Departments of 1 Pediatric Critical Care, 2 Infectious Disease, and 4 Pulmonary Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262; and 3 Cell Therapeutics, Inc., Seattle, Washington 98119
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ABSTRACT |
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Lisofylline
[1-(5R-hydroxyhexyl)-3,7-dimethylxanthine]
decreases lipid peroxidation in vitro and in vivo suppresses
proinflammatory cytokine expression in models of lung injury due to
sepsis, blood loss, and oxidative damage. In the present experiments,
we used a murine hyperoxia model to examine the effects of lisofylline on the activation of nuclear transcriptional regulatory factors [nuclear factor-B and cAMP response element binding protein
(CREB)], the expression of proinflammatory cytokines in the
lungs, and the circulating levels of oxidized free fatty acids as well
as on hyperoxia-induced lung injury and mortality. Treatment with lisofylline inhibited hyperoxia-associated increases in tumor necrosis
factor-
, interleukin-1
, and interleukin-6 in the lungs as well as
decreased the levels of hyperoxia-induced serum-oxidized free fatty
acids. Although hyperoxic exposure produced activation of both nuclear
factor-
B and CREB in lung cell populations, only CREB activation was
reduced in the mice treated with lisofylline. Lisofylline diminished
hyperoxia-associated increases in lung wet-to-dry weight ratios and
improved survival in animals exposed to hyperoxia. These results
suggest that lisofylline ameliorates hyperoxia-induced lung
injury and mortality through inhibiting CREB activation, membrane
oxidation, and proinflammatory cytokine expression in the lungs.
cytokine expression; nuclear factor-B; adenosine
3',5'-cyclic monophosphate response element binding
protein; lipid oxidation
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INTRODUCTION |
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HYPEROXIA-INDUCED LUNG INJURY is characterized by an
intense inflammatory response initiated and exacerbated by the
generation of reactive oxygen species (ROS) (14, 21).
Histological changes include alveolar and interlobular septal edema,
neutrophil and macrophage infiltration, type II cell hyperplasia, and
fibroblastic proliferation (12, 25). Increased expression of
proinflammatory cytokines accompanies hyperoxia and appears to
contribute to the development of lung injury in this setting. Tumor
necrosis factor (TNF)- is present in the lung in increased amounts
early in the course of hyperoxia, even before histological changes are
seen (38, 43). Survival during hyperoxia was improved when an
anti-TNF-
antibody was given during hyperoxic exposure (22, 43).
Interleukin (IL)-1
and IL-6 levels are also elevated in the lung
during exposure to high concentrations of oxygen (23, 28).
Intracellular signaling pathways leading to the activation of
transcriptional regulatory factors such as nuclear factor-B (NF-
B) and cAMP response element (CRE) binding protein (CREB) can be
affected by ROS (36, 39). The ability of ROS to modulate the activity
of NF-
B or CREB may be particularly important in the setting of
hyperoxia where increased amounts of ROS are generated in the lungs
(21). NF-
B is activated in lung cells after as short a period of
hyperoxic exposure as 24 h (38). Binding sites for NF-
B and CREB are
present in the promoter regions of proinflammatory cytokine genes,
including TNF-
, IL-1
, and IL-6 (27, 31, 39).
Therefore, increased proinflammatory cytokine expression as a result of
ROS-induced activation of transcriptional factors may play an important
role in initiating lung injury during hyperoxia.
Lisofylline [LSF; 1-(5R-hydroxyhexyl)-3,7-dimethylxanthine] reduces lung free fatty acid ratios in in vitro models of IL-8-stimulated neutrophils and lung injury (16, 17). Patients with acute respiratory distress syndrome or those who are at risk for developing acute lung injury have increased serum levels of free fatty acids, which are reduced by treatment with LSF (6).
The addition of high concentrations (100 µM) of LSF to peripheral
blood mononuclear cells stimulated with lipopolysaccharide decreased
the release of TNF-, IL-1
, and IL-6 (33). Nevertheless, LSF
improves survival from endotoxemia at doses that produce concentrations substantially below 100 µM even when administered 4 h after the endotoxin challenge (32). In murine and porcine models of
acute lung injury induced by ischemia-reperfusion or sepsis,
LSF treatment decreased proinflammatory cytokine levels in the lungs
and diminished the severity of inflammatory lung injury (1, 17).
