Departments of 1 Internal Medicine, 3 Biomedical Research, and 4 Oriental Medicine, Kitasato Institute Hospital, Tokyo 108-8642; and 2 Department of Medicine, School of Medicine, Keio University, Tokyo 160-8582, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intercellular
adhesion molecule-1 (ICAM-1) of the vascular endothelium plays a key
role in the development of pulmonary oxygen toxicity. We studied the
effect of steroid on hyperoxia-induced ICAM-1 expression using cultured
endothelial cells in vitro. Human pulmonary artery endothelial cells
(HPAECs) were cultured to confluence, and then the monolayers were
exposed to either control (21% O2-5% CO2) or
hyperoxic (90% O2-5% CO2) conditions with and
without a synthetic glucocorticoid, methylprednisolone (MP). MP reduced hyperoxia-induced ICAM-1 and ICAM-1 mRNA expression in a dose-dependent manner. Neutrophil adhesion to hyperoxia-exposed endothelial cells was
also inhibited by MP treatment. In addition, MP attenuated hyperoxia-induced H2O2 production in HPAECs as
assessed by flow cytometry. An electrophoretic mobility shift assay
demonstrated that hyperoxia activated nuclear factor-B (NF-
B) but
not activator protein-1 (AP-1) and that MP attenuated hyperoxia-induced
NF-
B activation dose dependently. With Western immunoblot analysis, I
B-
expression was decreased by hyperoxia and increased by MP treatment. These results suggest that MP downregulates
hyperoxia-induced ICAM-1 expression by inhibiting NF-
B activation
via increased I
B-
expression.
intercellular adhesion molecule-1; nuclear factor-B; inhibitory
protein I
B-
; glucocorticoid
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PULMONARY OXYGEN TOXICITY is an important clinical problem that occurs in patients on long-term mechanical ventilation requiring a high inspired oxygen concentration. The lungs of these patients are chronically exposed to a hyperoxic environment. The most common histopathological evidence of pulmonary oxygen toxicity is pulmonary edema, with neutrophil infiltration into the lung parenchyma (13). The mechanism by which hyperoxia causes lung injury has been shown to be through the increased formation of reactive oxygen species (ROS) (14). ROS then produce cellular damage directly through lipid peroxidation and indirectly by increasing the expression of proinflammatory cytokines, adhesion molecules, and other inflammatory mediators. The endothelial cell inflammatory response is believed to play a central role in the pathogenesis of oxidant lung injury and is also considered pivotal in mediating neutrophil migration into the lung parenchyma.
A variety of adhesion molecules on the surfaces of endothelial cells
and neutrophils mediate adherence to the vascular endothelium, which
must occur for neutrophils to migrate into an inflammatory site. Suzuki
et al. (29) previously reported that the exposure of human
pulmonary artery endothelial cells (HPAECs) and human umbilical vein
endothelial cells (HUVECs) to hyperoxia selectively induces
intercellular adhesion molecule-1 (ICAM-1) and ICAM-1 mRNA expression.
ICAM-1 induction, which requires de novo protein synthesis, and the
interaction with its receptors on neutrophils play important roles in
regulating the retention, migration, and activation of neutrophils in
the lungs. It has recently been shown (17) that transcription factors
such as nuclear factor-B (NF-
B) and activator protein-1 (AP-1)
participate in regulating the gene expression of many modulators of
inflammatory and immune responses, including ICAM-1. In NF-
B
activation, a ROS such as H2O2 is considered to
serve as one of the key messengers (26).
Pulmonary oxygen toxicity is different from other inflammatory lung diseases such as endotoxin-induced lung injury because the stimuli mediated by oxygen are relatively subacute and moderate compared with those mediated by endotoxin or inflammatory cytokines. Therefore, the interaction between the endothelium and neutrophils in the development of pulmonary oxygen toxicity is not fully understood, and currently, no commonly accepted treatment exists for patients suffering from hyperoxia. Although the effectiveness of steroids in oxidant lung injury remains controversial, several kinds of steroids are used clinically in the treatment of a variety of inflammatory diseases. In particular, a synthetic glucocorticoid, methylprednisolone (MP), has been clinically and widely used as steroid pulse therapy for immunoreactive diseases including rheumatoid arthritis (30), acute allograft rejection after kidney transplantation (21), bronchial asthma (8), idiopathic pulmonary fibrosis (10), and bronchiolitis obliterans after lung transplantation (28). In experimental studies, MP inhibited the endotoxin-induced lung vascular permeability in sheep (5), ROS-induced increase in microvascular permeability of isolated rat lungs (15), and zymosan-induced pulmonary damage in rabbits (6). The molecular mechanism of MP in the treatment of oxidant lung injury such as pulmonary oxygen toxicity has not been fully characterized.
