1 Department of Internal Medicine, Seoul National University College of Medicine, and 3 Lung Institute, Seoul National University Medical Research Center, Chongno-Gu, Seoul 110-799; and 2 Clinical Research Institute, Seoul National University Hospital, Chongno-Gu, Seoul 110-744, Korea
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ABSTRACT |
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The anti-inflammatory effect of
acetylsalicylic acid (ASA) has been thought to be secondary to the
inhibition of prostaglandin synthesis. Because doses of ASA necessary
to treat chronic inflammatory diseases are much higher than those
needed to inhibit prostaglandin synthesis, a prostaglandin-independent
pathway has been emerging as the new anti-inflammatory mechanism of
ASA. Here, we examined the effect of ASA on the interleukin
(IL)-1- and tumor necrosis factor (TNF)-
-induced
proinflammatory cytokine expression and evaluated whether this effect
is closely linked to the nuclear factor (NF)-
B/I
B-
pathway. A
high dose of ASA blocked IL-1
- and TNF-
-induced TNF-
and IL-8
expression, respectively. ASA inhibited TNF-
-induced activation of
NF-
B by preventing phosphorylation and subsequent degradation of
I
B-
in a prostanoid-independent manner. TNF-
-induced
activation of I
B kinase was also suppressed by ASA pretreatment.
These observations suggest that the anti-inflammatory effect of ASA in
lung epithelial cells may be due to suppression of I
B kinase
activity, which thereby inhibits subsequent phosphorylation and
degradation of I
B-
, activation of NF-
B, and proinflammatory cytokine expression in lung epithelial cells.
nuclear factor-B; interleukin-8
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INTRODUCTION |
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OVER THE LAST DECADE, many studies of basic biological characteristics of inflammation and tissue injury have implicated proinflammatory cytokine-mediated tissue injury in the pathogenesis of a wide variety of inflammatory disorders including sepsis, acute respiratory distress syndrome, and multiorgan dysfunction syndrome. As a result, anti-inflammatory agents, which inhibit the expression of proinflammatory cytokines, have been tried as the specific therapy for these diseases (4).
Acetylsalicylic acid (ASA) is a nonsteroidal anti-inflammatory drug (NSAID) used in the treatment of many inflammatory diseases. It is rapidly deacetylated to salicylate in the intact organism. Its ability to inhibit arachidonic acid metabolites by blocking cyclooxygenase (COX) and prostaglandin H synthase has been regarded as the main anti-inflammatory mechanism. However, doses of ASA necessary to treat chronic inflammatory diseases are much higher than those needed to inhibit prostaglandin synthesis (21, 25). In addition, nonacetylated salicylates, which do not interfere with prostaglandin synthesis, are still effective anti-inflammatory agents when used in high doses (21, 25). These findings have led to the speculation that the anti-inflammatory effect of ASA may be mediated by a prostaglandin-independent pathway.
Nuclear factor (NF)-B is a ubiquitous inducible transcription factor
involved in immune, inflammatory, stress, and developmental processes.
It is sequestered in the cytoplasm in an inactive state by association
with the inhibitory molecule I
B-
. NF-
B is rapidly activated in
response to various stimuli including viral infection, lipopolysaccharide, ultraviolet (UV) irradiation, and proinflammatory cytokines such as tumor necrosis factor (TNF)-
and interleukin (IL)-1
(2, 3, 11). TNF-
leads to the sequential
activation of the downstream NF-
B-inducing kinase (NIK) and the
recently isolated TNF-
-inducible I
B kinase (IKK) complex
(9, 15, 23, 26, 29). When activated, IKK directly
phosphorylates Ser32 and Ser36 of I
B-
,
triggering ubiquitination at Lys21 and Lys22
and rapid degradation of I
B-
in 26S proteasomes (2, 3, 11). This process liberates NF-
B, allowing it to translocate to the nucleus. In the nucleus, NF-
B binds to its cognate
B site and transactivates the downstream genes. Most genes for
inflammatory mediators such as TNF-
, IL-2, IL-6, IL-8, lymphotoxin,
granulocyte-macrophage colony-stimulating factor, interferon-
, and
adhesion molecules have
B sites in the 5'-flanking region (2,
3, 11).
