0022-3565/00/2932-0370$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 293:370–375, 2000 /2218/817859
Vol. 293, No. 2
Printed in U.S.A.
Selective Inhibitor of p38 Mitogen-Activated Protein Kinase
Inhibits Lipopolysaccharide-Induced Interleukin-8 Expression in
Human Pulmonary Vascular Endothelial Cells
SHU HASHIMOTO, YASUHIRO GON, KEN MATSUMOTO, SHUICHIRO MARUOKA, IKUKO TAKESHITA,
SHINICHI HAYASHI, YASUKIYO ASAI, YOSHITO ASAI, ITSURO JIBIKI, TATSUYA MACHINO, and TAKASHI HORIE
First Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan
Accepted for publication December 23, 1999
This paper is available online at http://www.jpet.org
Adult respiratory distress syndrome (ARDS), a form of
acute lung injury, which is characterized by permeability
edema, is observed in severe insults such as bacteremia sepsis (Nogare, 1989; Matthay, 1990; Bernard et al., 1994;
Hashimoto and Horie, 1994). Although understanding of the
basic mechanisms in the production of ARDS has increased,
little therapeutic progress has been made and mortality remains high. The pathogenesis of ARDS is complex and involves multiple inflammatory cells and mediators (Tate and
Repine, 1983; Nogare, 1989; Matthay, 1990; Hashimoto and
Horie, 1994). Although ARDS occurs in neutropenic patients
(Mauder et al., 1986; Ognibene et al., 1986), it has been
shown that neutrophils play an important role in the production of acute lung injury (Tate and Repine, 1983; Weiland et
al., 1986; Hogg, 1987; Nogare, 1989; Matthay, 1990; Hashimoto and Horie, 1994). The extravasation and accumulation
of neutrophils at the sites of injury depend on adhesion to and
migration through endothelial linings (Hashimoto and Horie,
1994). Interleukin (IL)-8 that displays a chemotactic activity
for neutrophils participates in this process through the reReceived for publication October 20, 1999.
the present study, we therefore attempted to clarify these issues. The results showed that LPS induced p38 MAP kinase
phosphorylation and activity, and SB 203580 as a selective
inhibitor of p38 MAP kinase activity inhibited p38 MAP kinase
activity and IL-8 expression in LPS-stimulated pulmonary vascular endothelial cells. These results indicate that p38 MAP
kinase regulates LPS-induced IL-8 expression in pulmonary
vascular endothelial cells. Although it is currently not known
whether SB 203580 is capable of producing beneficial effects
on ARDS, a strategy of inhibiting p38 MAP kinase activity by a
selective p38 MAP kinase inhibitor may apply to the therapy for
ARDS.
cruitment of neutrophils (Mantovani and Dejana, 1989; Huber et al., 1991; Baggiolini et al., 1994). IL-8 also activates
neutrophils to generate several toxic products such as reactive oxygen species, proteases, and arachidonic acid metabolites (Mantovani and Dejana, 1989; Baggiolini et al., 1994),
each of which may initiate endothelial cell damage leading to
an increase in endothelial permeability (Zimmerman et al.,
1983; Idell et al., 1985; Tagan et al., 1991). Several studies
have shown that IL-8 plays an important role in the production of ARDS (Miller et al., 1992; Matsumoto et al., 1997;
Mukaida et al., 1998). ARDS is often seen in bacteremia
sepsis and bacterial endotoxin, a lipopolysaccharide (LPS),
can stimulate vascular endothelial cells to produce IL-8
(Mantovani and Dejana, 1989; Strieter et al., 1989; Baggiolini et al., 1994). Therefore, it is important to clarify the
mechanism of IL-8 expression in LPS-stimulated pulmonary
vascular endothelial cells.
Recent studies, which explored the intracellular signal regulating cytokine expression, have shown that p38 mitogenactivated protein (MAP) kinase that belongs to MAP kinase
superfamily (Davis, 1994) is involved in cytokine expression
(Cuenda et al., 1995; Shapiro and Dinarello, 1995; Matsu-
ABBREVIATIONS: ARDS, adult respiratory distress syndrome; IL, interleukin; LPS, lipopolysaccharide; MAP, mitogen-activated protein; HPAEC,
human pulmonary artery endothelial cell; PAGE, polyacrylamide gel electrophoresis; ATF, activating transcription factor.
