Departments of 1 Physiology and 2 Biochemistry, Mercer University School of Medicine, Macon, Georgia 31207
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ABSTRACT |
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Alterations in
alveolar macrophage (AM) function during sepsis-induced hypoxia may
influence tumor necrosis factor (TNF) secretion and the progression of
acute lung injury. Nuclear factor (NF)-B is thought to regulate the
expression of endotoxin [lipopolysaccharide (LPS)]-induced
inflammatory cytokines such as TNF, and NF-
B may also
be influenced by changes in O2
tension. It is thus proposed that acute changes in
O2 tension surrounding AMs alter
NF-
B activation and TNF secretion in these lung cells. AM-derived
TNF secretion and NF-
B expression were determined after acute
hypoxic exposure of isolated Sprague-Dawley rat AMs. Adhered AMs
(106/ml) were incubated (37°C
at 5% CO2) for 2 h with LPS
(Pseudomonas aeruginosa, 1 µg/ml) in
normoxia (21% O2-5%
CO2) or hypoxia (1.8% O2-5%
CO2). AM-derived TNF activity
was measured with a TNF-specific cytotoxicity assay. Electrophoretic
mobility shift and supershift assays were used to determine NF-
B
activation and to identify NF-
B isoforms in AM extracts. In
addition, mRNAs for selected AM proteins were determined with RNase
protection assays. LPS-exposed AMs in hypoxia had higher levels of TNF
(P < 0.05) and enhanced expression
of NF-
B (P < 0.05); the
predominant isoforms were p65 and c-Rel. Increased mRNA bands for
TNF-
, interleukin-1
, and interleukin-1
were also observed in
the hypoxic AMs. These results suggest that acute hypoxia in the lung
may induce enhanced NF-
B activation in AMs, which may result in
increased production and release of inflammatory cytokines such as TNF.
transcription factor; nuclear factor-B; oxygen; acute lung
injury; cytokines
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INTRODUCTION |
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WITH ITS LOCATION in the O2-rich environment of lung airways, the alveolar macrophage (AM) depends primarily on energy provided by oxidative phosphorylation for its secretory function in a normoxic environment (28). The relative activities of enzymes used in oxidative phosphorylation and glycolysis in AMs are best suited for environments that are well oxygenated, and acute changes in environmental O2 may perturb AM function (21, 28). In sepsis-induced acute lung injury (ALI), airway edema and atelectasis, which may result in low ventilation-perfusion conditions, could result in reduced O2 tension around AMs in the affected lung areas (16, 19, 30). Because AMs play a central role in cytokine secretion and phagocytosis of bacteria and particles in the lung, low O2-induced alterations in macrophage function during ALI may influence the progression of this lung injury (19-21).
Tumor necrosis factor- (TNF), an important early-phase mediator of
ALI, is avidly produced by AMs in response to bacterial endotoxin
[lipopolysaccharide (LPS)] (13, 16, 20, 30). Previously,
investigators (8, 13, 22) demonstrated that TNF secretion in human
mononuclear cells and macrophage cell lines may be increased by
exposing these cultured cells to hypoxia for 18-24 h. These
authors suggested that hypoxia may be a stimulus that induces
macrophages to release higher levels of TNF and also of interleukin
(IL)-1 during conditions where O2
tension reaches very low levels. Members of the transcription factor
nuclear factor (NF)-
B family are thought to be involved
in the production of inflammatory cytokines such as TNF in macrophages
(1, 18, 26). An initial event in the transcriptional regulation of
cytokine production is the dissociation of NF-
B proteins from I
B
inhibitory proteins (1, 12, 17, 24). This allows NF-
B to migrate to
the nucleus where it binds to specific promoter sites, initiating activation of cytokine gene transcription (1, 12, 18, 24). Inhibition
of NF-
B activation and attenuation of cytokine production have been
observed after treatment with corticosteroids and antioxidants, but
regulation of this transcription factor and the inflammatory cytokines
induced by NF-
B is a multifaceted area of investigation (1, 10, 12,
17, 18, 23, 24).