Recent reports (14, 16, 20, 24) suggest that high doses of
salicylates show an anti-inflammatory effect through the inhibition of
NF-B activation in monocytic, lymphocytic, and endothelial cells.
However, the anti-inflammatory effect of ASA and its mechanism of
action in lung epithelial cells are poorly understood. In the present
study, we investigated the effect of ASA on proinflammatory cytokine
expression and evaluated whether this effect is closely linked to
NF-
B/I
B-
regulation in lung epithelial cells. First, we found
that a high dose of ASA blocked TNF-
-induced IL-8 mRNA and protein
expression. Second, ASA pretreatment inhibited TNF-
-induced
activation of NF-
B by preventing the degradation of I
B-
.
Finally, TNF-
-induced activation of endogenous IKK and subsequent
phosphorylation of I
B-
were suppressed by ASA pretreatment. These
observations suggest that the anti-inflammatory effect of ASA in lung
epithelial cells may be due to the blocking of I
B-
phosphorylation by suppressing IKK activity, thereby inhibiting
subsequent degradation of I
B-
, activation of NF-
B, and
proinflammatory cytokine expression.
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METHODS |
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Cell culture. BEAS-2B cells, representing normal human bronchial epithelial cells, were maintained as a monolayer in keratinocyte growth medium (Clonetics, Walkersville, MD), and A549 cells, representing type II alveolar epithelial cells, were maintained in RPMI 1640 medium containing 10% fetal bovine serum, 60 µg/ml of penicillin, and 100 µg/ml of streptomycin at 37°C under 5% CO2.
Reagents.
Recombinant human TNF- and an ELISA kit for IL-8 were purchased from
R&D Systems (Minneapolis, MN). A stock solution of TNF-
was prepared
in distilled water, and aliquots were stored at
70°C until used.
Rabbit polyclonal anti-I
B-
, anti-p65, and anti-IKK-
antibodies
and recombinant glutathione S-transferase (GST)-I
B-
were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit
polyclonal anti-phosphorylated I
B-
antibody (Ser32)
was supplied by New England Biolabs (Beverly, MA). Goat anti-rabbit secondary antibody conjugated with horseradish peroxidase and T4
polynucleotide kinase were purchased from Promega (Madison, WI).
Rhodamine isothiocyanate-conjugated goat anti-rabbit immunoglobulin G
antibody was obtained from Jackson ImmunoResearch (West Grove, PA).
Protein G Sepharose beads and an enhanced chemiluminescence kit were
supplied by Amersham Pharmacia Biotech (Uppsala, Sweden). Protease
inhibitors were obtained from Roche (Mannheim, Germany). ASA,
indomethacin, and prostaglandin E2 were obtained from Sigma (St. Louis, MO). The proteasome inhibitor
N-carbobenzoxyl-Leu-Leu-Leu-leucinal (MG-132) was purchased
from the Peptide Institute (Osaka, Japan). TRIzol reagent was obtained
from GIBCO BRL (Life Technologies, Gaithersburg, MD).
[
-32P]dCTP and [
-32P]ATP were
supplied by ICN Pharmaceuticals (Costa Mesa, CA). A random-priming kit
was purchased from Stratagene (La Jolla, CA).
Northern blot analysis.
Total cellular RNA was isolated with TRIzol reagent. Equal amounts of
total RNA (20 µg/lane) from each sample were loaded into each lane of
1.0% agarose-2% formaldehyde gels and capillary transferred to nylon
membrane. The RNA was cross-linked to the nylon membrane by 1,500-J UV
irradiation in a UV cross-linker (Stratagene). The human cDNA for IL-8
was radiolabeled with [-32P]dCTP with a random-priming
kit. After prehybridization of the membranes for 2 h at 45°C in
hybridization buffer, radiolabeled cDNA probe (1 × 106
counts · min
1 · ml
1 final
concentration) was added and incubated overnight at 45°C. The
membranes were then washed at 45, 50, and then 55°C. The membranes were exposed to X-ray film in a cassette with an intensifying screen at
70°C.