370
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ABSTRACT
Adult respiratory distress syndrome (ARDS) characterized by
permeability edema is observed in severe insults such as bacteremia sepsis. Interleukin (IL)-8, which chemoattracts and activates neutrophils, has been suggested to play an important
role in the production of ARDS. Therefore, the inhibition of IL-8
production is an important strategy for the treatment of ARDS.
Recent studies have revealed the role of p38 mitogen-activated
protein (MAP) kinase in cytokine expression and the inhibition
by a selective inhibitor of p38 MAP kinase activity of cytokine
expression in a variety of cell types. However, little is known
about the role of p38 MAP kinase in lipopolysaccharide (LPS)induced IL-8 expression in pulmonary vascular endothelial cells
and the effect of a selective p38 MAP kinase inhibitor on it. In
2000
p38 MAP Kinase Inhibitor and ARDS
moto et al., 1998; Hashimoto et al., 1999a,b). We have previously shown that p38 MAP kinase regulates tumor necrosis
factor-␣-induced IL-8 expression in human pulmonary vascular endothelial cells (Hashimoto et al., 1999a); however,
the role of p38 MAP kinase in LPS-induced IL-8 expression in
pulmonary vascular endothelial cells has not been determined. In the present study, therefore, we studied the role of
p38 MAP kinase in IL-8 expression in pulmonary vascular
endothelial cells and the production of ARDS more precise by
examining the role of p38 MAP kinase in LPS-stimulated
IL-8 expression in these cells. To this end, we attempted to
examine p38 MAP kinase phosphorylation and activation,
and the effect of SB 203580 as the specific inhibitor of p38
MAP kinase activity on p38 MAP kinase activity and IL-8
expression in LPS-stimulated human pulmonary vascular
endothelial cells.
Reagents. LPS was obtained from Sigma Chemical Co. (St. Louis,
MO). The pyridinyl imidazole SB 203580, a selective inhibitor of p38
MAP kinase activity (Lee et al., 1994), was kindly provided by
SmithKline Beecham (King of Prussia, PA) and was dissolved in
dimethyl sulfoxide.
Cell Culture. Normal human pulmonary arterial endothelial
cells (HPAECs) used as pulmonary vascular endothelial cells in this
study were obtained from Clonetics (San Diego, CA). The cells (1 ⫻
104 cells/ml) were placed onto a 24-well flat-bottom tissue culture
plate (Corning, Corning, NY) for determination of cytokine production. Cells were placed in a tissue culture dish (Falcon 1007; Falcon
Labware, Oxnard, CA) for Western blot analysis. For Northern blot
analysis (Falcon 1005), cells were placed with vascular endothelial
growth medium-2 (Clonetics) containing 0.2% fetal bovine serum,
gentamycin-amphotericin B, epidermal growth factor, insulin-like
growth factor, fibroblast growth factor, vascular endothelial growth
factor, ascorbic acid, heparin, and hydrocortisone. The cells were
grown until they were subconfluent and the medium was changed.
To examine the effect of LPS on threonine and tyrosine phosphorylation of p38 MAP kinase in HPAECs, the cells that had been
pretreated with endothelial growth medium-2 without fetal bovine
serum, epidermal growth factor, fibroblast growth factor, insulinlike growth factor, vascular endothelial growth factor, ascorbic acid,
and hydrocortisone (growth factor-free medium) for 16 h were stimulated with various concentrations of LPS and incubated for the
desired times. To examine the effect of SB 203580 on LPS-induced
p38 MAP kinase activity and IL-8 expression in HPAECs, the cells
that had been preincubated with growth factor-free medium with or
without SB 203580 for 1 h were stimulated with LPS and cultured
for the desired times at 37°C in humidified 5% CO2. The cells for
analysis of p38 MAP kinase activity were lysed at 10 min after
stimulation with LPS and the culture supernatants for determination of IL-8 protein were harvested at 24 h, centrifuged, and the
supernatants were collected, filtrated with a Millipore filter, and
stored at ⫺80°C until assay. The cells for analysis of IL-8 mRNA
expression were collected at 6 h and stored at ⫺80°C until analysis
of mRNA expression.