Because LSF decreases membrane peroxidation, inhibits proinflammatory intracellular signaling, and diminishes proinflammatory cytokine expression, we hypothesized that it may have protective effects on hyperoxia-induced lung injury. To evaluate this question, we examined the effects of LSF on the transcriptional regulation of proinflammatory cytokine genes in the lungs and the oxidation of membrane lipids as well as on the severity of lung injury and mortality during hyperoxic exposure.
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METHODS |
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Materials. LSF was
provided by Cell Therapeutics (Seattle, WA). Methoxyflurane was
obtained from Pittman-Moore (Mundelein, IL). Percoll and
poly(dI-dC) · poly(dI-dC) were obtained from Pharmacia (Uppsala, Sweden). The colorimetric protein assay kit, bovine
serum albumin standard,
N,N,N',N'-tetramethylethylenediamine (TEMED), and ammonium persulfate were purchased from Bio-Rad
Laboratories (Hercules, CA). The conserved CRE and B
oligonucleotides were synthesized by Operon (Alameda, CA) with
previously published sequences (Roesler/Zabel). All murine PCR primers
[TNF-
, IL-1
, IL-6, and hypoxanthine
phosphoribosyltransferase (HPRT)] were obtained from Clontech
(Palo Alto, CA). Moloney murine leukemia virus (MMLV) reverse
transcriptase, 0.1 M dithiothreitol, and 5× first-strand buffer
were all purchased from Life Technologies (Grand Island, NY).
Recombinant RNasin and random hexamers were obtained from Promega
(Madison, WI). AmpliTaq DNA polymerase
and 10× PCR buffer were purchased from Perkin-Elmer (Branchburg,
NJ). [
-32P]dATP was
obtained from Dupont-NEN Life Science Products (Boston, MA). Sequenase
version 2.0 T7 DNA polymerase was obtained from United States
Biochemical (Cleveland, OH). Antibodies to the p50 and p65 subunits of
NF-
B were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
The antibody to phosphorylated (phospho)-CREB was obtained from Upstate
Biotechnology (Lake Placid, NY). NucTrap purification columns were
obtained from Stratagene (La Jolla, CA). Rabbit anti-murine polyclonal
antibody, recombinant TNF-
standard, and the ELISA kit for murine
IL-6 were purchased from Endogen (Woodburn, MA). The hamster
anti-murine TNF-
monoclonal antibody was obtained from Genzyme
Diagnostics (Cambridge, MA). Biotin and ruthenium (II) trisbipyridae
chelate were purchased from Igen (Gaithersburg, MD). The
streptavidin-coated paramagnetic beads were obtained from Dynal (Lake
Success, NY). Bis-acrylamide (2% solution) was purchased from Fisher
(Pittsburgh, PA). Biomax MS film was obtained from Eastman Kodak
(Rochester, NY). All HPLC grade reagents were purchased from
Burdick-Jackson (Seattle, WA). Standards for HPLC,
9-(S)- and
13-(S)hydroxyoctadecadienoic acid (HODE), 9-(S)- and
13-(S)hydroperoxyoctadecadienoic
acid (HPODE), and 5-hydroxyeicosatetraenoic acid lactone were obtained
from Cayman Chemical (Ann Arbor, MI). RPMI 1640 medium with 25 mM
HEPES-L-glutamine, penicillin,
streptomycin, fetal calf serum, collagenase, deoxyribonuclease, and all
other chemicals were all obtained from Sigma (St. Louis, MO).
Animals. The study protocol was approved by the University of Colorado (Denver, CO) Health Sciences Center Animal Subject Protection Committee, and all protocols followed National Institutes of Health guidelines for the use of laboratory animals. Male BALB/c mice, 4 wk of age, were obtained from The Jackson Laboratories (Bar Harbor, ME) and then allowed to acclimate to Denver's altitude for at least 2 wk before study. All mice were 6-7 wk old when used for experiments. The mice were kept on a 12:12-h light-dark cycle and provided mice chow (Agway Prolab 3000, Syracuse, NY) and water ad libitum. BALB/c mice were used in these experiments because of their intermediate sensitivity to hyperoxic injury compared with that of other mouse strains (20).