IL-8 ELISA.
Cells (1 × 104) were grown in 96-well culture plates
in equal numbers. The supernatants were collected and stored at
70°C until analyzed. IL-8 concentrations were quantified with an
ELISA kit according to the manufacturer's specifications.
Western blot analysis.
Cytoplasmic, nuclear, and whole cell extracts were prepared as
previously described (28). Twenty micrograms of protein
were resolved by 10% SDS-PAGE and transferred to nitrocellulose. The membranes were blocked with 5% skim milk-PBS-0.1% Tween 20 for 1 h before overnight incubation at room temperature with rabbit polyclonal anti-p65 antibody, anti-IB-
antibody, or antibody specific for phosphorylated I
B-
diluted 1:1,000 in 5% skim
milk-PBS-0.1% Tween 20. The membranes were washed three times in 1×
PBS-0.1% Tween 20 and incubated with goat-anti-rabbit horseradish
peroxidase-conjugated antibody diluted 1:2,000 in 5% skim
milk-PBS-0.1% Tween 20 for 1 h. After successive washes, the
membranes were developed with an enhanced chemiluminescence kit.
Immunofluorescent staining for NF-B.
The cells grown in two-well chamber slides were fixed and permeabilized
as previously described (28). The cells were incubated with rabbit polyclonal anti-p65 antibody diluted 1:100 in 1% BSA for
30 min. The cells were incubated with rhodamine
isothiocyanate-conjugated goat anti-rabbit immunoglobulin G antibody
diluted 1:100 in 1% BSA for 30 min. After being mounted with 50%
glycerol, the slides were analyzed with a fluorescence light microscope.
Electrophoretic mobility shift assays.
NF-B DNA binding activity was assessed as previously described
(28). Briefly, nuclear extracts were incubated for 20 min at room temperature with a radiolabeled NF-
B consensus sequence in
the
light chain enhancer in B cells
(5'-AGTTGAGGGGACTTTCCCAGGC-3'). In competition experiments,
a 50-fold molar excess of unlabeled oligonucleotide was added to the
binding reaction. In supershift experiments, 0.4 µg of anti-p65 or
anti-p50 antibody was added and allowed to react for 45 min at room
temperature. DNA-protein complexes were resolved on 4% nondenaturing
polyacrylamide gels. The gels were dried and autoradiographed at
70°C.
IKK assay.
IKK activity was assessed with an in vitro kinase assay as previously
described (28). In brief, the IKK complex was
immunoprecipitated with an anti-IKK- antibody diluted 1:100. The
immunoprecipitates were incubated at 30°C for 30 min in a kinase
buffer containing 0.5 µg of GST-I
B-
(containing amino acids
1-317) and 10 µCi of [
-32P]ATP. Kinase reaction
products were subjected to SDS-PAGE in 10% gels followed by transfer
to a nitrocellulose membrane and autoradiography.
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RESULTS |
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ASA blocks IL-1- and TNF-
-induced proinflammatory cytokine
expression.
To determine whether ASA shows anti-inflammatory effects in lung
epithelial cells, we first analyzed the effect of ASA on proinflammatory cytokine expression. To evaluate whether ASA inhibits cytokine production dose dependently, A549 cells were pretreated with
medium or various amounts of ASA (2.5, 5, 10, or 20 mM) for 2 h
and then stimulated with IL-1
or TNF-
. IL-8 concentrations in the
culture supernatants were assayed by ELISA after 18 h of TNF-
stimulation. TNF-
increased IL-8 production in the absence of ASA.
Both IL-1
- and TNF-
-induced IL-8 production were reduced by
pretreatment with a high dose of ASA (Fig.
1A). To evaluate whether the
reduction in TNF-
-induced IL-8 production was due to the decrease in
mRNA expression, the cells were pretreated with 20 mM ASA for 2 h
and then stimulated with IL-1
or TNF-
for 4 h.