Measurement of IL-8. The concentrations of IL-8 in the culture
supernatants from HPAECs were measured by commercially available enzyme-linked immunosorbent assay kits (Amersham International, Aylesbury, UK). Enzyme-linked immunosorbent assay was
performed according to the manufacturer’s instructions. All samples
were assayed in duplicate.
Western Blot Analysis of p38 MAP Kinase Phosphorylation.
Threonine and tyrosine phosphorylation of p38 MAP kinase was
analyzed by commercially available kits (PhosphoPlus p38 MAP
kinase antibody kit; New England Biolabs, Inc., Beverly, MA). The
kit uses antiphospho-p38 MAP kinase that is specific for phosphorylated threonine and tyrosine kinase of p38 and does not cross-react
with phosphorylated threonine and tyrosine of extracellular signalregulated kinase-1 or -2 or c-Jun-NH2-terminal kinase. Analysis of
threonine and tyrosine phosphorylation of p38 MAP kinase was
performed according to the manufacturer’s instructions. Briefly, after separating proteins from the cell lysate by 15% SDS-polyacrylamide gel electrophoresis (PAGE), the cell lysate containing 10 ␮g of
protein was electophoretically transferred to nitrocellulose membrane and the membrane was blotted with a specific antibody to
phosphorylated threonine and tyrosine of p38 MAP kinase. To show
the amounts of p38 MAP kinase immunoblotted, blots were stripped
and reprobed with phosphorylation-state independent p38 MAP kinase-specific antibody to determine total p38 MAP kinase levels.
p38 MAP Kinase Assay. The activity of p38 MAP kinase was
analyzed by commercially available kits (p38 MAP kinase assay kit;
New England Biolabs Inc.). The kit uses two different antibodies,
anti-p38 MAP kinase antibody that is specific for p38 MAP kinase
and dose not cross-react with extracellular signal-regulated kinase-1
or -2 or c-Jun-NH2-terminal kinase, and antiphospho-specific activating transcription factor (ATF)-2 antibody to detect p38 MAP
kinase-induced phosphorylation of ATF-2. p38 MAP kinase activity
was analyzed according to the manufacturer’s instructions. Briefly,
the cell lysate containing 200 ␮g of protein was incubated with
anti-p38 MAP kinase antibody to selectively immunoprecipitate p38
MAP kinase from the cell lysates and the immunoprecipitates were
incubated with ATF-2 fusion protein in the presence of ATP, which
allowed immunoprecipitated active p38 MAP kinase to phosphorylate its substrate, ATF-2. The samples were separated by a 15%
SDS-PAGE, transferred to membranes, and blotted with antiphospho-specific ATF-2 antibody.
Northern Blot Analysis. Total RNA was prepared with an RNA
extraction kit (RNA zol B; Cinna, Friendswoods, TX) with the acid
guanidine thiocyanate-phenol-choloroform extraction methods. Total
RNA (10 ␮g) was denatured in a solution containing formaldehyde
and formamide, and electrophoresed in a 1% agarose gel containing
formaldehyde (Thomas, 1983). Then it was cappillary-transferred
onto a nylon membrane (Hybond N; Amersham International). The
membrane was prehybridized with rapid hybribuffer (Amersham
International) and then hybridized with 32P-labeled probes for 2 h at
65°C. The probe used in this study were the PstI-PstI fragments of
cDNA for ␤-actin and the full length of cDNA for IL-8, which was
kindly provided by Dr. Koji Matsushima (Tokyo University School of
Medicine, Department of Hygiene; Mukaida et al., 1989). After hybridization, the membrane was washed with standard saline citrate
containing SDS and then autoradiographed with Kodax XAR film at
⫺70°C.
Statistical Analysis. Statistical significance was analyzed with
ANOVA. P values ⬍.05 were considered significant.
Results
LPS Induces IL-8 Production by Pulmonary Vascular Endothelial Cells. First, we examined a dose-dependent effect of LPS on IL-8 production by HPAECs. To this
end, the culture supernatants from HPAECs stimulated with
various concentrations of LPS were harvested at 24 h after
cultivation (Fig. 1). The concentrations of IL-8 in the culture
supernatants from LPS-stimulated culture increased in a
dose-dependent manner.