Interventions. Study groups of mice
were placed in hyperbaric chambers lined with lime soda. The chambers
were pressurized to 760 mmHg and, when needed, depressurized over 30 min. Timed hyperoxic exposures (inspired
O2 fraction 1.0, 760 mmHg) lasted 24-72 h. Test groups of animals
(n = 5-8) were treated
intraperitoneally with either LSF (100 mg · kg1 · dose
1)
in 0.2-ml total volume or the same volume of sterile phosphate-buffered saline (PBS) carrier every 8 h. This dose of LSF has been used in
previous experiments (1, 32) that examined the effects of LSF in
modifying the development of acute inflammatory lung injury after
endotoxemia or hemorrhage in mice. The first dose of LSF or PBS was
given immediately before the mice were put into the hyperbaric
chambers. Room air control animals were housed in chambers at
atmospheric pressure and did not receive any interventions. For
survival studies, mice received either PBS
(n = 20) or LSF (n = 20) for the first 72 h of
hyperoxia, then were observed under hyperoxic conditions until all
animals had expired. The 72-h period of treatment was used because
after 72 h, depressurizing the chambers appeared to affect mouse
mortality, with increased animal deaths occurring during the
depressurization period.
Bronchoalveolar lavage. The animals
were first anesthetized with methoxyflurane, then killed by cervical
dislocation. The thoracic skin and fascia were opened by a midline
incision. The trachea was exposed by blunt dissection. Bronchoalveolar
lavage (BAL) fluid was obtained as previously described (34). In brief, the lungs were lavaged three times with the same 1-ml aliquot of cold,
sterile PBS. The final returned volume was consistently >0.75 ml. The
fluid was immediately centrifuged at 2,500 rpm for 30 s. The
supernatant was removed and stored at 70°C until used for
cytokine measurements.
Cytokine protein measurements. TNF-
protein was measured with an electrochemiluminescence method (11).
Briefly, a protein A-purified immunoglobulin G preparation from a
rabbit anti-murine polyclonal antibody was labeled with ruthenium (II)
trisbipyridae chelate as per the manufacturer's instructions (Igen).
Twenty-five microliters of ruthenylated antibody (2 µg/ml) were
combined with 25 µl of a biotinylated hamster anti-murine TNF-
monoclonal antibody (1 µg/ml) and diluted in an
electrochemiluminescence buffer (PBS, pH 7.4, with 0.25% bovine serum
albumin, 0.5% Tween 20, and 0.01% sodium azide). The antibody
solution was combined with 25 µl of a recombinant TNF-
standard or
25 µl of a BAL fluid sample in a 6-ml polypropylene tube for
incubation at room temperature overnight. Twenty-five microliters of a
1 mg/ml solution of streptavidin-coated paramagnetic beads were added
to each tube, and the tubes were agitated for 15 min at room
temperature. The reaction was quenched by adding 200 µl/tube of PBS,
pH 7.4. The sample and standards were quantitated with an Origen 1.5 analyzer (Igen). The sensitivity of the assay was 60 pg TNF-
/ml.
IL-6 levels were determined with an ELISA assay specific for murine IL-6 (Endogen) and performed as per manufacturer's instructions. The sensitivity for this assay was 50 pg/ml.
Semiquantitative PCR from whole lung
homogenates. Groups of five mice, with results obtained
from individual mice, were used for each experimental condition.
Semiquantitative PCR was used in these studies because the amount of
RNA obtained from each mouse was insufficient to prepare Northern blots
for several cytokines. The animals were anesthetized and prepared for
dissection as described in Bronchoalveolar
lavage. The thorax was opened with two
lateral incisions along the rib cage. The right heart was injected with cold, sterile PBS (1-2 ml) until the lungs had been thoroughly flushed. The lungs were excised with care to avoid the peritracheal lymph nodes and rinsed in PBS. The lungs were briefly blotted, then
snap-frozen in liquid nitrogen. The lungs were homogenized for 30 s on
ice in a denaturing solution containing 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol
as per Chomczynski and Sacchi (9); then mRNA was phenol extracted. cDNA
was synthesized from 1 µg of mRNA with MMLV reverse transcriptase and
random hexamer oligonucleotide primers as described by Kawasaki (24).
Semiquantitative PCR was performed with primers specific for murine
TNF-, IL-1
, and IL-6. A single PCR master mix was prepared.