IL-1
-induced TNF-
and IL-1
- and TNF-
-induced IL-8 mRNA
expression were assayed by Northern blot analysis. Although TNF-
mRNA was hardly detectable in untreated cells, IL-1
induced a marked
increase in TNF-
mRNA 4 h after stimulation, and this increase
was blocked completely in the presence of ASA (Fig. 1B). Both IL-1
- and TNF-
-induced IL-8 mRNA expression were also
suppressed by ASA pretreatment (Fig. 1B). To exclude the
possibility that this effect of ASA is due to its cytotoxicity, the
cells were incubated in the presence of 2.5, 5, 10, or 20 mM ASA for
2.5 h. Cell viability was evaluated by MTT assay. Cell viability
did not change in both cells at all doses used (data not shown). These observations indicate that ASA shows anti-inflammatory effects in lung
epithelial cells by inhibiting proinflammatory cytokine production.
|
NF-B activation is inhibited by ASA.
Because most of the proinflammatory cytokine genes including IL-8
contain
B-binding motifs in their promoter regions, we questioned
whether the inhibition of proinflammatory cytokine expression by ASA is
due to the blocking of TNF-
-induced activation of NF-
B. NF-
B
activation was assayed by two approaches: one measured the nuclear
translocation of NF-
B and the other assessed the NF-
B-DNA binding
activity by electrophoretic mobility shift assay. The expression of
NF-
B was assayed by Western blot analysis for the p65 subunit of
NF-
B in cytoplasmic and nuclear extracts from cells stimulated with
TNF-
in the presence and absence of ASA. Although the majority of
p65 was located in the cytoplasmic fraction in the basal state, p65
increased in the nuclear fraction 30 min after TNF-
stimulation,
which was completely blocked by ASA pretreatment (Fig.
2A). Total cellular expression
of p65 was not affected by ASA pretreatment (Fig. 2A). We
next investigated the subcellular localization of NF-
B by
immunofluorescent staining. There was a strong nuclear staining of p65
30 min after stimulation with TNF-
in both BEAS-2B and A549 cells
compared with the cytoplasmic distribution in unstimulated cells. This
nuclear translocation of p65 by TNF-
was blocked by ASA pretreatment
as demonstrated by the cytoplasmic staining pattern (Fig.
2B). We next evaluated the effect of ASA on the NF-
B-DNA
binding activity by electrophoretic mobility shift assay. Nuclear
extracts from TNF-
-stimulated cells had more active NF-
B
available to bind to the
B probe compared with extracts from
untreated cells. This TNF-
-induced increase in NF-
B-DNA binding
activity was inhibited by 20 mM ASA (Fig. 2C). When a
50-fold molar excess of unlabeled double-strand NF-
B oligonucleotide
was added to the binding reaction, the retarded band disappeared,
suggesting the specificity of binding. Supershift assay showed the
presence of the p50 and p65 subunits of NF-
B (Fig. 2C).
These results indicate that suppression of proinflammatory cytokine
expression by ASA is due to the blockade of NF-
B activation.
|
ASA suppresses IB-
degradation in a prostanoid-independent
manner.
Because NF-
B exists as an inactive form bound to the inhibitory
protein I
B-
in the cytoplasm, the degradation of I
B-
must
occur in order for NF-
B to translocate to the nucleus. We next
analyzed the effect of ASA on the IL-1
- and TNF-
-induced degradation of I
B-
. IL-1
- and TNF-
-induced degradation of I
B-
was blocked by high doses of ASA (Fig.
3A). ASA is known to be a weak
inhibitor of COX activity in respiratory epithelial cells. To evaluate
whether the inhibition of COX activity stabilizes I
B-
, we
examined the effect of indomethacin, which is a potent COX inhibitor,
on the TNF-
-induced degradation of I
B-
. I
B-
degradation
by TNF-
was not blocked by indomethacin pretreatment at all doses
used (10, 50, 100, and 500 µM; Fig. 3B). To evaluate whether ASA stabilizes I
B-
in a prostanoid-dependent
manner, we next examined the effect of exogenously applied
prostaglandin E2 on the I
B-
stabilizing effect of
ASA. Exogenously applied prostaglandin E2 did not reduce
the blocking effect of ASA on the TNF-
-induced degradation of
I
B-
(Fig. 3C). These observations suggest that
ASA-induced blocking of NF-
B activation is due to the stabilization
of I
B-
in a prostanoid-independent manner.
|
IB-
phosphorylation is blocked by ASA.