This prompted us to study the mechanisms by which MP exerts its
anti-inflammatory activity against pulmonary oxygen toxicity in terms
of adhesion molecules with the use of cultured endothelial cells. The
purpose of this study was to investigate the effect of MP on ICAM-1
expression, ICAM-1 mRNA expression, neutrophil adhesion to endothelial
cells, and the DNA binding activity of NF-B and AP-1 in
hyperoxia-exposed endothelial cells.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Endothelial cell culture. HPAECs (Kurabo, Osaka, Japan) were cultured in an endothelial cell growth medium supplemented with 10% fetal calf serum, penicillin G (100 U/ml), and streptomycin (100 µg/ml; GIBCO BRL, Life Technologies, Grand Island, NY) at 37°C and 5% CO2 in a humidified incubator. The cells used for experiments were from passages 8 to 12. The endothelial cells were grown on 25-cm2 tissue culture flasks (Corning, New York, NY) and then subcultured with 0.025% trypsin-0.05 mM EDTA (GIBCO BRL). The endothelial cells were identified by their characteristic cobblestone monolayer appearance, typical ultrastructure, and the presence of von Willebrand factor antigen as confirmed by indirect immunofluorescence staining (DAKO Japan, Tokyo, Japan). Cell viability always exceeded 95% as determined by the trypan blue exclusion test.
Exposure of endothelial cell monolayers to hyperoxia.
Endothelial cell monolayers cultured to confluence were exposed to
control (21% O2-5% CO2, 1 atm) or hyperoxic
(90% O2-5% CO2, 1 atm) conditions for various
periods at 37°C in a humidified multigas incubator (APM-36, ASTEC,
Fukuoka, Japan). In some experiments, endothelial cells were exposed to
hyperoxia plus various concentrations of MP
(11,17,21-trihydroxy-6
-methyl-1,4-pregnadiene-3,20-dione-21-sodium succinate, C26H33NaO8, mol wt 497;
Upjohn, Kalamazoo, MI). MP dissolved in sterile normal saline was added
to the culture medium just before hyperoxic exposure. The oxygen
concentration was monitored continuously and maintained at 90 ± 0.5%
during the entire hyperoxic exposure.
PO2 and
PCO2 in the medium were measured in
preliminary experiments with a blood gas analyzer (model 178, Corning).
After a 15-min exposure to hyperoxia,
PO2 in the medium was consistently
>580 Torr. The culture medium had the same pH value under control and
hyperoxic conditions. Viability of the endothelial cells under
hyperoxic conditions exceeded 85% within 72 h as measured by the
trypan blue exclusion test. Under control conditions, endothelial cell
viabilities were >95% up to 48 h without MP and 91 ± 6% at 24 h
and 68 ± 5% at 48 h with 10 mM MP. Under hyperoxic conditions, the
viabilities were 96 ± 8% at 24 h and 92 ± 7% at 48 h
without MP and 89 ± 9% at 24 h and 62 ± 7% at 48 h with 10 mM MP.
Under phase-contrast microscopy, endothelial monolayers appeared
morphologically normal at 72 h of exposure to hyperoxia.
Flow cytometric analysis. The level of ICAM-1 expression in the endothelial cells was measured by flow cytometry. Endothelial cells with and without MP were detached by treatment with 0.1% EDTA (Sigma, St. Louis, MO) for 1 min at 37°C and washed with Dulbecco's phosphate-buffered saline (DPBS). The suspended cells were incubated with phycoerythrin-conjugated anti-human ICAM-1 monoclonal antibody (LB-2, Becton Dickinson, San Jose, CA) for 30 min at 4°C. The cells were washed three times with DPBS, fixed with 1% paraformaldehyde (Sigma), and then analyzed. The intensity of fluorescence and the light-scattering properties of the cells were determined with a FACScan flow cytometry system equipped with an argon laser (488-nm emission, 15-mW output; Becton Dickinson). Phycoerythrin red fluorescence was collected between 564 and 606 nm with a band-pass filter. All analyses were run simultaneously with a mouse isotype (IgG2) control antibody (Becton Dickinson), and the values obtained were subtracted. In each sample, 10,000 endothelial cells were examined. The list mode was evaluated with the Lysis II program (Becton Dickinson). All experiments were done in quadruplicate, and the results are expressed as the percent intensity of fluorescence compared with that at time 0.