Although there is evidence that 24 h of exposure to hypoxia may
increase TNF secretion in human mononuclear cells and that hypoxia may
activate transcription factors in certain cell lines, the NF-B and
secretory activities of AMs during very acute stages of hypoxic
exposure have not been defined. We hypothesized that reduction in the
O2 tension surrounding AMs acutely
induces alterations in NF-
B activation and TNF production in these
lung cells. We proposed to examine AM-derived TNF production after
exposure to hypoxia and to determine whether the nuclear transcription
factor NF-
B is affected by exposure of AMs to low
O2. To test this, TNF activity was
monitored in conditioned medium, NF-
B activation was determined in
cell extracts, and cytokine mRNA was measured in isolated rat AMs
exposed to acute hypoxia. Electrophoretic mobility shift assays (EMSAs)
and RNase protection analyses were performed on cellular extracts of
these AMs to determine the NF-
B activation and induction of selected
mRNAs in these lung cells after acute alterations in
O2 tension.
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MATERIALS AND METHODS |
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Experimental protocol with isolated
AMs. Pulmonary AMs were collected from the
bronchopulmonary lavage fluid of male Sprague-Dawley rats
(n = 8) with methods similar to those
previously described (20, 21). Before lavage, the rats were
exsanguinated after intraperitoneal anesthesia with pentobarbital
sodium (50 mg/kg; Abbott, Chicago, IL). The lungs were lavaged 10 times
in situ with 10-ml aliquots of
Ca2+- and
Mg2+-free phosphate-buffered
saline (Mediatech, Washington, DC). The cell fractions were washed in
phosphate-buffered saline, counted, and suspended
(106 cells/ml) in Dulbecco's
culture medium (DM; Mediatech) containing 1% penicillin-streptomycin
(Mediatech) and 5% fetal bovine serum (Sigma, St. Louis, MO). The
cells were allowed to adhere (37°C at 5%
CO2) to plastic culture dishes
(Falcon, Lincoln Park, NJ) for 18 h, the DM and nonadherent cells were
then decanted, and 1 ml of fresh DM was added to the adhered AMs. In
certain control experiments, AMs were adhered for only 1 h before the
experimental protocols to determine whether adherence time affected
NF-B activation. Adherence yielded >95% viable adhered AMs as
determined with trypan blue exclusion and nonspecific esterase stain
(Sigma) on selected preparations (20). The adhered AMs were incubated
(37°C at 5% CO2) in
normoxia (21% O2-5%
CO2) or hypoxia (1.8%
O2-5%
CO2) for 2 h with and without
LPS (Pseudomonas aeruginosa, 1 µg/ml; List Laboratories, Campbell, CA) added at
time 0. The 2-h time point was
selected for these studies because previous experiments performed in
this laboratory indicated that LPS-induced TNF activity from AMs was
measurable and stable at this time (20). Incubations of AMs in normoxia
or hypoxia were performed in modular incubation chambers (21) as
detailed in Incubation of AMs in normoxia and hypoxia. At the 2-h time point, the
cell-free conditioned DM was removed and the adhered AM preparations
were rinsed once with phosphate-buffered saline. With the use of trypan
blue exclusion (20) on selected AM preparations, cell viability was
determined to be >90% in all experimental conditions examined. The
0-time DM samples, the 2-h conditioned DM samples, and the AM
preparations were stored at
70°C until time of analysis.
Each experiment was performed on two to eight individual rat AM populations.
Incubation of AMs in normoxia and hypoxia. All isolation and adherence procedures for AMs were done in room air. The culture dishes containing adhered AMs with and without LPS were placed in humidified modular incubation chambers (Flow Laboratories, McLean, VA). The chambers were sealed and flushed with the appropriate gas mixture for 10 min, and the sealed modular chambers were incubated (37°C) for 2 h (21). In all experiments, exposure of the cell preparations to 95% air-5% CO2 is referred to as normoxic, whereas hypoxic exposure refers to the preparations in an environment of 95% N2-5% CO2. The percentages of O2 and CO2 in the modular incubation chambers were tested with Beckman gas analyzers (21). In the chambers incubated in normoxia, the O2 level was 21.0 ± 0.3% and CO2 was 5.0 ± 0.4%; in the hypoxic chambers, O2 was 1.8 ± 0.4% and CO2 was 5.0 ± 0.3%.
Preparation of whole cell extracts. Whole cell extracts were prepared by a modification of the extraction method described by Dent and Latchman (6). The following extraction buffer (0.05 ml) was added to each of the frozen, adhered cell preparations: 20 mM HEPES buffer (pH 7.8), 450 mM NaCl, 0.4 mM EDTA, 0.5 mM dithiothreitol (Sigma), 25% glycerol, 50 µg/ml of antipain, 40 µg/ml of bestatin, 50 µg/ml of chymotrypsin, 10 µg/ml of E64, 0.5 µg/ml of leupeptin, 0.7 µg/ml of pepstatin, 100 µg/ml of phosphoramidon, 1 mg/ml Prefabloc, and 2 µg/ml of aprotinin (Boehringer Mannheim, Indianapolis, IN). The cellular material was scraped into Eppendorf tubes, treated to two more freeze-thaw cycles (thaw at 37°C), and then centrifuged (13,000 g for 10 min; Marathon MicroA). The supernatants were immediately analyzed for the presence of transcription factors by EMSA (6). The protein content of each of the extracts was determined with the Bio-Rad assay kit (Melville, NY), which is based on the Bradford assay (3).