Aliquots used for each sample contained 1× PCR buffer, 0.188 mM
each deoxyribonucleotide triphosphate, 0.4 µM each
single-strand DNA primer, 0.04 U of AmpliTaq DNA polymerase, and cDNA from
0.25 µg of mRNA, and the final volume was adjusted to 50 µl with
sterile deionized water. After an initial 2-min denaturation step at
85°C, between 26 and 38 cycles of PCR were performed as follows: 1 min, 95°C denaturation; 1 min, 60°C anneal; and 1 min, 72°C
extension. Coamplification of the housekeeping gene HPRT
was used to standardize the PCR products. PCR products were
electrophoresed on a 1.6% agarose gel and stained with ethidium
bromide. The number of PCR cycles was selected so that the
ethidium-stained amplified DNA products were below the level of
saturation. Analysis of the gel was performed with a gel-documentation
system (ImageStore 5000 with GelBase Windows Software,
Ultraviolet Products, San Gabriel, CA). Absorbance for each cytokine
was normalized to the respective HPRT absorbance.
Preparation of nuclear extracts.
Isolation of intrapulmonary monocytes and neutrophils has previously
been described by our laboratory (2). The mean number of cells isolated
per mouse after 24 h of hyperoxic exposure was 9.26 ± 1.22 × 105 in PBS-treated
animals and 7.76 ± 2.56 × 105 in LSF-treated animals. After
isolation, the intrapulmonary monocytes and neutrophils pooled from 10 mice were washed with PBS, and the nuclear proteins were isolated as
described by Hillman et al. (19). Briefly, 2-3 × 107 cells were resuspended
in 250 µl of buffer A (13),
incubated on ice for 15 min, and homogenized by 15 passages through a
25-gauge needle. After centrifugation for 6 min at 600 g at 4°C, the nuclear pellet was
resuspended in 50 µl of buffer C
(13) and incubated on ice for 15 min. The nuclear extracts were
centrifuged for 10 min at 12,000 g at
4°C. The supernatant was collected and stored in aliquots at
70°C. Protein concentrations were determined with a
colorimetric assay (Bio-Rad protein assay, Bio-Rad Laboratories) and
standardized with bovine serum albumin.
Electrophoretic mobility shift assay.
This procedure has been previously described by our laboratory (39).
The B site of the immunoglobulin gene (44) and the CRE conserved
site (35) were used for identifying DNA-protein complexes. Synthetic
double-stranded oligonucleotides of the following sequences (enhancer
motif underlined) were fill-in labeled with
[
-32P]dATP with T7
DNA polymerase:
B,
5'-TTTTCGAGC
GAGC-3' and
3'-GCTCG
GGCTCGTTTT-5'
and CRE,
5'-TTTTCGAGC
CAGAGC-3' and
3'-GCTCG
CTCGTTTT-5'.
The DNA binding reaction was performed at room temperature for 20 min
with a total volume of 20 µl. The reaction mixture contained 2.5 µg
of nuclear extract, 10 mM Tris-Cl, pH 7.5, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1 mM MgCl2, 4%
glycerol, 0.08 µg poly(dI-dC) · poly(dI-dC)/µg
nuclear extract, and 32P-labeled
double-stranded oligonucleotides at 0.7 fmol/µg nuclear extract. For
supershift reactions, antibodies to the p50 (5 µl) or p65 (1 µl)
subunits of NF-B or phospho-CREB (1 µl) were added to the reaction
mixture just before the 20-min incubation period. After incubation, 2 µl of 10× gel loading buffer (250 mM Tris-Cl, pH 7.5, 0.2%
bromphenol blue, 0.2% xylene cyanol, and 40% glycerol) were added to
each sample reaction; then each sample was loaded onto a 4%
polyacrylamide gel (acrylamide-bis-acrylamide 80:1, 2.5% glycerol in
0.5× Tris-borate-EDTA, TEMED, and ammonium persulfate) and
electrophoresed at 10 V/cm. The gel was then dried and analyzed by autoradiography.
Determination of lipid oxidation from serum by
reverse-phase HPLC. Serum was obtained from control
unmanipulated mice and from mice exposed to hyperoxia for 48 h and
treated with either PBS or LSF. The serum was transferred to silanized
(100 µl of methanol and 0.0375% butylated
hydroxytoluene) glass vials and immediately placed at
70°C before lipid analysis was performed. For analysis of 9- and 13-HODEs and 9- and 13-HPODEs, serum was transferred to a
pre-argoned tube, and 10,000 counts/min of
13-[14C]HODE and
13-[14C]HPODE in 50 µl of absolute ethanol were added to each sample. Chloroform-methanol
(2:1) followed by PBS (pH 6.5) was added to each sample, and the
partitioned phases were separated by centrifugation. The lower
(organic) phase was collected and dried completely under nitrogen.
