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,
*
Department of Human Genetics, National Institute of Genetics, Yata, Mishima, Shizuoka; and
First Department of Internal Medicine and
Department of Clinical Chemistry and Laboratory Medicine, Faculty of Medicine, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B,
nuclear factor-IL-6, and activator protein-1 have been shown to be
involved in this process. However, to date it has been thought that no
known STAT molecule plays a role in the activation of monocytes by LPS.
In this study we examined whether some known STAT molecule is
stimulated by LPS, based on the finding that a GAS motif sequence is
conserved in the promoter regions of human, mouse, and rat
cyclo-oxygenase-2 (COX-2) genes. Consequently, LPS induced activation
of STAT5 in human monocytes, and this STAT5 activation occurred in an
indirect way via granulocyte-macrophage CSF (GM-CSF) secreted by
LPS-stimulated monocytes. Expression of COX-2 protein was partially
reduced by treatment of anti-human GM-CSF Ab. Activation of STAT5
was inhibited by either IL-10 or dexamethasone (Dex), but not by
aspirin. IL-10 blocked activation of STAT5 indirectly by suppressing
GM-CSF production, while Dex inhibited this activation both directly
and indirectly. Taken together, these results suggest that in addition
to other transcription factors, STAT5 plays an important role in
activation of monocytes by LPS, and that STAT5 is another target for
IL-10 and Dex to inhibit COX-2 expression in activated monocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the details of how this molecular mechanism works remain
largely unknown, transcription factors such as nuclear factor-B
(NF-B),3 NF-IL-6, and
activator protein-1 (AP-1) have been previously shown to be involved in
this process (3, 4, 5, 6, 7, 8). Studies in mice of gene targeting of NF-B, and
NF-IL-6 have revealed that these mice are susceptible to some
particular pathogens. However, production of monokines was not
significantly impaired, although the promoter regions of their genes
have binding elements to these transcription factors (9, 10). These
results may indicate that other transcription factors are involved in
the LPS-induced activation of monocytes, and that they may coordinate
this process.
Latent cytoplasmic transcription factors, STATs, which were initially
identified as activated transcriptional factors by IFN-
and IFN-
,
are now believed to be involved in the signal pathways of many
cytokines along with some growth factors (11). To date, it has been
supposed that LPS does not induce activation of known STAT molecules in
monocytes, but it has been noted that a STAT-like molecule, LPS-induced
and IL-1-induced (LIL) STAT, is stimulated by LPS and binds to LPS and
IL-1-responsive element (LILRE) of the human pro-IL-1ß gene (12, 13).
IL-10 and dexamethasone (Dex) are known to exhibit similar
anti-inflammatory effects on activated monocytes. It has been
previously shown that IL-10 and Dex inhibited cytokine production in
LPS-stimulated monocytes at both transcriptional and
post-transcriptional levels (14, 15, 16, 17). In addition, we and others have
recently demonstrated that both IL-10 and Dex suppressed the expression
of the inducible cyclo-oxygenase (COX-2), which is believed to provide
substantial amounts of prostanoids at inflamed sites, in LPS-stimulated
monocytes (18, 19). The inhibitory mechanism of IL-10 remains unclear;
however, it has been shown that IL-10 evokes inactivation of NF-B
(20). Dex has been shown to repress activation of AP-1, NF-B,
NF-ATp, and Oct-1 through ligand-bound steroid receptors (21).