ICAM-1 mRNA expression with RT-PCR. The effect of MP on hyperoxia-induced ICAM-1 mRNA expression was analyzed with RT-PCR. Total RNA was extracted from the endothelial cells. First-strand cDNA was synthesized from 10 µg of RNA by SuperScript RT (GIBCO BRL, Life Technologies, Gaithersburg, MD) following the manufacturer's instructions. We amplified synthesized first-strand cDNA with PCR (Perkin-Elmer Cetus, Norwalk, CT) with 50 pmol of the 5'and 3' primers with 2.5 U of Taq polymerase (Takara Biomedicals, Kyoto, Japan) in a total volume of 50 µl. The reaction buffer consisted of 10 mM Tris · HCl, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, and 10 mM deoxynucleotide triphosphates. PCR cycles were allowed to run for 30 s at 94°C, followed by 30 s at 55°C and 1 min at 72°C, and a final extension at 72°C for 10 min. The 5' and 3' primers were 5'-TGACCATCTACAGCTTTCCGCC-3' and 5'-GTCTGAGGTTACACGGTCCGA-3', respectively (24). Human glyceraldehyde-3-phosphate dehydrogenase primers (Clontech Laboratories, Palo Alto, CA) were used as an internal control. The 5' and 3' primers were 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and 5'-CATGTGGGCCATGAGGTCCACCAC-3', respectively. A 10-µl aliquot of the amplified DNA reaction mixture was fractionated by 2.0% agarose gel electrophoresis, and the amplified product was then visualized by ultraviolet fluorescence after being stained with ethidium bromide. To quantify the levels of mRNA, a standard curve was obtained by titration of RNA from hyperoxia-exposed endothelial cells. The difference in mRNA expression among test samples was determined from the standard curve obtained by running the same number of cycles as unknown samples. The specificity of the amplified products was validated by their predicted size on agarose gel.
Neutrophil adhesion assay. To determine the effects of MP on neutrophil adherence to hyperoxia-exposed endothelial cells, we conducted adhesion assays. Human peripheral blood neutrophils were obtained from healthy adult volunteers and separated on a discontinuous gradient consisting of Histopaque 1077 and 1119 (Sigma). Neutrophils were resuspended in DPBS at a final concentration of 5 × 105 cells/ml. Neutrophil purity exceeded 98% as confirmed by a modified Wright's stain (Diff-Quik Stain Set, American Scientific Products, McGaw Park, IL). Cell viability by trypan blue exclusion exceeded 98%. One hundred microliters of isolated neutrophils (5 × 105 cells/ml) were applied to HPAEC monolayers in six-well tissue culture plates (Corning) that had previously been exposed to either control (21% O2-5% CO2) or hyperoxic (90% O2-5% CO2) conditions in a humidified multigas incubator (APM-36, ASTEC) for 48 h. In some experiments, endothelial monolayers were exposed to hyperoxia with anti-ICAM-1 monoclonal antibody (KM972, Kyowa Medex, Tokyo, Japan) that was added to the culture medium just before the hyperoxic exposure. The plates were then incubated for 60 min at 37°C under 5% CO2 in a humidified incubator. Nonadherent neutrophils were removed by gently washing the plates three times with prewarmed DPBS. Ten randomly selected fields were read at ×200 magnification under a light microscope. Neutrophil adhesion was evaluated by counting the number of neutrophils adhering to the endothelial cell monolayer.
Intracellular H2O2 production in
endothelial cells. A ROS such as H2O2 is
considered to serve as one of the key messengers in NF-B activation
(26). Therefore, to evaluate the effects of MP on intracellular
H2O2 production in HPAECs, we used
2',7'-dichlorofluorescin diacetate (DCFH-DA; Molecular
Probes, Eugene, OR). DCFH-DA is freely permeable across cell membranes
and is oxidized by intracellular H2O2, then
converted to green fluorescent 2',7'-dichlorofluorescein (DCF) (29). Endothelial cell monolayers cultured to confluence were
incubated with 10 µM DCFH-DA for 60 min at 37°C, washed, and then
exposed to either control (21% O2-5% CO2) or
hyperoxic (90% O2-5% CO2) conditions for 4 h
at 37°C in a humidified multigas incubator (APM-36). In some
experiments, endothelial cells were exposed to hyperoxia plus various
concentrations of MP. The endothelial cells were washed with DPBS twice
and collected with rubber scraper. Intracellular
H2O2 production in HPAECs was assessed by
measuring DCF fluorescence with flow cytometry. In each sample, 10,000 cells were examined. Fluorescence was collected at 530 nm by employing a band-pass filter, and the list mode was analyzed with the Lysis II
program system. All experiments were performed in quadruplicate, and
the results are expressed as the percent intensity of fluorescence compared with the control value.