LPS Induces p38 MAP Kinase Phosphorylation. To
determine whether LPS could induce the threonine and tyrosine phosphorylation of p38 MAP kinase, HPAECs were
stimulated for the desired times and p38 MAP kinase was
immunoblotted. Immunoblot study showed that stimulation
of the cells with LPS caused increases in the threonine and
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Materials and Methods
371
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Hashimoto et al.
tyrosine phosphorylation of p38 MAP kinase in a dose-dependent manner (Fig. 2a, top). To determine the time course of
tyrosine phosphorylation of p38 MAP kinase, HPAECs were
stimulated with 1000 ng/ml LPS for the desired times as
indicated. Amounts of LPS-induced threonine and tyrosine
phosphorylation of p38 MAP kinase increased at 5 min, sustained between 10 and 30 min, and returned to near basal
levels at 60 min (Fig. 2b, top). Figure 2, a and b (bottom),
showed that equal amounts of p38 MAP kinase protein were
immunoblotted with phosphorylation-independent p38 MAP
kinase-specific antibody regardless of dose of LPS and time of
culture periods, indicating that LPS stimulation-induced increases in the threonine and tyrosine phosphorylation of p38
MAP kinase occurred in the absence of changes in p38 MAP
kinase protein levels.
SB 203580 Inhibits LPS-Induced p38 MAP Kinase
Activity. Activation of p38 MAP kinase is mediated by dual
phosphorylation of the threonine residues and tyrosine residues of p38 MAP kinase (Raingeaud et al., 1995). In addition
to analysis of phosphorylation of p38 MAP kinase, we examined whether LPS could induce p38 MAP kinase activity. p38
MAP kinase activity was analyzed by a specific immunoprecipitation with anti-p38 MAP kinase antibody after an in
vitro kinase assay of its substrate ATF-2. As shown in Fig. 3,
LPS induced p38 MAP kinase activity as demonstrated by
the increased phosphorylation of ATF-2. SB 203580 inhibited
LPS-induced increases in p38 MAP kinase activity.
SB 203580 Inhibits LPS-Induced IL-8 Production.
LPS induced IL-8 production and SB 203580 inhibited LPSinduced p38 MAP kinase activity. These results suggested
that LPS stimulation-induced IL-8 production might be mediated through p38 MAP kinase-dependent pathway. To test
this possibility, HPAECs that had been preincubated with or
without SB 203580 were stimulated with LPS, and the concentrations of IL-8 in the culture supernatants were determined at 24 h after cultivation. The concentrations of IL-8 in
the culture supernatants from the cells cultured with LPS in
the presence of SB 203580 were lower than those from the
cells cultured with LPS in the absence of the SB 203580 (Fig.
4), indicating that SB 203580 inhibited LPS-induced IL-8
production.
SB 203580 Inhibits LPS-Induced IL-8 mRNA Expression. An inhibitory effect of SB 203580 on LPS-induced IL-8
protein production by HPAEC suggested that this action
might have resulted from an inhibitory effect of this inhibitor
on IL-8 gene expression. To test this possibility, HPAEC that
had been preincubated with or without SB 203580 were
stimulated with LPS and IL-8 mRNA expression was ana-
Fig. 2. LPS induces the threonine and tyrosine phosphorylation of p38 MAP kinase. HPAECs were stimulated with various concentrations of LPS for
5 min (a) and were stimulated with LPS (1000 ng/ml) for the desired times as indicated (b). The lysates from HPAECs were separated by 15%
SDS-PAGE, transferred to membranes, and blotted with a specific antibody to phosphorylated threonine and tyrosine of p38 MAP kinase (p38
MAPK-P; a and b, top). Blots shown in a and b (top) were stripped and reprobed with a phosphorylation-independent p38 MAP kinase-specific antibody
to show the amounts of p38 MAP kinase blotted (p38 MAPK; a and b, bottom). Lane P, positive protein prepared from C-6 glioma cells stimulated with
anisomycin for phosphorylated threonine and tyrosine of p38 MAP kinase; and lane N, negative protein prepared from C-6 glioma cells unstimulated
with anisomycin. The amounts of p38 MAP kinase phosphorylation were quantified by NIH Image analyzer and are presented as the amounts of p38
MAP kinase phosphorylation relative to control cells treated without agonist (1.0; bottom graphs). Three identical experiments independently
performed gave similar results.
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Fig. 1. LPS induces IL-8 production. HPAECs were cultured either with
medium or various concentrations of LPS, and the concentrations of IL-8
in the culture supernatants were determined at 24 h after cultivation.