In the present study we demonstrate that LPS activates STAT5 through inducing GM-CSF production in human monocytes, and GM-CSF production affects the expression of COX-2. Furthermore, we demonstrate that IL-10 and Dex, but not aspirin, abrogate STAT5 activation. These results suggest that STAT5 is involved in LPS-induced activation of monocytes, and that IL-10 and Dex exert inhibitory effects on COX-2 expression through deactivation of STAT5.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS was purchased from Difco Laboratories (Detroit, MI). Dex, aspirin, and cyclohexamide were purchased from Sigma Chemical Co. (St. Louis, MO). Herbimycin A was purchased from Wako (Osaka, Japan). Anti-STAT1, -2, -3, and -6 Abs were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-STAT4 Ab was given by Dr. Koh Yamamoto (Tokyo Medical and Dental University, Tokyo, Japan). Anti-STAT5 Ab and the oligonucleotide probe, GRR (5'-GTATTTCCCAGAAAAGGAAC-3'), were provided by Dr. Hiroshi Wakao (Tokyo University, Tokyo, Japan). Anti-COX-2 Ab was purchased from Transduction Laboratories (Lexington, KY). Anti-phosphotyrosine mAb (4G10) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Recombinant human GM-CSF (hGM-CSF) was a gift from Japan Schering-Plough Corp. (Osaka, Japan). Anti-hGM-CSF Abs were either given by Dr. Toshio Kitamura (DNAX Research Institute, Palo Alto, CA) or purchased from Genzyme (Cambridge, MA). Recombinant human IL-10 (hIL-10) was a gift from Dr. Satish Menon (DNAX Research Institute).
Isolation and culture of human monocytes
The procedures of isolation and culture of human monocytes were previously described (18). Briefly, buffy coats from healthy donors were provided by the Numazu Red Cross Center (Numazu, Japan). Mononuclear cells were separated over Ficoll-Hypaque. Subsequently, these mononuclear cells were adhered to culture dishes for 2 h with a culture medium composed of RPMI 1640 with 10% FCS, then washed three times vigorously with warmed PBS to remove nonadherent cells. The remaining adherent cells consisted of >90% nonspecific esterase positive and >99% viable, as determined by trypan blue exclusion. These cells were further cultured with the culture medium for the experiments described below.
Extraction of nuclear proteins and electrophoretic mobility shift assay (EMSA)
The procedures of extraction of nuclear proteins and EMSA were previously described (22). Human monocytes were stimulated by 1 µg/ml LPS for the indicated times, by hGM-CSF at the indicated concentrations for 15 min, or by 10 ng/ml hIL-10 for 30 min and then lysed to extract nuclear proteins. In some experiments, monocytes were pretreated with 2 µg/ml herbimycin A for 2 h before the stimulation. In pretreatment experiments, 10 ng/ml hIL-10 for 30 min, 1.0 µM Dex for 2 h, 50 mM aspirin for 2 h, or 5 µg/ml cyclohexamide for 1 h was added before monocyte stimulation. Some experiments were performed in the presence of 10 µg/ml anti-hGM-CSF Ab. The amounts of loaded nuclear extracts were normalized before mixing.
Nuclear extract was mixed with binding buffer (20 mM HEPES-NaOH (pH 7.9), 2 mM EDTA, 100 mM NaCl, 10% glycerol, and 0.2% Nonidet P-40), poly(dI-dC), and 32P-labeled oligonucleotide probe. The mixtures were incubated at room temperature for 30 min. To detect supershifted bands, anti-STAT Abs or anti-phosphotyrosine Ab were added on ice for 30 min to the assay mixtures. The reaction mixtures were loaded on a 4% polyacrylamide gel and run with a running buffer of 0.25x TBE. The gel was then dried onto Whatman 3MM paper (Whatman, Clifton, NJ). The DNA/protein complexes were visualized by autoradiography.
The oligonucleotide probes used were: COX-2-sp3 (5'-TCTCTTTCCAAGAAACAAG-3'), COX-2-sp4 (5'-ATTTCTTCTGTTGAAAGCAA-3'), and GRR.
Immunoprecipitation and Western blotting
The procedures of immunoprecipitation and Western blotting of STAT5 were previously described (22). Human monocytes were incubated with 1.0 µM Dex for 2 h, then lysed in the lysis buffer containing 1% Triton X-100, and the proteins were immunoprecipitated from clarified cell lysates by adding 30 µl of protein A-Sepharose preconjugated with anti-STAT5 Ab (5 µl) and incubated for at least 2 h at 4°C. Sepharose beads were washed four times with the same lysis buffer. Sepharose-bound proteins were then eluted by boiling with SDS-PAGE sample buffer, applied to SDS-PAGE, and transferred electrophoretically to nitrocellulose filters. Proteins were probed with anti-STAT5 Ab and visualized by enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL).