Nuclear protein extracts and electrophoretic mobility shift
assay. Endothelial cell monolayers were exposed to either control or hyperoxic conditions and treated with and without MP. The cells were
washed with ice-cold DPBS, scraped with a cell scraper, and then
collected and resuspended in 2 ml of cold DPBS. The cells were pelleted
for 10 s and resuspended in 400 µl of cold buffer A [10
mM HEPES-KOH, pH 7.9, 10 mM NaCl, 1.5 mM MgCl2,
0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride
(PMSF)] by flicking the tube. The cells were allowed to swell on
ice for 10 min and were then vortexed for 10 s. The samples were
centrifuged for 10 s, and the pellet was resuspended in 20 µl of cold
buffer C (20 mM HEPES-KOH, pH 7.9, 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF, and
25% glycerol) and incubated on ice for 20 min. Cellular debris was
removed by centrifugation for 5 min at 15,000 rpm and 4°C, and the
supernatant fraction was stored at 80°C. The protein
concentration was determined with a bicinchoninic acid protein assay
(Pierce, Rockford, IL) with BSA (Sigma) as a standard. The yield was
10-20 µg protein/106 cells. Double-stranded NF-
B
and AP-1 consensus oligonucleotide probes were
5'-AGTT
AGGC-3'
(19) and
5'-CG- CTT
GCCGGAA-3', respectively (18); the binding sites are underlined. The
oligonucleotides were annealed with the complementary strand to produce
5'-overhanging ends (BamH I), which enabled labeling by
Klenow polymerase (Amersham, Tokyo, Japan) in the presence of
deoxynucleotide triphosphates and [32P]dCTP
(3,000 Ci/mmol; Amersham). The labeled oligonucleotide was purified on
push columns (Stratagene, La Jolla, CA). The typical binding reaction
of 20 µl contained 10,000 counts/min of 32P-labeled
double-stranded oligonucleotide, 10 µg of nuclear extraction in
buffer C, 20 µg of BSA, and 2 µg of poly(dI-dC) (Sigma) in gel shift binding buffer. Specific binding was confirmed by competition with a 100-fold excess of unlabeled double-stranded oligonucleotide competitor. For the supershift assay, the antibody against p65 (Santa
Cruz Biotechnology, Santa Cruz, CA) was added to the nuclear extracts
for 10 min before the addition of radiolabeled probe. After incubation
for 20 min at room temperature, samples were analyzed on 4% native
acrylamide gels run at 170 V for 1.5 h in 0.5× Tris-borate-EDTA
buffer. The gels were then dried and visualized by autoradiography.
Western immunoblot analysis. For immunoblot analysis, 100-mm
dishes of confluent cells with and without MP were exposed to either
control conditions or hyperoxia for various periods, and then the cells
were washed with DPBS and lysed on ice in modified radioimmunoprecipitation assay buffer containing PMSF solution and
aprotinin. In some experiments, 1 µg/ml of lipopolysaccharide (LPS;
955:B5; Sigma) was added as a positive control. The lysate was
centrifuged at 15,000 rpm for 20 min, and the protein concentration was
determined with a bicinchoninic acid protein assay. Samples with equal
amounts of protein were mixed with SDS sample buffer consisting of 20%
glycerol, 4% SDS, 0.16 M Tris (pH 6.8), 4% -mercaptoethanol, and
0.5% bromphenol blue and then boiled for 90 s. Ten micrograms of each
sample were fractionated on a 10% SDS-polyacrylamide gel, transferred
onto an immobilon-polyvinylidene difluoride membrane (Immobilon-P,
Millipore, Bedford, MA), and incubated with an antibody against
I
B-
(C-21; Santa Cruz). The primary antibody was
counterstained with horseradish peroxidase-conjugated rabbit IgG
antibody, visualized with an enhanced chemiluminescence detection kit
(Pierce) according to the manufacturer's instructions, and then
exposed to photographic films. Hybridization signals on films were
quantified by scanning densitometry (NIH Image Program, National
Institutes of Health, Bethesda, MD). All experiments were done in
quadruplicate, and the results are expressed as the percent density
compared with the control value.
Statistical analysis. All data are presented as means ± SD. Two-way analysis of variance and Fisher's paired least significant difference test were used to detect differences among groups (StatView II, Abacus Concepts, Berkeley, CA). A P value of <0.05 indicated significant differences between the means.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The effect of MP on hyperoxia-induced ICAM-1 expression was studied
with flow cytometry (Fig. 1). ICAM-1
expression increased in hyperoxia-exposed HPAECs (167.4 ± 25.5%)
compared with the control value (100.0 ± 15.2%; P < 0.01).
Treatment with MP (1 and 10 mM) attenuated hyperoxia-induced ICAM-1
expression in HPAECs (124.7 ± 8.8 and 56.5 ± 11.3%, respectively;
P < 0.01).
|
Hyperoxic exposure for 48 h upregulated ICAM-1 mRNA expression in
HPAECs as previously reported (29). We therefore examined the effect of
MP on hyperoxia-induced ICAM-1 mRNA expression (Fig. 2). Hyperoxia-induced ICAM-1 mRNA was
attenuated by MP dose dependently.
|
We studied whether the treatment of endothelial cells with MP inhibited
neutrophil adhesion to endothelial cells that had been previously
exposed to hyperoxia for 48 h (Fig.