I
B-
degradation by a proteasome-dependent pathway is preceded by
phosphorylation on two serine residues (Ser32 and
Ser36) and ubiquitination. To address the
mechanism involved in the stabilization of I
B-
by ASA, we
examined IL-1
- and TNF-
-induced phosphorylation of I
B-
by
Western blot analysis. The cells were pretreated with the proteasome
inhibitor MG-132, which allows the phosphorylated I
B-
to
accumulate in the cell. As previously reported (8, 17,
22), MG-132 pretreatment stabilized the phosphorylated I
B-
in response to IL-1
and TNF-
, which was detectable as a slower
migrating band (Fig. 4A).
These slower migrating bands disappeared after ASA pretreatment (Fig.
4A). Because these slower migrating bands disappeared when
extracts were incubated with calf intestinal phosphatase, they were
considered to be phosphorylated I
B-
(data not shown). This was
further confirmed by immunoblotting with an antibody specific to
phosphorylated I
B-
on Ser32 (Fig. 4B).
These observations suggest that stabilization of I
B-
by ASA may
be due to the inhibition of I
B-
phosphorylation.
|
ASA blocks activation of IKK.
Cytokine-induced IB-
phosphorylation is mediated by the IKK
complex. To evaluate whether the decrease in phosphorylated I
B-
in ASA-treated cells is due to the inhibition of IKK activity, the
effect of ASA on IKK activity was assayed with GST-I
B-
as a
substrate after TNF-
stimulation with various doses of ASA. TNF-
induced a marked increase in phosphorylated GST-I
B-
after 5 and
10 min of stimulation in BEAS-2B and A549 cells, respectively, which
implies activation of the IKK complex. TNF-
-induced phosphorylation of I
B-
was completely blocked by ASA pretreatment at doses of 10 mM in BEAS-2B cells and 20 mM in A549 cells, suggesting different sensitivities to ASA in different cell lines (Fig.
5). This inhibition of IKK activity by
ASA was not due to a decrease in IKK-
protein levels because
immunoblot analysis demonstrated comparable IKK-
expression at all
conditions (data not shown). These results indicate that inhibition of
I
B-
phosphorylation by ASA may be through the inhibition of IKK
activation.
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DISCUSSION |
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Salicylates have been shown to inhibit the transcription of genes
such as adhesion molecules and inducible nitric oxide synthase, which
are involved in the inflammation process (10, 20). Because proinflammatory cytokine-mediated tissue injury has been implicated in
the pathogenesis of a wide variety of inflammatory disorders and
transcription of most proinflammatory cytokine genes is dependent on
NF-B activation, it is very likely that the anti-inflammatory effect
of salicylates is closely related to the suppression of proinflammatory
cytokine expression. Although ASA and sodium salicylates inhibit
NF-
B activation in monocytic, lymphocytic, and endothelial cells
(14, 16, 20, 24), TNF-
-induced activation of NF-
B was not blocked by salicylates in cultured cardiac fibroblasts (10). The effect of salicylates on the activation of
NF-
B differs according to cell type. At present, few data about the
anti-inflammatory effect of ASA and its mechanism of action in lung
epithelial cells have been presented, whereas the importance of the
role of lung epithelial cells in lung inflammation is increasing.
In this study, we demonstrated that ASA inhibited IL-1- and
TNF-
-induced proinflammatory cytokine expression by blocking NF-
B
activation in lung epithelial cells. This effect of ASA can be
generalized to respiratory epithelial cells because the same effects
were observed in both BEAS-2B and A549 cells, which represent bronchial
and alveolar epithelial cells, respectively. Considering the important
role of proinflammatory cytokines in the inflammatory process, this
result suggests an anti-inflammatory effect of ASA in lung epithelial
cells. We also found that blocking of NF-
B activation was due to the
stabilization of I
B-
. This result coincides with previous reports
in monocytic, lymphocytic, and endothelial cells (14, 16, 20,
24).