EMSA. Active NF-B isoforms present
in the AM extracts were detected with the EMSA (6). The oligonucleotide
containing the NF-
B consensus binding site was purchased from
Promega (Madison, WI). This NF-
B oligonucleotide contained the
sequence AGTTGAGGGGACTTTCCCAGGCTCAACTCCCCTGAAAGGGTCCG. The NF-
B
oligonucleotide was end labeled by incubation of the oligonucleotide
with [
-32P]ATP
(3,000 Ci/mmol; Amersham, Arlington Heights, IL) and T4 polynucleotide
kinase according to standard protocols (Maniatis Manual). Purification
of the labeled oligonucleotide was with the QIAquick Nucleotide Removal
kit (Qiagen) according to the manufacturer's instructions. Each
20-µl assay contained 5 µl of the prepared whole cell extract, 10 fmol of the NF-
B oligonucleotide end labeled with
32P, 0.25 µg of poly dI-dC
(Boehringer Mannheim), 0.5 mM dithiothreitol, 4% glycerol, and 20 mM
Tris (pH 7.5). For competition experiments, the assays also contained 1 pmol of unlabeled homologous or heterologous competitor oligonucleotide
that was added to the reaction mixture before the addition of labeled
oligonucleotide. After a 15-min incubation at room temperature, each of
the prepared whole cell extracts was loaded onto a nondenaturing 5%
polyacrylamide gel. The gels were run at 200 V for 1 h, 20 min in
0.5× Tris-borate-EDTA (0.045 M Tris borate and 1 mM EDTA). After
electrophoresis, the gels were dried and autoradiographed at
70°C with an intensifier screen. Bands corresponding to
NF-
B were identified by competition experiments: the homologous cold
oligonucleotide eliminated NF-
B binding, whereas the heterologous
one did not. Each EMSA gel contained all four conditions, with extracts
from a single rat AM population exposed to each condition on each gel.
For determining the densities corresponding to the NF-
B species, the
broadest band of NF-
B was analyzed in each lane. To control for
variable band intensity caused by changes in the specific activity of
the probe and/or exposure times, the intensity of the NF-
B band for
the normoxic condition was defined as 1 for each gel.
For supershift assays, NF-B antibodies to the isoforms p50, p52,
p65, Rel B, and c-Rel (Santa Cruz Biotechnology, Santa Cruz, CA) were
added to the binding reaction mixture and incubated for 20 min at room
temperature subsequent to the incubation of labeled oligonucleotide
probe with the whole cell extract. The antibodies used were specific to
the isoform indicated and specifically reactive to the rat proteins.
After incubation, the reaction mixture was electrophoresed as above.
The gel was dried and exposed for autoradiography. Autoradiographs were
scanned by densitometry, and band densities were quantified as relative
densitometry units with the National Institutes of Health (NIH) Image program.
RNase protection assay. RNA was
extracted from AMs by the acid guanidinium-phenol-chloroform method of
Chomczynski and Sacchi (5). The DNA templates used in generating the
cytokine probes were the rCK-1 Multi-Probe Template Set (PharMingen,
San Diego, CA) that can be used to specifically target rat mRNAs
encoding IL-1, IL-1
, TNF-
, TNF-
, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-10, and interferon (IFN)-
as well as L32 and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The probes were
generated by transcribing with T7 RNA polymerase in the presence of
[
-32P]UTP (800 Ci/mmol). Hybridization (55°C for 18 h) and RNase protection were
carried out with the RPA II kit (Ambion, Austin, TX). After electrophoresis (200 V for 2 h) through a 7 M urea-5% acrylamide gel,
the gel was dried and autoradiographed. Quantification of band
densities was determined with the NIH Image program. For each cytokine,
the densitometry units under normoxia for each rat were compared with
the densitometry units under hypoxia for the same rat, enabling a
direct comparison of the increased RNA for each species. RNA from each
AM preparation was analyzed by RNase protection four times, giving
comparable results in each of the four rat AM populations.