The procedures of Western blotting of COX-2 were previously described (18). Monocytes (1 x 107 cells) were incubated for 24 h with 1 µg/ml LPS in the presence or the absence of 10 µg/ml anti-hGM-CSF Ab. After incubation, cells were lysed in the solubilization buffer (1% Tween-20, 10 mM PMSF, and 50 mM Tris-HCl, pH 8.0). Cell lysates were then sonicated for 30 s. The clarified cell lysates were subjected to SDS-PAGE and transferred electrophoretically to a polyvinylidene fluoride membrane. Proteins were probed by anti-COX-2 Ab and visualized by enhanced chemiluminescence.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The search for COX-2 genes has revealed that the promoter region
of the human COX-2 gene has two GAS motif sequences (TTCNNN(N)GAA;
COX-2-sp3 and COX-2-sp4), and that COX-2-sp3 was well conserved in both
rat and mouse COX-2 genes (23, 24, 25) (Fig. 1
). This prompted us to investigate
whether some STAT molecule is activated by LPS in human monocytes,
leading to binding to these sequences. To test this hypothesis, we
performed EMSA using two kinds of probes, COX-2-sp3 and COX-2-sp4.
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A, lanes
1–3). On the other hand, the COX-2-sp4 probe did not
elicit any band in our system (data not shown). The appearance of this
band was inhibited by adding an excess of cold COX-2-sp3, but not cold
COX-2-sp4 (Fig. 2A, lanes 4 and5), demonstrating the specificity of the binding of this
molecule to COX-2-sp3 probe. Pretreatment of monocytes with herbimycin
A abrogated the band, and adding monoclonal anti-phosphotyrosine Ab
to the mixture diminished the band, proving that this molecule was
truly tyrosine phosphorylated (Fig. 2, B andC). The nuclear extracts of two human monocytic cell lines,
THP-1 and U937, and mouse monocytic cell lines, Raw264.7 and J774.1,
did not raise this band, suggesting that the formation of this band was
observed only in human peripheral blood monocytes (data not shown).
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, A and B).
Nonspecific bands appeared in the presence of anti-STAT4 and
anti-STAT5 Ab, which were observed even in the absence of the
nuclear extracts (Fig. 3A, lanes 6–9).
Furthermore, the mobility of the band induced by LPS was the same as
that of hGM-CSF-induced STAT5 (Figs. 4
and 5). These results showed that the molecule(s) activated by LPS in
human monocytes contained STAT5.
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Although activation of STAT5 was not detected 15 min after LPS
stimulation, activation of STAT5 was observed 15 min after stimulation
with hGM-CSF (Fig. 2
A, lane 2, and Fig. 4B, lane 2). This delayed profile of STAT5
activation by LPS raised the possibility that LPS indirectly activates
STAT5 by inducing some other molecules. To address this question, we
assessed the effect of cyclohexamide on STAT5 activation by LPS.
Treatment of LPS-stimulated monocytes with cyclohexamide resulted in
complete abrogation of STAT5 activation, indicating that this
activation is executed by some newly synthesized molecule (Fig. 4A).
It is known that LPS promotes generation of various kinds of cytokines,
such as TNF-, IL-1ß, IL-6, IL-8, IL-10, IL-12, G-CSF, M-CSF, and
GM-CSF, from human monocytes (26, 27, 28). It has been shown that among
these cytokines, GM-CSF causes STAT5 activation in human monocytes (12, 29, 30). To investigate whether STAT5 is activated by LPS through
GM-CSF production, we examined the effect of neutralizing
anti-hGM-CSF Ab on LPS-induced STAT5 activation. When monocytes
were incubated with hGM-CSF for 15 min, an activated molecule appeared,
and this molecule was ascertained to be STAT5 by supershift assay (Fig. 4B, lanes 1–3). LPS stimulation caused
the appearance of the same mobility band as STAT5, and this activation
was attenuated by approximately 42% in the presence of anti-GM-CSF
Ab as judged by image analyzer, compared with the presence of isotype
control Ab (six donors were tested for the analysis; Fig. 4B, lanes 4–6). Two kinds of
anti-hGM-CSF Abs equally inhibited the activation (data not shown).