3). Neutrophil adhesion
increased in hyperoxia-exposed HPAECs (17.1 ± 6.0 cells/mm2) compared with the control value (6.2 ± 2.3 cells/mm2; P < 0.01) and was attenuated
by treatment with anti-ICAM-1 monoclonal antibody (11.3 ± 2.5 cells/mm2). Treatment with 0.1 and 1 mM MP reduced the
number of neutrophils adhering to hyperoxia-exposed HPAECs (6.9 ± 2.5 and 3.8 ± 2.1 cells/mm2, respectively; P < 0.01).
|
The effect of MP on hyperoxia-induced H2O2
production in HPAECs as assessed by using DCFH-DA is shown in Fig.
4. The DCF fluorescence was increased by
hyperoxic exposure for 4 h (167 ± 19%) compared with the control
value. MP reduced hyperoxia-induced DCF fluorescence dose dependently.
One micromolar MP attenuated hyperoxia-induced H2O2 production (91 ± 16%; P < 0.05) compared with that with hyperoxia without MP.
|
Transcription factor NF-B and AP-1 binding sites were identified in
the promoter of the ICAM-1 gene, which participates in regulating
ICAM-1 expression (17). We therefore investigated the effect of
hyperoxia on NF-
B and AP-1 DNA binding activities by electrophoretic
mobility shift assay (Fig. 5). NF-
B was
activated by hyperoxic exposure for 2 h and returned to baseline level
for 4 h. On the other hand, AP-1 was not activated by hyperoxia during the experiment.
|
Because it has been shown that hyperoxia selectively activated NF-B,
we studied the effect of MP on hyperoxia-induced NF-
B DNA binding
activity (Fig. 6). Endothelial cells were
exposed to hyperoxia for 2 h with and without MP and subjected to
electrophoretic mobility shift assay. Hyperoxia-induced NF-
B
activation was reduced by MP dose dependently.
|
IB-
is an inhibitory protein that prevents nuclear transport and
activation of the transcription factor NF-
B. At first, the time
course of I
B-
expression under hyperoxic conditions was studied
with Western immunoblot analysis and then compared with LPS treatment
as a positive control (Fig. 7). The
expression of I
B-
under hyperoxic conditions was unchanged up to
2 h but was attenuated at 4 h and then returned to the baseline level at 6 h. In contrast, I
B-
expression was decreased by treatment with LPS at 1-2 h and then partially returned to the baseline level at 4-6 h.
|
We further examined the effect of MP on hyperoxia-induced IB-
expression in endothelial cells (Fig. 8).
Endothelial cell monolayers were exposed to either control or hyperoxic
conditions for 4 h with and without MP. The level of I
B-
expression was decreased by hyperoxia compared with that under control
conditions and was increased by MP treatment. On the other hand,
I
B-
expression was strongly inhibited by LPS and recovered by MP
treatment.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, we showed that the synthetic glucocorticoid MP
attenuated hyperoxia-induced ICAM-1 and ICAM-1 mRNA expression and
reduced neutrophil adhesion to endothelial cells (Figs. 1-3). Hyperoxia-increased neutrophil adhesion was inhibited by treatment with
an anti-ICAM-1 monoclonal antibody, suggesting that, at least partially, neutrophil adhesion was mediated by ICAM-1. In addition, we
have shown that MP attenuated hyperoxia-induced intracellular H2O2 production in HPAECs by applying DCFH-DA
(Fig. 4). Suzuki et al. (29) previously showed that the exposure of
cultured HPAECs and HUVECs to hyperoxia for 48-72 h increased
ICAM-1 and ICAM-1 mRNA expression followed by increased neutrophil
adhesion. An antioxidant, N-acetylcysteine, inhibits
hyperoxia-induced ICAM-1 expression in endothelial cells by increasing
extracellular glutathione concentration in vitro (1). Taken
together, these results suggest that increased ROS production,
including H2O2, by hyperoxia enhances ICAM-1
expression and thus stabilizes and prolongs neutrophil adhesion.
Decreases in ICAM-1, ICAM-1 mRNA, and neutrophil adhesion to
hyperoxia-exposed endothelial cells by MP suggested that this may be a
mechanism by which MP ameliorates oxidant lung injury including
pulmonary oxygen toxicity (5, 6, 15). MP might thus be useful in the
treatment for oxidant lung injury such as pulmonary oxygen toxicity at
some clinical condition. Koizumi et al. (16) have shown that
dexamethasone improved survival and decreased lung damage if given when
exposure to hyperoxia was to be soon terminated; however, dexamethasone
worsened lung damage and diminished survival if given early during
exposure to hyperoxia. On the other hand, Halpern et al. (11) reported that rats receiving MP (10-60
mg · kg1 · day
1)
survived for less time than control rats.