The data are expressed as the mean ⫾ S.D. of five different experiments.
*P ⬍ .01 compared with IL-8 concentrations in HPAECs cultured with
medium.
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2000
p38 MAP Kinase Inhibitor and ARDS
Fig. 3. LPS induces p38 MAP kinase activity and SB 203580 inhibits p38
MAP kinase activity. HPAECs that had been preincubated with or without SB 203580 (10 ␮M) for 1 h were stimulated with LPS (1000 ng/ml) for
10 min. p38 MAP kinase activity was analyzed as described in Materials
and Methods. The cells were cultured with medium (lane 1), SB 203580
(lane 2), LPS (lane 3), and LPS ⫹ SB 203580 (lane 4). The concentrations
of dimethyl sulfoxide used in this study were 0.01%, which had no effect.
Three identical experiments independently performed gave similar results.
Discussion
In the present study, we examined a role of p38 MAP
kinase and the effect of SB 203580 as a selective inhibitor of
p38 MAP kinase activity on LPS-induced IL-8 expression in
HPAECs. The results showed that LPS induced IL-8 expression and the phosphorylation and activity of p38 MAP kinase
in HPAECs. SB 203580 inhibited LPS-induced increases in
p38 MAP kinase activity, IL-8 protein and mRNA expression
in HPAECs. These results indicate that p38 MAP kinase
plays an important role in LPS-activated signaling pathway
that regulates IL-8 expression in HPAECs, and that SB
203580 might inhibit LPS-induced IL-8 expression at the
level of transcription.
Fig. 5. SB 203580 inhibits LPS-induced IL-8 mRNA expression. HPAECs
that had been preincubated with or without SB 203580 (10 ␮M) for 1 h
were cultured either with medium or LPS (1000 ng/ml) for 6 h, and the
IL-8 mRNA and ␤-actin mRNA expression were analyzed by Northern
blot analysis. The cells were cultured with medium (lane 1), SB 203580
(lane 2), LPS (lane 3), and LPS ⫹ SB 203580 (lane 4). The concentrations
of dimethyl sulfoxide used in this study were 0.01%, which had no effect.
Three identical experiments independently performed gave similar results.
p38 MAP kinase is activated by various environmental
stresses such as hyperosmotic shock, heat shock, cold shock,
UV-irradiation, and inflammatory cytokines, and it plays an
important role in apoptosis and cytokine expression. (Davis,
1994, Cuenda et al., 1995; Raingeaud et al., 1995; Shapiro
and Dinarello, 1995; Matsumoto et al., 1998; Hashimoto et
al., 1999a,b). In addition, LPS stimulates human peripheral
blood monocytes (Manthey et al., 1998) and human vascular
umbilical vein endothelial cells (Schumann et al., 1996) to
activate p38 MAP kinase. In the present study, we showed
that LPS induced the phosphorylation and activity of p38
MAP kinase in HPAECs. Collectively, these results indicated
that p38 MAP kinase activation is a general event in LPSsignaling pathway in these cell types.
The specific inhibitor of p38 MAP kinase has been identified (Lee et al., 1994), providing effective tool for investigating the role of p38 MAP kinase in cellular signaling (Cuenda
et al., 1995; Shapiro and Dinarello, 1995; Matsumoto et al.,
1998; Hashimoto et al., 1999a,b). In the present study, SB
203580 was used as the specific inhibitor of p38 MAP kinase
activity to elucidate the biologic function of p38 MAP kinase
in LPS-induced IL-8 expression. SB 203580 inhibits p38
MAP kinase activity and IL-8 expression in LPS-stimulated
cells, whereas SB 203580 did not affect p38 MAP kinase
Fig. 4. SB 203580 inhibits LPS-induced IL-8 production. HPAECs that had been preincubated with medium or various concentrations of SB 203580 for 1 h
were cultured either with medium or LPS (1000 ng/ml),
and the concentrations of IL-8 in the culture supernatants were determined at 24 h after cultivation. The
concentrations of dimethyl sulfoxide used in this study
were 0.01%, which had no effect. The data are expressed as the mean ⫾ S.D. of five different experiments. *P ⬍ .01 compared with IL-8 concentrations in
HPAECs cultured without SB 203580.