These results suggest that secreted GM-CSF contributes, at least in
part, to STAT5 activation in LPS-stimulated monocytes. Treatment of
GM-CSF-stimulated monocytes with cyclohexamide did not affect STAT5
activation, proving that STAT5 is activated directly by GM-CSF (Fig. 4C). Given that anti-hGM-CSF Ab did not
completely prevent LPS-induced STAT5 activation, we could not exclude
the possibility that some cytokine other than GM-CSF induced by LPS
would also lead to STAT5 activation. Treatments of neither Abs against
G-CSF or M-CSF, which have been shown to activate STAT5 in other cells,
influenced LPS-induced STAT5 activation (31, 32) (data not shown).
Next, to evaluate the amount of secreted GM-CSF triggered by LPS, we
compared the intensity of the STAT5 band induced by 1 µg/ml LPS with
that of bands induced by various concentrations of hGM-CSF. Stimulation
of monocytes with hGM-CSF at a concentration of 10 pg/ml up to 10 ng/ml
showed STAT5 activation in a dose-dependent manner (Fig. 4, lanes
3–6). The intensity of the LPS-induced band was less than
that of 10 pg/ml hGM-CSF, indicating that the level of secreted GM-CSF
induced by LPS is <10 pg/ml (Fig. 5).
This idea is further supported by our findings that tyrosine
phosphorylation of STAT5 could not be detected upon stimulation of
either 1 µg/ml LPS or 10 pg/ml hGM-CSF by Western blotting (data not
shown). In addition, GM-CSF in the supernatant of LPS-stimulated
monocytes was below the lowest level detectable by ELISA (8 pg/ml; data
not shown).
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The fact that STAT5 was activated by LPS through secretion
of GM-CSF prompted us to test whether STAT5 activation by GM-CSF
contributes to COX-2 expression. When monocytes were coincubated with
anti-hGM-CSF Ab, LPS-induced COX-2 expression was decreased by
approximately 60% compared with that after treatment with the isotype
control Ab, as analyzed by the National Institutes of Health Image 1.55
program (four donors were tested for the analysis; Fig. 6). These results indicate that GM-CSF
secretion contributes, at least in part, to LPS-induced COX-2
expression. Together with the results of STAT5 deactivation and COX-2
down-regulation by anti-hGM-CSF Ab, it is supposed that the
regulation mechanisms of both STAT5 activation and COX-2 expression are
parallel. However, stimulation of hGM-CSF alone did not cause any COX-2
expression (data not shown). Thus, these results raise the possibility
that GM-CSF-stimulated STAT5, in concert with other transcription
factors, such as NF-B, NF-IL-6, and AP-1, might regulate COX-2
expression in LPS-stimulated monocytes.
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It has been shown that IL-10 and Dex effectively abrogate
PGE2 production in LPS-stimulated monocytes by
suppressing the transcription of COX-2 gene (18, 19, 33, 34). To
investigate whether IL-10 and Dex influence LPS-induced STAT5
activation, we pretreated monocytes with either hIL-10 or Dex before
LPS stimulation. As a result, hIL-10 and Dex completely blocked STAT5
activation by LPS, whereas aspirin did not evoke any effect (Fig. 7A). In view of
previous findings that both IL-10 and Dex significantly inhibited
GM-CSF production in LPS-stimulated monocytes, it was assumed that
abrogation of LPS-induced STAT5 activation by hIL-10 and Dex is, at
least partly, ascribable to the suppression of GM-CSF production in
LPS-stimulated monocytes (27, 35).