NF-B was originally identified as a heterodimeric complex consisting
of 55-kDa (p55) and 65-kDa (p65) subunits. NF-
B is present in
cytoplasm complexed with its inhibiting protein I
B, which prevents
NF-
B from being translocated to the nucleus and binding to DNA. Many
inducers of NF-
B, including tumor necrosis factor-
, LPS, and
interleukin-1, phosphorylate I
B-
at Ser32 and
Ser36 contained within the NH2 terminus of the
protein. Phosphorylated I
B-
is then ubiquitinated on
Lys21 and Lys22 and subsequently degraded by
the 26S proteasome. The degradation of I
B unmasks the nuclear
localization sequences in the remaining NF-
B dimer, which, in turn,
translocates to the nucleus, binds to the specific promoter sites, and
activates gene transcription. We demonstrated that hyperoxia increased
the NF-
B DNA binding activity in endothelial cells (Fig. 5),
although we did not identify NF-
B as the molecule responsible for
regulating ICAM-1 expression in the present study. Increased ROS
production such as H2O2 by hyperoxia is
believed to serve as the messenger for NF-
B activation. Schreck et
al. (26) showed, using H2O2-treated Jurkat T
cells, that NF-
B was rapidly activated within 30-60 min in
vitro. Shea et al. (27) reported in mice that it took 24 h for NF-
B
activation in pulmonary lymphocytes with hyperoxic (100%
O2) exposure in vivo. The difference in onset of NF-
B
activation between cell culture and the animal model may be related to
the time required to overwhelm the endogenous antioxidants such as
superoxide dismutase and glutathione present in the lungs.
AP-1, a heterodimer of protooncogene proteins c-Fos and c-Jun, is
induced by many types of stimuli, including
12-O-tetradecanoylphorbol 13-acetate (TPA), growth factors,
cytokines, and ultraviolet irradiation (9). AP-1 DNA binding activity
in endothelial cells was not affected by hyperoxic exposure (Fig. 5).
Our result suggested that hyperoxia induces ICAM-1 expression through
activating NF-B but not AP-1. The types of transcription factors
involved in target gene expression appear to depend on the experimental
conditions such as cell type and type of stimuli. For example, Roebuck
et al. (23) reported that H2O2 activates ICAM-1
expression in HUVECs through AP-1. Bradley et al. (4) observed that
H2O2 increases ICAM-1 expression in HUVECs
without activating NF-
B. Meyer et al. (20) reported that AP-1
responded weakly in H2O2-treated HeLa cells,
whereas NF-
B was strongly activated.
Under the control conditions, we detected IB-
, an inhibitory
protein that prevents nuclear transport and activation of transcription factor NF-
B (Figs. 7 and 8). Pulmonary endothelial cells in this in
vitro system thus express I
B-
and then regulate NF-
B
activation. Treating cells with NF-
B inducers rapidly disperses
I
B-
, which reappears at later time points. Rapid I
B-
degradation was responsible for initially releasing NF-
B to the
nucleus, whereas subsequent I
B-
resynthesis terminated NF-
B
transcription in induced cells. In our study, the expression of
I
B-
was suppressed by treatment with LPS at 1-2 h (Fig. 7).
In contrast with LPS, hyperoxia moderately attenuated I
B-
expression at 4 h. The difference in I
B-
degradation between
hyperoxia and LPS may depend on the intensity of stimuli or the signal
transduction pathway.
Glucocorticoids exert their potent anti-inflammatory action by binding
to the intracellular glucocorticoid receptor (GR), which belongs to the
steroid/thyroid hormone receptor superfamily. After ligand binding, GR
is translocated to the nucleus and is thus able to activate the
transcription of several genes by binding to a specific DNA sequence,
termed glucocorticoid response element (3). Glucocorticoids are
believed to inhibit NF-B activation in at least two ways. First, GRs
interact directly with p65 to inhibit DNA binding via a protein-protein
interaction (7, 22). Second, glucocorticoids increase I
B-
production, which, in turn, prevents NF-
B translocation to the
nucleus. We showed in this study that MP attenuated hyperoxia-induced
NF-
B activation and increased I
B-
expression (Fig. 8). Our
results suggested that MP attenuated ICAM-1 expression by inhibiting
NF-
B activation through I
B-
synthesis. Auphan et al. (2)
reported that Jurkat T cells treated with TPA partially dispersed
I
B-
and that dexamethasone increased I
B-
expression.
Scheinman et al. (25) also observed that dexamethasone enhanced
I
B-
synthesis and attenuated NF-
B activation stimulated by
tumor necrosis factor-
. On the other hand, Heck et al. (12) reported
that a GR mutant, which does not enhance I
B-
, represses NF-
B
activity and that glucocorticoid analogs, which competently enhance
I
B-
synthesis, do not repress NF-
B activity. They concluded
that glucocorticoid-mediated I
B-
induction and
glucocorticoid-mediated NF-
B repression are two events that are not
related each other.