Mobile-phase solvent [400 µl of 10 parts 0.15% acetic acid, 7 parts acetonitrile, and 5 parts tetrahyrofuran (vol/vol)] was
added, and the tubes were vortexed and sonicated. The suspended lipid
residue was collected and separated on a
C18 reverse-phase HPLC Jones
Genesis column (4 µm, 25 cm × 4.6 mm) with a flow rate of 1 ml/min, then analyzed by the integrated absorbance signal between 195 and 300 nm monitored via a photodiode array (Shimadzu, Columbia, MD).
Standards for each chromatogram included
9-(S)HODE, 9-(S)HPODE,
13-(S)HODE, and
13-(S)HPODE. Quantitation was
accomplished by interpolating the peak area response of a sample within
the peak area dose-response curve for the standards.
Wet-to-dry lung weight ratios. All mice used for lung wet-to-dry weight ratios were of identical ages. The lungs were excised, rinsed briefly in PBS, blotted, then snap-frozen in liquid nitrogen to obtain the "wet" weight. The lungs were then dried in an oven at 60°C for 24 h to obtain the "dry" weight.
Statistical analysis. Due to the inherent variability between groups of mice, for each experiment the entire group of animals had the same birth date, were exposed to either hyperoxia or room air in the identical chambers, and were prepared and studied at the same time. Data from semiquantitative PCR were obtained from individual mice and analyzed individually before group means and SE were calculated. Data presented are means ± SE and were analyzed by one-way analysis of variance with a Student-Newman-Keuls test of multiple comparisons. Survival curve data were analyzed by log-rank analysis. A P value < 0.05 was considered significant.
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RESULTS |
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Effects of LSF on hyperoxia-induced proinflammatory
cytokine expression in the lung. Representative
semiquantitative PCR results are shown in Fig.
1. Levels of TNF- mRNA in the lung were
significantly increased after 24 h of hyperoxic exposure and continued
to be elevated at 48 h (P < 0.01;
Fig. 2). IL-1
and IL-6 mRNA levels were
not increased in the lung at 24 h of hyperoxia but were significantly increased after 48 h of exposure (P < 0.01; Figs. 3 and
4).
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TNF- protein in BAL fluid was significantly elevated versus the
control levels after 48 h of hyperoxia
(P < 0.001; Fig.
5). No IL-1
protein was found in BAL
fluid from control or hyperoxia-exposed mice. IL-6 protein levels were
increased in BAL fluid after 48 h of hyperoxia
(P < 0.05; Fig.
6).
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Therapy with LSF completely prevented hyperoxia-induced increases in
lung TNF- and IL-1
mRNA levels and suppressed the
hyperoxia-induced increase in IL-6 mRNA levels by 38% (Figs.
2-4). Treatment with LSF also reduced TNF-
and IL-6 protein
levels in BAL fluid by 20 and 38%, respectively, after 48 h of
exposure to hyperoxia compared with treatment with PBS (Figs. 5 and 6).
Effects of LSF on hyperoxia-induced activation of
nuclear transcription factors NF-B and CREB in the
lung. To explore the effects of LSF on transcriptional
regulatory events that precede hyperoxia-induced increases in lung
cytokine protein, we examined the activation of the transcriptional
factors NF-
B and CREB in lung cells of mice exposed to hyperoxia for
24 h and treated with either LSF or PBS during that period. In previous
experiments, Shea et al. (38) demonstrated that 24 h of
hyperoxia results in increased activation of the transcriptional factor
NF-
B in lung cell populations. Such activation of NF-
B occurs
before increased levels of TNF-
protein are detectable in the lungs, and activated NF-
B continues to be present in lung cells for periods
of hyperoxic exposure > 24 h in length.
NF-B was constitutively expressed in lung cells from control animals
(Fig.
7A).
Increased activation of NF-
B was present in mice exposed to
hyperoxia for 24 h (Fig. 7A, 24P).
Specificity for the observed DNA-protein complex was confirmed by the
complete ablation of the NF-
B band when a 500-fold excess of
unlabeled
B DNA oligonucleotide was added to the reaction (Fig.
7A, 24P+cold
B). The
hyperoxia-induced NF-
B complex contained both the p65 and p50
subunits as shown by the supershifts when antibodies to p65 or p50 were
added to the reaction mixture (Fig.
7A, 24P+anti-p65 and 24P+anti-p50).