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lyzed at 6 h after cultivation (Fig. 5). IL-8 mRNA expression
was up-regulated when HPAEC were stimulated with LPS
(lane 3), whereas SB 203580 inhibited LPS-induced up-regulation of IL-8 mRNA expression (lane 4; LPS ⫹ SB 203580).
The levels of ␤-actin mRNA expression did not vary significantly with culture conditions. The total number of cells and
cell viability at the end of culture period of each experiment
determined by trypan blue exclusion assay did not differ with
culture conditions. Figs. 1, 4, and 5 suggest that LPS-induced
IL-8 expression and the inhibition by SB 203580 of IL-8
expression did not result from cell cytotoxicity.
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Hashimoto et al.
controlling ARDS. Further investigations are needed to clarify this point.
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activity and IL-8 production in unstimulated cells, showing
that the lack of effect of SB 203580 on basal levels of p38
MAP kinase activity and IL-8 production. SB 203580 inhibits
the catalytic activity of p38 MAP kinase by binding to the
ATP site and subsequently phosphorylating its substrate
(Young et al., 1997). SB 203580 might exert an inhibitory
effect on the induced p38 MAP kinase activity and subsequent IL-8 production, but not on basal levels of p38 MAP
kinase activity and IL-8 production under our experimental
conditions. Alternatively, the lack of effect of this compound
on basal levels of p38 MAP kinase activity and IL-8 production might result from the sensitivity of SB 203580 to inhibit
p38 MAP kinase activity and subsequent IL-8 production.
Regardless, these results may indicate a favored effect of SB
203580 on controlling p38 MAP kinase-mediated IL-8 production in inflammatory diseases.
ARDS is frequently seen in conjunction with Gram-negative bacteremia sepsis (Nogare, 1989). Several studies have
suggested that IL-8 and neutrophils play an important role
in the production and progression of ARDS and animal models of acute lung injury (Weiland et al., 1986; Hogg, 1987;
Nogare, 1989; Matthay, 1990; Miller et al., 1992; Bernard et
al., 1994; Hashimoto and Horie, 1994; Matsumoto et al.,
1997; Mukaida et al., 1998). Although an anti-inflammatory
treatment aimed at inhibiting and ameliorating the production of ARDS and animal models of acute lung injury have
been studied, there is no established treatment to halt the
production of ARDS (Metz and Sibbald, 1991; Meduri, 1996;
Brett et al., 1998). The administration of a neutralizing antibody against IL-8 is effective in prevention of endotoxemiainduced ARDS-like lung injury (Mukaida et al., 1998). Consequently, the inhibition of IL-8 production and the
attenuation of neutrophil recruitment into the site of inflammation are important strategies for controlling acute lung
injury. IL-8 produced by vascular endothelial cells has been
suggested to contribute to the production of acute lung injury
through recruitment of neutrophils into the site of inflammation. A selective inhibitor of p38 MAP kinase activity has
been identified, providing effective tool for investigating the
role of p38 MAP kinase in cytokine expression (Davis, 1994;
Cuenda et al., 1995; Shapiro and Dinarello, 1995; Matsumoto
et al., 1998; Hashimoto et al., 1999a,b). It has been reported
that the administration of SB 203580 reduces mortality in
murine model of LPS-induced endotoxin shock (Badger et al.,
1996). In this study, we demonstrated that SB 203580 inhibited LPS-induced IL-8 expression in HPAECs. Thus, our
results may indicate a favorite effect of SB 203580 on ARDS
seen in Gram-negative bacteria sepsis. ARDS has high mortality and an effective treatment of ARDS has not been established (Metz and Sibbald, 1991; Meduri, 1996; Brett et al.,
1998). In the present study, we shed light on elucidating the
role of p38 MAP kinase in LPS-induced IL-8 expression in
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kinase regulates LPS-induced IL-8 expression and SB
203580 inhibited IL-8 expression in HPAECs. Although it is
currently not known whether SB 203580 is capable of producing beneficial effects on ARDS, a strategy of inhibiting
signal cascade, p38 MAP kinase, may apply to the therapy
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2000
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Send reprint requests to: Dr. Shu Hashimoto, First Department of Internal
Medicine, Nihon University School of Medicine, 30-1 Oyaguchikamimachi,
Itabashi-Ku Tokyo 173-8610, Japan. E-mail: [email protected]
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