TNF assay. The conditioned DM samples were assayed for TNF activity with the L929 cytotoxicity assay (19, 20). Briefly, L929 mouse fibroblast cells in DM were grown to confluence in flat-bottom 96-well plates, and fresh DM with actinomycin D (5 µg/ml) was added to each well. One hundred microliters of each of the following were added to duplicate wells of L929 cells: DM alone to represent 0% cytotoxicity, AM-conditioned medium samples, and DM over blank wells to represent 100% cytotoxicity. The plates were incubated (37°C at 5% CO2) for 20 h, and the remaining L929 cells were stained for 10 min with 0.5% crystal violet in 20% methanol, rinsed, and air-dried. With a microplate reader, the optical density (550 nm) of the stained cells and the percent L929 cytoxicity were determined. One unit of TNF activity equals 50% L929 cytotoxicity (19, 20).
Statistical analysis. One-way analysis of variance and paired t-tests were used with Tukey's range tests to test for significant differences between groups (29). The level of significance was assigned at P < 0.05 (29).
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RESULTS |
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Hypoxia induces increased TNF
activity. Compared with the time
0 TNF activity of the AM preparations (0.5 ± 0.1 and 0.3 ± 0.4 U/ml for control and LPS-exposed AMs, respectively),
the conditioned medium of control, non-LPS-exposed AMs in normoxia for
2 h showed no significant increase in TNF activity (Fig.
1). The medium of control AMs exposed to
hypoxia for 2 h showed a trend for increased TNF activity compared with
the activity at time 0 and 2 h in
control AMs incubated in normoxia. In AM preparations exposed to either normoxia or hypoxia, TNF activity was significantly increased in
conditioned medium of AMs exposed to LPS. Compared with the LPS-induced
TNF activity of AM preparations exposed to normoxia for 2 h, the
conditioned medium of AMs exposed to LPS and hypoxia for 2 h
demonstrated significantly increased TNF activity.
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Hypoxia induces enhanced NF-B DNA
binding. LPS-stimulated AMs demonstrated increased
NF-
B binding, and the binding was completely inhibited by
preincubation of nuclear extracts with an excess of unlabeled consensus
oligonucleotide competitor (Fig. 2). The binding was unchanged when cold heterologous competitor activator protein-1 oligonucleotide was included. Compared with the
LPS-induced NF-
B binding observed in AMs in normoxia, 2 h of hypoxic
exposure enhanced the LPS-induced NF-
B binding. To confirm the
identity of the NF-
B isoforms, nuclear extracts from the LPS-exposed
AMs were treated with antibodies to the p50, p52, p65, Rel B, and c-Rel
protein components of NF-
B (Fig.
2B). The nuclear extracts showed
shifts with antibodies to p65 and c-Rel, with p65 inducing the most
dramatic shift of the NF-
B complex. In a series of experiments examining control, non-LPS-exposed AMs incubated for 2 h in normoxia or
hypoxia, NF-
B binding activity in the AMs exposed to hypoxia alone
showed enhanced expression of NF-
B binding (Fig.
3).
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Hypoxia induces enhanced cytokine
mRNA. Incubation for 2 h with LPS induced mRNA bands
for IL-1, TNF-
, and IL-1
in AMs exposed to normoxia or
hypoxia, but control, non-LPS-exposed AMs in either normoxia or hypoxia
showed no mRNA bands for these cytokines (Fig.
4A).
Compared with the LPS-induced cytokine mRNA of AMs exposed to normoxia
for 2 h, AMs exposed to LPS and hypoxia for 2 h demonstrated enhanced
mRNA bands for IL-1
, IL-1
, and TNF-
(Fig. 4). In addition,
compared with the mRNA bands of normoxia-exposed AMs, the bands for
IFN-
, L32, and GAPDH appeared enhanced in the hypoxia-exposed AMs
incubated with and without LPS (Fig.
4A).