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B). The expression levels of STAT5 and GM-CSF
receptor were invariable by pretreatment with Dex, as estimated by
Western blotting and flow cytometry, respectively (Fig. 7C
and data not shown). The expression level of STAT5 was constant during
treatment with hIL-10, as evaluated by Western blotting (data not
shown). These results indicate that IL-10 indirectly inhibits STAT5
activation by abrogating GM-CSF production, whereas Dex inhibits this
activation in both direct and indirect ways. Specificity of binding of IL-10-activated STAT1
The finding that pretreatment of hIL-10 did not cause the
appearance of any band using the COX-2-sp3 probe was unexpected,
because it had been previously reported that IL-10 activates both STAT1
and STAT3 using the GRR probe (12, 36). To explore the possibility that
this discrepancy was due to the difference in probes, we subjected both
the COX-2-sp3 probe and the GRR probe to EMSA to detect IL-10-activated
STAT molecules. The hIL-10-stimulated nuclear extract did not elicit
any band using the COX2-sp3 probe, whereas the same extract did elicit
a clear band using GRR (Fig. 8A, lanes
1–4). This band was strongly inhibited by adding an excess
of the GRR probe rather than by adding an excess of the COX-2-sp3 probe
(Fig. 8A, lanes 5–8). Anti-STAT1 Ab, but
not anti-STAT3 and STAT6 Abs, supershifted this IL-10-induced band
(Fig. 8B). To date, it is unclear why activation of
STAT3 was not detected in our system. These results implied that a GAS
motif-containing sequence preferentially binds to an activated STAT
molecule, with the consequence that IL-10-activated STAT1 has higher
affinity for the GRR sequence than for the COX-2-sp3 sequence.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Our
failure to detect either tyrosine phosphorylation of STAT5 by Western
blotting or GM-CSF protein by ELISA suggests that for short periods of
incubation (90 min), LPS-stimulated monocytes secrete very small
amounts of GM-CSF. In this regard, our EMSA system is much more
sensitive than the above two systems in detecting LPS-induced GM-CSF
production via STAT5 activation in monocytes.
Previous studies suggested that no known STAT molecule was involved in
LPS-induced monocyte activation (12, 13). Consistent with a previous
report (12), we did not detect any STAT activation 15 min after LPS
stimulation; however, during the longer period (90 min), STAT5
activation was clearly induced by secreted GM-CSF (Fig. 2). It has been
reported that LPS induces formation of the oligonucleotide complex
coding the LILRE of the human pro-IL-1ß gene with a STAT-like factor;
this factor is referred to as LIL-STAT (13). However, it is thought
that the LIL-STAT molecule is distinct from STAT5 because of the
following differing characteristics. 1) The band of LIL-STAT was not
supershifted by anti-STAT5 Ab. 2) LIL-STAT was activated 15 min
after LPS stimulation, whereas STAT5 was not. 3) LIL-STAT activation
was observed in a human monocytic cell line, THP-1, whereas STAT5
activation was not. 4) A G residue at position 8 of LILRE
(TTCCTGAGA) was critical for binding to LIL-STAT,
whereas COX-2-sp3 did not have a G residue at position 8. These results
may suggest that LPS activates more than two STAT molecules, either
directly or indirectly.
It has been shown that LPS induces the secretion of TNF-, IL-1ß,
IL-6, IL-8, IL-10, IL-12, G-CSF, and GM-CSF from monocytes, and that
IL-6, IL-10, IL-12, and G-CSF cause activation of STAT1, -3, and -4
among these cytokines (11, 26, 27). In addition, i.p. injection of LPS
leads to STAT3 activation in mouse hepatocytes, probably by IL-6
secretion (37). However, we could not detect any activation of these
STAT molecules in the present study (Fig. 3). This may have been due to
the low level of secretion of these cytokines or to inappropriate probe
selection. Further studies are needed to resolve this point.