Treatment with LSF during exposure to hyperoxia for 24 h did not affect
NF-
B activation (Fig. 7A, 24P and
24L) as confirmed by densitometric analysis (Fig.
7B).
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CREB was constitutively expressed in lung cells from control animals in
both the phosphorylated and unphosphorylated forms (Fig.
8A).
Specificity for the observed DNA-protein complexes was confirmed by
complete ablation of both CREB bands when a 500-fold excess of
unlabeled CREB-specific DNA oligonucleotide was added to the reaction
(Fig. 8A, 24P+cold CREB). Increased
phospho-CREB was present in mice exposed to 24 h of hyperoxia (Fig.
8A, 24P). Treatment with LSF
consistently reduced the hyperoxia-associated increase in phospho-CREB
by 45-50% (Fig. 8A, 24L) as
confirmed by densitometric analysis (Fig. 8B).
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Effects of LSF on hyperoxia-induced generation of
oxidized free fatty acids. A representative HPLC
tracing demonstrating oxidized lipid analysis from a mouse exposed to
hyperoxia for 48 h and treated with PBS and a room air control mouse is
shown in Fig. 9. Exposure to 48 h of
hyperoxia significantly increased serum levels of the oxidized products
of linoleic acid (HODE and HPODE) as well as the total amount of
oxidized linoleic acid and arachadonic acid products (total diene
chromophores) compared with room air control mice
(P < 0.05, P < 0.001, and
P < 0.001, respectively; Figs.
10-12).
No alterations in serum levels of these oxidized lipids was found after
24 h of hyperoxia.
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Treatment with LSF prevented hyperoxia-associated increases in HODE and total diene chromophore levels (Figs. 10 and 12). Although HPODE levels were significantly increased in LSF-treated animals after 48 h of hyperoxia compared with that in room air control animals, these levels were significantly less than those in animals treated with PBS and exposed to hyperoxia for 48 h (P < 0.05; Fig. 11).
Effect of LSF on lung injury and survival during
hyperoxic exposure. Lung wet-to-dry weight ratios were
significantly increased in animals exposed to 72 h of hyperoxia
compared with control, unmanipulated animals
(P < 0.05; Fig.
13). Treatment with LSF prevented the
hyperoxia-induced increase in lung edema. Wet-to-dry lung weight ratios
in mice that received LSF did not differ significantly from that in
unmanipulated control animals not exposed to hyperoxia (Fig. 13). The
effects of LSF on hyperoxia-associated mortality was examined by
treating mice with either LSF or PBS during the first 72 h of hyperoxic
exposure (Fig. 14). LSF treatment
significantly prolonged survival (P < 0.02 by log-rank analysis).
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DISCUSSION |
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In the present experiments, treatment with LSF diminished the severity
of hyperoxia-induced lung injury and improved survival. Although
hyperoxia resulted in activation of the transcriptional regulatory
factors NF-B and CREB in lung cells, only CREB activity was
decreased by LSF, suggesting that LSF may affect intracellular signaling pathways leading to CREB phosphorylation. Functional CRE
sites are present in the promoter regions of proinflammatory cytokine
genes, including TNF-
, IL-1
, and IL-6, and can modulate the
transcription and expression of these cytokines. The ability of LSF to
inhibit CREB activation may therefore be a mechanism by which this
agent prevents proinflammatory cytokine expression and the development
of lung injury during hyperoxic exposure.
ROS can enhance the activation of transcriptional factors such as
NF-B and CREB (4, 36, 39). This interaction between ROS and
transcriptional factors may be due, in part, to the stimulatory effects
of ROS on intracellular kinases or phosphatases that regulate phosphorylation and downstream activation of transcription factors. In
particular, ROS appear to contribute to the activation of NF-
B (4)
through enhancing phosphorylation of serine residues
Ser32 and
Ser36 of the inhibitory peptide
I
B-
via a specific I
B ubiquitination-dependent protein kinase
(8). After phosphorylation, I
B-
is degraded and the
transcriptionally active NF-
B heterodimer translocates to the
nucleus (8). Kinases known to be affected by ROS and involved in the
activation of CREB include p38 mitogen-activated protein kinase (41)
and the extracellular signal-regulated kinases (ERK1 and ERK2) (30).