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DISCUSSION |
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The results of our experiments indicate that compared with AMs exposed
to normoxia, AMs exposed for 2 h to hypoxia secrete higher levels of
TNF when stimulated in vitro with LPS. In a previous study,
Leeper-Woodford and Mills (21) reported that rabbit AMs had reduced ATP
levels and decreased retention of phagocytosed particles within the
first 30-60 min of hypoxic exposure. Data from this previous study
demonstrated that the ATP response to hypoxia is rapid and that AM
function may be dramatically altered by low
O2. The results of our present
studies now indicate that the early-response mechanisms of AMs to
hypoxia may also induce altered cytokine secretion in AMs and that very
acute hypoxic exposure upregulates LPS-induced TNF release from these
lung macrophages. Previously, investigators have suggested that hypoxia
upregulates TNF production by macrophages. Kisala et al. (16) noted
enhanced TNF and IL-1 production from AMs after pulmonary atelectasis
and associated the enhanced fever response to these elevated cytokine levels from AMs in collapsed areas of the lung. These authors suggested
that hypoxia was the most likely cause for the changes in AM
activation. Ertel et al. (7) demonstrated that 60 min of hypoxemia
induced release of TNF and other proinflammatory cytokines in mouse
plasma and found that peritoneal macrophages and Kupfer cells from
these mice produced increased levels of TNF, IL-1, and IL-6 in
vitro. Hempel et al. (13) and other investigators (8, 22)
have demonstrated that in vitro exposure to 24 h of hypoxia increased
the LPS-stimulated release of TNF in human mononuclear cells
and a macrophage cell line. Our results not only support these previous
observations that hypoxia induces increased TNF release but also
provide evidence that hypoxia induces alterations in TNF production
and secretion in AMs at very acute time points.
To further characterize the nature of the effect of hypoxia and LPS on
TNF production in AMs, we analyzed the effect of acute hypoxic exposure
on NF-B activation. In our present studies, NF-
B activation was
increased by exposure of AMs to acute hypoxia. These results suggest
that the stimulatory effects of hypoxia on TNF activity may be mediated
in part through increased activation of NF-
B, a transcription factor
that may represent a central pathway for regulation of the expression
of multiple proinflammatory mediators including TNF, IL-1, IL-2, IL-6,
IL-8, and certain stress-response proteins. These factors resulting
from NF-
B activation are responsible for many early responses to
bacterial infection or LPS exposure and may play essential roles in
conditions such as ALI (1, 18, 26).
NF-B is a heterodimeric complex containing the Rel isoforms p50,
p52, p65, Rel B, and c-Rel, and in resting cells, NF-
B is
sequestered in the cytoplasm bound with the I
B inhibitor (1, 10, 12,
23, 24). With LPS stimulation, I
B is phosphorylated and degraded in
the cell cytoplasm so that free NF-
B dimers can then translocate to
the nucleus and bind with high-affinity sites in the promoter regions
of target genes that stimulate transcription of those genes (1, 10, 12,
26). Although studies have indicated that free NF-
B binds to DNA as
heterodimers of the p50 and p65 subunits to initiate transcription,
Schmitz and Baeuerle (24) found that transient expression of p65 alone
could also result in gene transcription. In addition, these authors and
others (1, 10, 12, 17) have suggested that p50 dimers could act as
regulators of NF-
B activity and actually suppress the transcriptional activity of p65 subunits. With respect to the subunit
composition required for activation of gene transcription, the time
course of transcription factor activation may also be crucial (1, 24).
It has been proposed that appearance of the p50 isoform may be a later
activation or downregulator event and that activation of specific
transcription factor isoforms may vary depending on the times examined
after stimulation of the cells (1, 24). Dimer composition of the
transcription factor isoforms may determine the fine DNA-binding
specificity of a given NF-
B complex in that varying isoforms may be
a way to selectively control transcriptional activation (1).
In the present studies, we found increased p65 and c-Rel isoforms of
NF-B in the LPS-stimulated AMs exposed to acute hypoxia, with the
p65 isoform of NF-
B appearing to be the predominant one in this
macrophage system. Hansen et al. (10) have also noted that c-Rel may be
an important subunit in NF-
B activation in that c-Rel may complex
with p65 to initiate transcription. Our results agree with these
findings and indicate that in the AM system, the LPS-inducible
protein-DNA complexes assembled on the NF-
B binding sites contain
p65 and possibly c-Rel proteins. Because our studies examined very
early LPS- and hypoxia-induced activation of AMs, it is possible that
activation of the specific isoforms of NF-
B is time dependent and
that our findings indicating early activation of p65 and c-Rel may
reflect the immediate-response elements of LPS- and hypoxia-induced
NF-
B activation.
The present studies also demonstrate increased mRNA for TNF, IL-1,
and IL-1
in the AMs exposed to acute hypoxia. Because the TNF,
IL-1
, and IL-1
genes contain NF-
B binding sites in their
promoters (1, 18, 26), the increased intensity of these RNA bands in
the LPS-induced, hypoxia-exposed AMs is consistent with the increased
NF-
B activity detected in these lung cells. These results are also
consistent with previous reports (18, 30) that LPS in vivo induces TNF
and IL-1 as the earliest responders from the lung in sepsis and
supports the proposal that these macrophage cytokines play a central
role in the early pathogenesis of LPS- and bacterial-induced injury to
the lung.
