-stimulated iNOS
mRNA production in fetal hepatocytes (55). Cycloheximide was
partially able to reduce A42-stimulated iNOS mRNA production. The partial reduction of iNOS mRNA may reflect the fact that the 1 µM cycloheximide concentration is not enough to
completely inhibit protein synthesis. Higher concentrations of
cycloheximide were not used because they proved to be toxic to the
primary astrocyte cultures; the 1 µM cycloheximide
concentration did not promote a strong inflammatory response or toxic
response in the astrocytes. A slight increase in iNOS mRNA was
detected in cells treated with control buffer alone in the presence of
cycloheximide, but this weak stimulation has been seen before in other
systems with a similar concentration of cycloheximide (55). Similarly,
the observation that A42-stimulated nitrite could not be inhibited completely with increasing concentrations of IL-1ra is consistent with
previous studies using human fetal astrocytes stimulated with IL-1
(56), where the effective concentration of receptor antagonist needed
to completely inhibit IL-1-stimulated iNOS mRNA production was
several orders of magnitude higher than the concentration of stimulus.
The ability of dnTRAF2-C to inhibit TNF- but not IL-1-stimulated
NFB activation in cultured astrocytes, and the ability of dnTRAF6-C
to inhibit IL-1- but not TNF-stimulated NFB activation in
cultured astrocytes are consistent with previous studies with 293 cells, which demonstrate TRAF-specificity in cytokine stimulation of
NFB (27). In addition, we have extended our investigations beyond
the NFB response element reporter construct, and using the iNOS
promoter reporter construct, we show, as has recently been shown for
TRAF2 and the IL-8 promoter (45), that TRAF- and
NIK-dependent signaling can also produce direct
consequences in models of inflammation, emphasizing the need to
characterize further how TRAFs and NIK interact in signal transduction mechanisms.
There are other potential signal transduction pathways activated during
inflammation, which may also participate in A42-stimulated iNOS
production in astrocytes. For example, the p38 MAPK and c-Jun N-terminal kinase/stress-activated protein kinase stress kinase pathways that target the AP-1 transcription factor could contribute to
iNOS promoter activity. In mixed glial cultures, it has been reported
(57) that p38 MAPK can mediate TNF- and IL-1-stimulated iNOS
activation in astrocytes, based on the observation that the p38
inhibitor SB203580 is able to inhibit cytokine-stimulated iNOS activity
and nitrite production. Whether or not p38 MAPK plays a role in
A42-stimulated IL-1 production in microglia or regulates iNOS
expression in astrocytes in cooperation with NFB has not been
addressed. A recent study showed that p38 MAPK was important in
LPS-stimulated IL-1 transcription in murine macrophages (58), so it
is possible that in a mixed glial culture containing both microglia and
astrocytes, suppressing p38 MAPK leads indirectly to iNOS inhibition
via an IL-1 inhibition in microglia. Indeed, it has recently been
demonstrated that activated p38 MAPK can be immunolocalized to
microglia that are associated with amyloid plaques in AD brain (59),
and amyloid fibrils can activate p38 MAPK in microglia in
vitro (60).
Altogether, our data substantiate a model of inflammation observed in
AD where A42 activates microglia to produce pro-inflammatory cytokines such as IL-1 and TNF. These cytokines in turn activate surrounding astrocytes, which exacerbate the inflammation with the
production of neurotoxic mediators such as iNOS. The resultant peroxynitrite ultimately damages local neurons and contributes to the
neurodegeneration observed in AD.
-Amyloid Stimulation of Inducible Nitric-oxide Synthase in
Astrocytes Is Interleukin-1
(TNF
B-inducing
Kinase-dependent Signaling Mechanism*
and
TOP
ABSTRACT
INTRODUCTION