STAT5 was initially purified from sheep mammary gland as a
prolactin-induced transcription factor regulating expression of the
ß-casein gene and was subsequently shown to be encoded by two
distinct genes, STAT5A and STAT5B (29, 38). To date, STAT5 activation
has been proved to be induced by IL-2, IL-3, IL-5, IL-7, IL-15, G-CSF,
M-CSF, GM-CSF, erythropoietin, thrombopoietin, growth hormone,
epidermal growth factor, platelet-derived growth factor, and engagement
of B cell receptor (29, 31, 32, 39, 40, 41, 42, 43). Our present study revealed
that in addition to these stimuli, LPS causes STAT5 activation
indirectly. The role of STAT5 as a proliferation signal is still
controversial; however, it has been verified that activation of STAT5
is essential for growth hormone-induced expression of hepatic serine
protease inhibitor 2.1; oncostatin M expression stimulated by IL-2,
IL-3, and erythropoietin; and IL-3-induced expression of cis,
osm, and pim-1 (29, 44, 45, 46, 47). In the present study, we
demonstrated that treatment of anti-hGM-CSF Ab partially reduced
LPS-induced COX-2 expression (Fig. 6). This indicates that GM-CSF
secretion contributes to COX-2 expression induced by LPS, and that it
may be due to STAT5 activation induced by secreted GM-CSF. It has been
shown that deletion of the GAS motif sequence of the human COX-2 gene
does not influence the expression level of COX-2 induced by LPS using a
transient transfection assay in bovine arterial endothelial cells (48).
However, it may be that GM-CSF is not secreted from endothelial cells
stimulated by LPS. These results suggest that STAT5 plays some role,
such as expression of COX-2 in activated human monocytes.
The role of LPS-induced GM-CSF production in monocyte activation has been well investigated to date, demonstrating that GM-CSF leads to enhancement of proliferation, Ag presentation, killing of parasites, oxidative metabolism, and antitumor immunity of monocytes (49). In these biologic activities of GM-CSF, it is not known which signal pathway is transduced through STAT5. Studies of STAT5 gene-targeting mice and analyses of the dominant negative phenotype of STAT5 would be helpful in clarifying this point. Furthermore, the studies in gene-disrupted mice of GM-CSF or IL-3/GM-CSF/IL-5 ßc receptor have exhibited abnormal pulmonary pathologic features consisting of lymphocytic infiltration and areas resembling alveolar proteinosis in steady state, indicating that GM-CSF is essential for normal pulmonary physiology and resistance to local infection (50, 51, 52). However, it remains to be examined what LPS-induced signal pathway is triggered by secreted GM-CSF. Further studies aimed at analyses of the impaired function in monocytes of these mice in response to LPS would be useful in this regard.
IL-10 and Dex were previously shown to preferentially inhibit
transcription of the COX-2 gene, resulting in remarkable suppression of
PGE2 production in LPS-stimulated monocytes and neutrophils
(18, 33, 34, 53). On the other hand, aspirin alleviates
PGE2 production by acetylating the serine residue, followed
by inactivation of both COX-1 and COX-2 (19). In the present study,
hIL-10 and Dex significantly inhibited STAT5 activation in
LPS-stimulated monocytes, while aspirin did not. At present, it is
unclear how IL-10 and Dex regulate transcription in the COX-2 gene. It
has been demonstrated that IL-10 selectively inhibits NF-B
activation in LPS-stimulated monocytes (20). On the other hand, Dex has
been shown to inhibit activation of AP-1, NF-B, NF-ATp, and Oct-1
through ligand-bound steroid receptors (21). In view of the finding
that the promoter region of the human COX-2 gene contains the binding
motifs of NF-B, NF-IL-6, and AP-1, it is of interest to determine
which of these transcription factors is a target for IL-10 and Dex. The
present study offers the possibility that in addition to these
transcription factors, STAT5 is another target for IL-10 and Dex to
inhibit COX-2 expression in either a direct or an indirect
way.
In this study we reported that IL-10-activated STAT1 has a higher
affinity for the GRR probe than for the COX-2-sp3 probe (Fig. 8).
Furthermore, we have confirmed that IFN--activated STAT1 and STAT5
can bind to the COX-2-sp3 probe (K. Yamaoka, unpublished observations).