Prostanoids are important mediators of airway inflammation, and their
synthesis is mediated by COX. NSAIDs, which potently inhibit COX
activity, reduced IL-8 production in a prostanoid-dependent manner in
airway smooth muscle cells (18), and a study
(19) has shown that inhaled NSAIDs are protective in
airway inflammation. Because ASA is known to inhibit COX activity, we
examined whether the IB-
stabilizing effect of ASA is prostanoid
dependent. Indomethacin, which potently inhibits COX-induced
prostaglandin E2 production, did not block TNF-
-induced
degradation of I
B-
, and the I
B-
stabilizing effect of ASA
was not overcome by exogenously applied prostaglandin E2.
The most likely explanation for this result is that the
anti-inflammatory effect of ASA is prostanoid independent in lung
epithelial cells.
Because NF-B is sequestered in the cytoplasm by I
B-
,
activation of NF-
B requires degradation of I
B-
. The first step of degradation involves phosphorylation of I
B-
by the IKK
complex. The IKK complex is made up of several kinases including
IKK-
and IKK-
, and it requires phosphorylation by NIK to
become activated. Phosphorylated I
B-
undergoes ubiquitination and
finally degradation through a proteasome pathway. Because
TNF-
-induced phosphorylation of I
B-
was not observed in
the presence of the proteasome inhibitor in this study, the I
B-
stabilizing effect of ASA is likely to occur at the level of I
B-
phosphorylation by either inhibition of IKK activity or activation of
phosphatase. Because our immune complex kinase assay showed that ASA
suppressed endogenous IKK activity, it is likely that ASA blocks
I
B-
phosphorylation by suppressing IKK activation rather than by
activating a phosphatase.
The TNF-- and IL-1
-induced NF-
B/I
B signaling pathway
involves distinct pathways. TNF-
stimulation recruits TNF
receptor-associated factor (TRAF)-2 and the receptor-interacting
protein (12, 13), whereas IL-1
uses the IL-1 receptor
(IL-1R) accessory protein and the IL-1R-associated kinase to transmit
signals to TRAF-6 (6, 7). The TNF-
and IL-1
pathways
converge on NIK to activate the IKK complex. Thus the target to block
cytokine-induced degradation of I
B-
could be the
receptor-interacting protein, TRAF-2, TRAF-6, NIK, or IKK. However,
because ASA pretreatment in this study blocked both TNF-
- and
IL-1
-induced phosphorylation of I
B-
by inhibiting the
activation of IKK, it seems likely that ASA interferes with a common
signal upstream or parallel to IKK.
It has been reported that in vitro treatment with salicylates or ASA
inhibited IKK- but failed to affect IKK-
(27). In this study, our results demonstrated that treatment of intact cells
with ASA inhibited TNF-
-induced endogenous IKK activity. Although we
used anti-IKK-
antibody for immunoprecipitation, it cannot be
concluded that ASA blocks the function of endogenous IKK-
because
IKK-
and IKK-
form a complex and IKK-
can be coimmunoprecipitated with anti-IKK-
antibody.
The concentrations of ASA that blocked TNF--induced IKK activation
in our experiments were 10 and 20 mM in BEAS-2B and A549 cells,
respectively, which is much higher than the usual therapeutic serum
concentration. Only high concentrations of ASA blocked nuclear translocation of NF-
B in endothelial cells (20, 24).
Therefore, it seems likely that this in vitro effect of ASA cannot be
applied to the anti-inflammatory effect in vivo. Salicylates accumulate in the mildly acidic environments prevailing at sites of inflammation (1, 5, 25). Salicylates are uncharged at low pH and can readily cross membranes (5). Therefore, the local
concentrations of ASA could be much higher than those of serum and
would be sufficient to suppress IKK activation and subsequent
NF-
B-dependent expression of proinflammatory cytokines.