Additional findings in our studies indicate that mRNA for IFN-,
GAPDH, and L32 may be slightly increased in the control and LPS-exposed
AMs incubated in hypoxia. It has been reported (27) that IFN-
expression may be increased through a TNF-dependent mechanism. It is
therefore possible that the enhanced TNF production we observed in AMs
exposed to hypoxia may induce IFN-
through activation of an
IFN-
-responsive factor that is also known to have an NF-
B site
(27). Our observations of slightly enhanced GAPDH and L32 bands for
mRNA may be consistent with the stress of hypoxia inducing cellular
activation (2, 7, 9, 14, 16, 17, 28). In AMs and other cells, the
glycolytic pathways are rapidly induced by a
low-O2 environment (9, 28), and under hypoxic conditions, the increased dependence on glycolysis as an
energy source is consistent with the increased levels of GAPDH mRNA in
our preparations. The ribosomal protein L32 is involved in translation
of mRNA in actively synthesizing ribosomes (15). There is evidence that
an ATP-dependent protein may be involved in ribosome activation, and it
is possible that this protein or low ATP-induced cellular stress may
influence L32 mRNA expression during the enhanced synthesis of cytokine
or stress proteins in the AMs exposed to hypoxia (9, 15). In addition,
it should be noted that mRNA stability may be affected by altered ATP
levels in that ATP may be required for mRNA degradation (11). In
hypoxic cells with low ATP, it may be that reduced mRNA degradation
occurs that allows for increased or prolonged expression of mRNA for certain cellular proteins (11).
Although Hempel et al. (13) proposed that hypoxia-induced production of
TNF and IL-1 was due to decreased
PGE2 synthesis during 24 h of
hypoxic exposure in macrophages, the mechanism for acute
hypoxia-mediated increased TNF production could possibly involve other
factors such as reactive oxygen species that could behave like second
messengers to activate transcription involving NF-B (2, 4, 9, 14,
17, 23-25, 27). In addition, there is evidence that
phosphorylation events, which may be involved in regulating
transcription, may be altered by hypoxia (17). An interesting study by
Koong et al. (17) demonstrated that hypoxia caused activation of
NF-
B by inducing tyrosine phosphorylation of I
B, an important
proximal step that precedes its dissociation from the NF-
B
cytoplasmic complex before transcriptional activation. Although no
conclusions can be made as to the mechanism of NF-
B activation and
increased TNF production and release in acute hypoxia, the dramatic
changes in oxidative metabolism and reduction in ATP levels in AMs
during acute exposure to low environmental
O2 may undoubtedly lead to complex
alterations in transcription, translation, and secretion in these lung cells.
The present studies demonstrate that acute hypoxic exposure may play a
role in the regulation of inflammatory genes through upregulation of
NF-B, a transcription factor critical to the activation of cytokine
genes that may be induced in acute inflammation. Our results also
provide evidence that hypoxia may be a potent regulatory mechanism of
lung cell activation during exposure to LPS and other acute
inflammatory states. Hypoxia may occur during ALI in patients with
sepsis, and increased cytokine production has been implicated as a
primary inducer of many facets of ALI. Attenuation of the activation of
NF-
B may be of great benefit in altering the acute cytokine
responses that are involved in ALI. Because NF-
B activation leads to
enhanced expression of many inflammatory cytokines, modulation of
NF-
B activation may provide a direct method of inhibiting
proinflammatory mediators. Manipulation of transcription factors such
as NF-
B may be useful for altering biological processes such as the
cytokine cascade that may induce ALI, and evaluation of potential
modulators of NF-
B activation is needed. Our results provide further
insight into the role of acute hypoxia in lung cell activation, and
these studies may lead to potential targets for intervention during disease states such as sepsis-induced ALI.
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ACKNOWLEDGEMENTS |
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S. K. Leeper-Woodford was supported in part by National Heart, Lung, and Blood Institute Grant HL-52917 and an American Lung Association of Georgia Research Grant.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. K. Leeper-Woodford, Dept. of Physiology, Mercer Univ. School of Medicine, Macon, GA 31207 (E-mail: leeper.sk{at}gain.mercer.edu).
Received 10 September 1997; accepted in final form 22 February 1999.
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