In conclusion, MP reduced hyperoxia-induced ICAM-1 and ICAM-1 mRNA
expression and neutrophil adhesion to endothelial cells. MP attenuated
hyperoxia-induced NF-B activation, with increased I
B-
expression. These results indicated that hyperoxia upregulates ICAM-1
expression through NF-
B activation by reducing I
B-
expression and that MP downregulates hyperoxia-induced ICAM-1 expression by
inhibiting NF-
B activation with increased I
B-
expression. MP
might be potent in the treatment of oxidant lung injury at some
clinical condition. Transcription factor NF-
B plays an important role in controlling gene expression of many modulators of inflammatory and immune responses, including ICAM-1. Because of its central role in
acute inflammation, the transcription factor NF-
B should provide a
key for modulating the endothelial adhesion molecule expression in
response to inflammatory stimuli.
![]() |
FOOTNOTES |
---|
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Y. Suzuki, Dept. of Internal Medicine, Kitasato Institute Hospital, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan (E-mail: suzukiyk{at}kitasato.or.jp).
Received 5 February 1999; accepted in final form 13 September 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aoki, T.,
Y. Suzuki,
K. Suzuki,
A. Miyata,
Y. Oyamada,
T. Takasugi,
M. Mori,
H. Fujita,
and
K. Yamaguchi.
Modulation of ICAM-1 expression by extracellular glutathione in hyperoxia-exposed human pulmonary artery endothelial cells.
Am. J. Respir. Cell Mol. Biol.
15:
319-327,
1996[Abstract].
2.
Auphan, N.,
J. A. DiDonato,
C. Rosette,
A. Helmberg,
and
M. Karin.
Immunosuppression by glucocorticoids: inhibition of NF-B activity through induction of I
B synthesis.
Science
270:
286-290,
1995[Abstract].
3.
Beato, M.,
P. Herrlich,
and
G. Schütz.
Steroid hormone receptors: many actors in search of a plot.
Cell
83:
851-857,
1995[ISI][Medline].
4.
Bradley, J. R.,
D. R. Johnson,
and
J. S. Pober.
Endothelial activation by hydrogen peroxide: selective increases of intercellular adhesion molecule-1 and major histocompatibility complex class I.
Am. J. Pathol.
142:
1598-1609,
1993[Abstract].
5.
Brigham, K. L.,
R. E. Bowers,
and
C. R. McKeen.
Methylprednisolone prevention of increased lung vascular permeability following endotoxemia in sheep.
J. Clin. Invest.
67:
1103-1110,
1981[ISI][Medline].
6.
Chiara, O.,
P. P. Giomarelli,
E. Borrelli,
A. Casini,
M. Segala,
and
A. Grossi.
Inhibition by methylprednisolone of leukocyte-induced pulmonary damage.
Crit. Care Med.
19:
260-265,
1991[ISI][Medline].
7.
De Bosscher, K.,
M. L. Schmitz,
W. V. Berghe,
S. Plaisance,
W. Fiers,
and
G. Haegeman.
Glucocorticoid-mediated repression of nuclear factor-B-dependent transcription involves direct interference with transactivation.
Proc. Natl. Acad. Sci. USA
94:
13504-13509,
1997
8.
Engel, T.,
A. Dirksen,
L. Frølund,
J. H. Heinig,
U. G. Svendsen,
B. K. Pedersen,
and
B. Weeke.
Methylprednisolone pulse therapy in acute severe asthma.
Allergy
45:
224-230,
1990[ISI][Medline].
9.
Foletta, V. C.,
D. H. Segal,
and
D. R. Cohen.
Transcriptional regulation in the immune system: all roads lead to AP-1.
J. Leukoc. Biol.
63:
139-152,
1998[Abstract].
10.
Gulsvik, A.,
F. Kjelsberg,
A. Bergmann,
S. S. Froland,
K. Rootwelt,
and
J. R. Vale.
High-dose intravenous methylprednisolone pulse therapy as initial treatment in cryptogenic fibrosing alveolitis.
Respiration
50:
252-257,
1986[ISI][Medline].
11.
Halpern, P.,
U. Teitelman,
and
A. Lanir.
Effect of methyl prednisolone on normobaric pulmonary oxygen toxicity in rats.
Respiration
48:
153-158,
1985[ISI][Medline].
12.
Heck, S.,
K. Bender,
M. Kullmann,
M. Göttlicher,
P. Herrlich,
and
A. C. B. Cato.
IB
-independent downregulation of NF-
B activity by glucocorticoid receptor.
EMBO J.
16:
4698-4707,
1997
13.
Jackson, R. M.
Molecular, pharmacologic, and clinical aspects of oxygen-induced lung injury.
Clin. Chest Med.
11:
73-86,
1990[ISI][Medline].