In this study, we have shown that high doses of ASA inhibit
proinflammatory cytokine production by blocking NF-B activation in
lung epithelial cells. We demonstrated that this inhibitory effect of
ASA on NF-
B activation is secondary to the stabilization of
I
B-
by blocking the phosphorylation of I
B-
and its
subsequent degradation. This blocking of I
B-
phosphorylation by
ASA was due to the inhibition of IKK activity. Because proinflammatory cytokines function in redundant and overlapping ways through the so-called cytokine "cascade" or "network," it would be
necessary to modulate the entire cytokine network at the same time to
achieve an anti-inflammatory response. Because transcription of most
proinflammatory cytokine genes is regulated by NF-
B activation, our
data showing that inhibition of IKK activation by ASA resulted in the
stabilization of I
B-
and inhibition of NF-
B activation suggest
that the IKK complex could be an excellent molecular target for a new
anti-inflammatory therapy.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Sarang Kim for proofreading this manuscript.
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FOOTNOTES |
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This work was supported by a 1998 National Research and Development Program (Ministry of Science and Technology).
Address for reprint requests and other correspondence: C. G. Yoo, Dept. of Internal Medicine, Seoul National Univ. College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Korea (E-mail: cgyoo{at}snu.ac.kr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 August 2000; accepted in final form 11 September 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abramson, SB,
and
Weissmann G.
The mechanisms of action of nonsteroidal antiinflammatory drugs.
Arthritis Rheum
32:
1-9,
1989[ISI][Medline].
2.
Baldwin, AS.
The NF-B and I
B proteins: new discoveries and insights.
Annu Rev Immunol
14:
649-681,
1996[ISI][Medline].
3.
Barnes, PJ,
and
Karin M.
Nuclear factor-B: a pivotal transcription factor in chronic inflammatory diseases.
N Engl J Med
336:
1066-1071,
1997
4.
Berthiaume, Y,
Ware LB,
and
Matthay MA.
Treatment of acute pulmonary edema and the acute respiratory distress syndrome
In: Pulmonary Edema, edited by Matthay MA,
and Ingbar DH.. New York: Dekker, 1998, p. 575-631.
5.
Brooks, PM,
and
Day RO.
Nonsteroidal antiinflammatory drugsdifferences and similarities.
N Engl J Med
324:
1716-1725,
1991[ISI][Medline].
6.
Cao, Z,
Henzel WJ,
and
Gao X.
IRAK: a kinase associated with the interleukin-1 receptor.
Science
271:
1128-1131,
1996[Abstract].
7.
Cao, Z,
Xiong J,
Takeuchi M,
Kurama T,
and
Goeddel DV.
TRAF6 is a signal transducer for interleukin-1.
Nature
383:
443-446,
1996[ISI][Medline].
8.
Chen, Z,
Hagler J,
Palombella VJ,
Melandri F,
Scherer D,
Ballard D,
and
Maniatis T.
Signal-induced site-specific phosphorylation targets IB
to the ubiquitin-proteasome pathway.
Genes Dev
9:
1586-1597,
1995[Abstract].
9.
DiDonato, JA,
Hayakawa M,
Rothwarf DM,
Zandi E,
and
Karin M.
A cytokine-responsive IB kinase that activates the transcription factor NF-
B.
Nature
388:
548-554,
1997[ISI][Medline].
10.
Farivar, RS,
and
Brecher P.
Salicylate is a transcriptional inhibitor of the inducible nitric oxide synthase in cultured cardiac fibroblasts.
J Biol Chem
271:
31585-31592,
1996
11.
Ghosh, S,
May MJ,
and
Kopp EB.
NF-B and Rel proteins: evolutionarily conserved mediators of immune responses.
Annu Rev Immunol
16:
225-260,
1998[ISI][Medline].
12.
Hsu, H,
Huang J,
Shu H-B,
Baichwal V,
and
Goeddel DV.
TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex.
Immunity
4:
387-396,
1996[ISI][Medline].
13.