Thus, each STAT molecule may have its preference to bind to the
specific nucleotide sequence containing the GAS motif. This idea is
supported by recent findings that the interposed or adjacent sequence
to the palindrome sequence plays a pivotal role in such a preference
(54, 55). Based on our findings, we speculate that different stimuli
cause different intramolecular modification of the same STAT molecule,
or association of different molecules with the same STAT molecule,
resulting in different abilities to form the DNA/protein complex.
Further analyses have to be conducted to resolve this point. The fact
that we could not detect the binding of IL-10-induced STAT1 to the
COX-2-sp3 probe might suggest that STAT5 deactivation is more important
than STAT1 activation for IL-10 to exert its anti-inflammatory
effect. This idea is further supported by a study of STAT1
gene-disrupted mice, in which the signal pathway of IFN is impaired but
the inhibitory effect of IL-10 on LPS-stimulated TNF production is not
affected (56). Taken together, these results may provide a clue to
elucidate the anti-inflammatory mechanism of IL-10.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kenji Izuhara, Department of Clinical Chemistry and Laboratory Medicine, Faculty of Medicine, Kyushu University, 3–1–1, Maidashi, Higashi-ku, Fukuoka, 812–82, Japan. E-mail address:
3 Abbreviations used in this paper: NF-B, nuclear factor-B; AP-1, activator protein-1; LIL, lipopolysaccharide-induced and interleukin-1-induced; LILRE, lipopolysaccharide- and IL-1-responsive element; Dex, dexamethasone; COX, cyclo-oxygenase; GM-CSF, granulocyte-macrophage colony-stimulating factor; h, human; EMSA, electrophoretic mobility shift assay.
Received for publication June 12, 1997. Accepted for publication September 30, 1997.
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 Y. Ohmori and T. A. Hamilton Requirement for STAT1 in LPS-induced gene expression in macrophages J. Leukoc. Biol., April 1, 2001; 69(4): 598 - 604. [Abstract] [Full Text]
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D. S. Whittaker, K. S. Bahjat, L. L. Moldawer, and M. J. Clare-Salzler Autoregulation of Human Monocyte-Derived Dendritic Cell Maturation and IL-12 Production by Cyclooxygenase- 2-Mediated Prostanoid Production J. Immunol., October 15, 2000; 165(8): 4298 - 4304. [Abstract] [Full Text] [PDF]
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 M. Mutoh, M. Takahashi, K. Fukuda, Y. Matsushima-Hibiya, H. Mutoh, T. Sugimura, and K. Wakabayashi Suppression of cyclooxygenase-2 promoter-dependent transcriptional activity in colon cancer cells by chemopreventive agents with a resorcin-type structure Carcinogenesis, May 1, 2000; 21(5): 959 - 963. [Abstract] [Full Text] [PDF]
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M. Barrios-Rodiles, G. Tiraloche, and K. Chadee Lipopolysaccharide Modulates Cyclooxygenase-2 Transcriptionally and Posttranscriptionally in Human Macrophages Independently from Endogenous IL-1{beta} and TNF-{alpha} J. Immunol., July 15, 1999; 163(2): 963 - 969. [Abstract] [Full Text] [PDF]
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J. L. E. Dean, M. Brook, A. R. Clark, and J. Saklatvala p38 Mitogen-activated Protein Kinase Regulates Cyclooxygenase-2 mRNA Stability and Transcription in Lipopolysaccharide-treated Human Monocytes J. Biol. Chem., January 1, 1999; 274(1): 264 - 269. [Abstract] [Full Text] [PDF]
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P. Delerive, P. Gervois, J.-C. Fruchart, and B. Staels Induction of Ikappa Balpha Expression as a Mechanism Contributing to the Anti-inflammatory Activities of Peroxisome Proliferator-activated Receptor-alpha Activators J. Biol. Chem., November 17, 2000; 275(47): 36703 - 36707. [Abstract] [Full Text] [PDF]
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