14.
Jornot, L.,
and
A. F. Junod.
Response of human endothelial cell antioxidant enzymes to hyperoxia.
Am. J. Respir. Cell Mol. Biol.
6:
107-115,
1992[ISI][Medline].
15.
Kjæve, J.,
L. Næss,
T. Ingebrigtsen,
J. Vaage,
and
L. Bjertnæs.
Toxic oxygen metabolites increase microvascular permeability in isolated perfused rat lungs: the effect of methylprednisolone.
Circ. Shock
33:
228-232,
1991[ISI][Medline].
16.
Koizumi, M.,
L. Frank,
and
D. Massaro.
Oxygen toxicity in rats: varied effect of dexamethasone treatment depending on duration of hyperoxia.
Am. Rev. Respir. Dis.
131:
907-911,
1985[ISI][Medline].
17.
Ledebur, H. C.,
and
T. P. Parks.
Transcriptional regulation of the intercellular adhesion molecule-1 gene by inflammatory cytokines in human endothelial cells.
J. Biol. Chem.
270:
933-943,
1995
18.
Lee, W.,
P. Mitchell,
and
R. Tjian.
Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements.
Cell
49:
741-752,
1987[ISI][Medline].
19.
Lenardo, M. J.,
and
D. Baltimore.
NF-B: a pleiotropic mediator of inducible and tissue-specific gene control.
Cell
58:
227-229,
1989[ISI][Medline].
20.
Meyer, M.,
R. Schreck,
and
P. A. Baeuerle.
H2O2 and antioxidants have opposite effects on activation of NF-B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor.
EMBO J.
12:
2005-2015,
1993[Abstract].
21.
Orta-Sibu, N.,
C. Chantler,
M. Bewick,
and
G. Haycock.
Comparison of high-dose intravenous methylprednisolone with low dose oral prednisolone in acute renal allograft rejection in children.
Br. Med. J.
285:
258-260,
1982[ISI][Medline].
22.
Ray, A.,
and
K. E. Prefontaine.
Physical association and functional antagonism between the p65 subunit of transcription factor NF-B and the glucocorticoid receptor.
Proc. Natl. Acad. Sci. USA
91:
752-756,
1994[Abstract].
23.
Roebuck, K. A.,
A. Rahman,
V. Lakshminarayanan,
K. Janakidevi,
and
A. B. Malik.
H2O2 and tumor necrosis factor- activate intercellular adhesion molecule 1 (ICAM-1) gene transcription through distinct cis-regulatory elements within the ICAM-1 promoter.
J. Biol. Chem.
270:
18966-18974,
1995
24.
Saito, I.,
K. Terauchi,
M. Shimura,
S. Nishiimura,
K. Yoshino,
T. Takeuchi,
K. Tsubota,
and
N. Miyasaka.
Expression of cell adhesion molecules in the salivary and lacrimal glands of Sjogren's syndrome.
J. Clin. Lab. Anal.
7:
180-187,
1993[ISI][Medline].
25.
Scheinman, R. I.,
P. C. Cogswell,
A. K. Lofquist,
and
A. S. Baldwin, Jr.
Role of transcriptional activation of IB
in mediation of immunosuppression by glucocorticoids.
Science
270:
283-286,
1995[Abstract].
26.
Schreck, R.,
P. Rieber,
and
P. A. Baeuerle.
Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-B transcription factor and HIV-1.
EMBO J.
10:
2247-2258,
1991[Abstract].
27.
Shea, L. M.,
C. Beehler,
M. Schwartz,
R. Shenkar,
R. Tuder,
and
E. Abraham.
Hyperoxia activates NF-B and increases TNF-
and IFN-
gene expression in mouse pulmonary lymphocytes.
J. Immunol.
157:
3902-3908,
1996[Abstract].
28.
Snell, G. I.,
D. S. Esmore,
and
T. J. Williams.
Cytolytic therapy for the bronchiolitis obliterans syndrome complicating lung transplantation.
Chest
109:
874-878,
1996
29.
Suzuki, Y.,
T. Aoki,
O. Takeuchi,
K. Nishio,
K. Suzuki,
A. Miyata,
Y. Oyamada,
T. Takasugi,
M. Mori,
H. Fujita,
and
K. Yamaguchi.
Effect of hyperoxia on adhesion molecule expression in human endothelial cells and neutrophils.
Am. J. Physiol. Lung Cell. Mol. Physiol.
272:
L418-L425,
1997
30.
Van den Brink, H. R.,
M. J. van Wijk,
R. G. Geertzen,
and
J. W. Bijlsma.
Influence of corticosteroid pulse therapy on the serum levels of soluble interleukin 2 receptor, interleukin 6 and interleukin 8 in patients with rheumatoid arthritis.
J. Rheumatol.
21:
430-434,
1994[ISI][Medline].