Hsu, H,
Shu H-B,
Pan M-B,
and
Goeddel DV.
TRADD-TRAF-2 and TRADD-FADD interactions define two distinct TNF receptor I signal transduction pathways.
Cell
84:
299-308,
1996[ISI][Medline].
14.
Kopp, E,
and
Ghosh S.
Inhibition of NF-B by sodium salicylate and aspirin.
Science
265:
956-959,
1994[ISI][Medline].
15.
Mercurio, F,
Zhu H,
Murray BW,
Schevchenko A,
Bennett BL,
Li JW,
Young DB,
Barbosa M,
Mann M,
Manning A,
and
Rao A.
IKK-1 and IKK-2: cytokine-activated IB kinases essential for NF-
B activation.
Science
278:
860-866,
1997
16.
Oeth, P,
and
Mackman N.
Salicylates inhibit lipopolysaccharide-induced transcriptional activation of the tissue factor gene in human monocytic cells.
Blood
86:
4144-4152,
1995
17.
Palombella, VJ,
Randa OJ,
Goldberg AL,
and
Maniatis T.
The ubiquitin-proteasome pathway is required for processing the NF-B1 precursor protein and the activation of NF-
B.
Cell
78:
773-785,
1994[ISI][Medline].
18.
Pang, L,
and
Knox AJ.
Bradykinin stimulates IL-8 production in cultured human airway smooth muscle cells: role of cyclooxygenase products.
J Immunol
161:
2509-2515,
1998
19.
Pang, L,
Pitt A,
Petkova D,
and
Knox AJ.
The COX-1/COX-2 balance in asthma.
Clin Exp Allergy
28:
1050-1058,
1998[ISI][Medline].
20.
Pierce, JW,
Read MA,
Ding H,
Luscinskas FW,
and
Collins T.
Salicylates inhibit IB-
phosphorylation, endothelial-leukocyte adhesion molecule expression, and neutrophil transmigration.
J Immunol
156:
3961-3969,
1996[Abstract].
21.
Rainsford, KD.
Aspirin and the Salicylates. London: Butterworths, 1984.
22.
Reed, MA,
Neish AS,
Luscinskas FW,
Palombella VJ,
Maniatis T,
and
Collins T.
The proteasome pathway is required for cytokine-induced endothelial-leukocyte adhesion molecule expression.
Immunity
2:
493-506,
1995[ISI][Medline].
23.
Regnier, CH,
Song HY,
Gao X,
Goeddel DV,
Cao Z,
and
Rothe M.
Identification and characterization of an IB kinase.
Cell
90:
373-383,
1997[ISI][Medline].
24.
Weber, C,
Erl W,
Pietsch A,
and
Weber PC.
Aspirin inhibits nuclear factor-B mobilization and monocyte adhesion in stimulated human endothelial cells.
Circulation
91:
1914-1917,
1995
25.
Weismann, G.
Aspirin.
Sci Am
264:
84-90,
1991[ISI][Medline].
26.
Woronicz, JD,
Gao X,
Cao Z,
Rothe M,
and
Goeddel DV.
IB kinase-
: NF-
B activation and complex formation with I
B kinase
and NIK.
Science
278:
866-869,
1997
27.
Yin, MJ,
Yamamoto Y,
and
Gaynor RB.
The anti-inflammatory agents aspirin and salicylate inhibit the activity of IB kinase-
.
Nature
396:
77-80,
1998[ISI][Medline].
28.
Yoo, CG,
Lee S,
Lee CT,
Kim YW,
Han SK,
and
Shim YS.
Anti-inflammatory effect of heat shock protein induction is related to stabilization of IB
through preventing I
B kinase activation in respiratory epithelial cells.
J Immunol
164:
5416-5423,
2000
29.
Zandi, E,
Rothwarf DM,
Delhase M,
Hayakawa M,
and
Karin M.
The IB kinase complex (IKK) contains two kinase subunits, IKK
and IKK
, necessary for I
B phosphorylation and NF-
B activation.
Cell
91:
243-252,
1997[ISI][Medline].