ERK1 and ERK2 have been shown to regulate early-response genes via signal transduction and activator of transcription (STAT) proteins (10) and to induce transcription by activating CREB through the phosphorylation of Ser133 (30). LSF inhibits IL-12-driven signaling, including STAT kinases, in differentiating T lymphocytes (5). Such inhibition of STAT kinases, which are downstream of ERK1 and ERK2, may decrease hyperoxia-associated activation of CREB, thereby preventing CREB-dependent transcription of proinflammatory cytokine genes.
In a previous report (7), LSF did not affect TNF--induced activation
of NF-
B in the macrophage cell line RAW 264.7. The present results
are consistent with a lack of effect of LSF on NF-
B activation. Even
though our experiments demonstrated that hyperoxia produced activation
of NF-
B in lung cells, treatment with LSF did not modify this effect.
Exposure to hyperoxia and ROS produces lipid peroxidation (29) as
demonstrated in the present study by increased circulating levels of
HODE, HPODEs, and total diene chromophores after 48 h of hyperoxia.
Oxidized linoleic acid derivatives are biologically active, inducing
IL-1 release in human monocytes (26, 42), and serve as modulators in
the mitogenic response induced by epidermal growth factor (15). In a
previous study (40) that investigated patients undergoing allogenic
bone marrow transplantation, LSF suppressed radiotherapy-induced
increases in serum HODE and HPODE. The present results show similar
inhibitory effects of LSF in preventing hyperoxia-associated generation
of oxidized linoleic acid products, including HODE, HPODE, and total
diene chromophores.
In vitro studies (3, 18) of endothelial cells demonstrated that
increased lipid peroxidation induced by ROS or the addition of linoleic
acid to cell cultures results in the activation of NF-B. It is
unknown at present whether oxidized lipids also induce activation of
CREB, but if so, then the prevention of lipid oxidation by LSF could
provide another mechanism for the LSF-associated inhibition of CREB
phosphorylation found in the present experiments. However, the effects
of oxidized lipids on CREB activation would have to be relatively
greater than those affecting NF-
B to explain the observed ability of
LSF to inhibit hyperoxia-induced activation of CREB, but not of
NF-
B, in the lungs.
In the present experiments, LSF significantly decreased TNF-,
IL-1
, and IL-6 expression in the lungs over the first 48 h of
hyperoxic exposure. However, even though the amounts of TNF-
and
IL-6 protein from mice exposed to hyperoxia and treated with LSF were
reduced compared with that in hyperoxia-exposed animals not treated
with LSF, cytokine levels were still increased in the LSF-treated mice
compared with those in the unmanipulated room air control animals.
These results indicate that hyperoxia produces activation of signaling
pathways that induce expression of proinflammatory cytokines that are
not affected by the administered dose of LSF. It is also possible that
hyperoxia activates other cytokine-inducing pathways that are not as
responsive to LSF. Transcriptional mechanisms associated with NF-
B
are possible candidates for this effect because NF-
B is important in
inducing the transcription of multiple proinflammatory mediators,
including TNF-
and IL-6, and remains activated in the lung during
hyperoxia despite LSF treatment. Other transcriptional factors involved in proinflammatory cytokine regulation, including activator
protein-1, are oxidant sensitive (37). The ability of LSF
to modulate the activation state of transcriptional factors other than
NF-
B and CREB after hyperoxic exposure is unknown. Cytokine protein
production may also be affected by posttranscriptional events, and the
effects of LSF on posttranscriptional regulation have not been examined.
Although LSF did significantly improve survival after hyperoxic exposure, all mice that received LSF during the first 72 h of hyperoxia still died by 140 h of exposure, demonstrating that the protection provided by LSF was incomplete. These survival results are consistent with the incomplete suppression of proinflammatory cytokine expression in the lungs achieved with LSF therapy with the present dose and schedule. Therefore, the present results, although encouraging in suggesting that LSF may have a therapeutic role in treating patients exposed to high concentrations of oxygen, do not indicate that LSF therapy alone will be entirely protective against the deleterious effects of hyperoxia. Combining LSF with other therapies able to protect cells from oxidant injury through mechanisms different from those of LSF or using LSF on a more intensive schedule might be expected to provide additional benefit.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the assistance of Debra Faulk, Merdad Parsey, and Robert Shenkar for guidance and mentoring.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-50284.
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: E. Abraham, Div. of Pulmonary Sciences and Critical Care Medicine, Univ. of Colorado Health Sciences Center, 4200 East Ninth Ave., Box C-272, Denver, CO 80262 (E-mail: Edward.Abraham{at}UCHSC.edu).
Received 30 June 1998; accepted in final form 28 January 1999.
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