1 Department of Surgery, University of Colorado Health Sciences Center and The Veterans Affairs Hospital, Denver 80262; and 2 Department of Pediatric Surgery, The Children's Hospital, Denver, Colorado 80218
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chemokines stimulate the influx of
leukocytes into tissues. Their production is regulated by nuclear
factor-B (NF-
B), an inducible transcription factor under the
control of inhibitory factor
B-
(I
B-
). We have previously
demonstrated that L-arginine (L-Arg) attenuates
neutrophil accumulation and pulmonary vascular injury after
administration of lipopolysaccharide (LPS). We hypothesized that
L-Arg would attenuate the production of lung chemokines by stabilizing I
B-
and preventing NF-
B DNA binding. We examined the effect of L-Arg on chemokine production, I
B-
degradation, and NF-
B DNA binding in the lung after systemic LPS. To
block nitric oxide (NO) production, a NO synthase inhibitor was given before L-Arg. LPS induced the production of chemokine
protein and mRNA. L-Arg attenuated the production of
chemokine protein and mRNA, prevented the decrease in I
B-
levels,
and inhibited NF-
B DNA binding. NO synthase inhibition abolished the
effects of L-Arg on all measured parameters. Our results
suggest that L-Arg abrogates chemokine protein and mRNA
production in rat lung after LPS. This effect is dependent on NO and is
mediated by stabilization of I
B-
levels and inhibition of NF-
B
DNA binding.
nitric oxide; nitric oxide synthase; cytokine-induced neutrophil
chemoattractant-1; macrophage inflammatory protein-2; acute lung
injury; nuclear factor-B; inhibitory factor
B
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
LIPOPOLYSACCHARIDE (LPS)-induced acute lung injury (ALI) is characterized by polymorphonuclear neutrophil (PMN) accumulation, pulmonary hypertension, pulmonary endothelial cell damage, and increased capillary permeability (17, 35). PMNs are important cellular effectors of this LPS-induced injury, and PMN depletion abrogates LPS-induced ALI (9, 50). Sheridan et al. (50) previously found that PMN depletion by vinblastine and rabbit anti-rat PMN antiserum significantly attenuated LPS-induced lung PMN accumulation, lung edema, and impairment of endothelium-dependent and -independent pulmonary vasorelaxation.
The accumulation of PMNs in tissue is a complex process that involves
chemoattractant cytokines (chemokines) (7, 13) and the
interplay of adhesion molecules of the selectin and integrin families
(3, 53). In general, the C-X-C (or ) chemokines mediate
the influx of PMNs and lymphocytes into tissue. Cytokine-induced neutrophil chemoattractant-1 (CINC-1) is an 8-kDa basic protein homologous to human interleukin (IL)-8 that is involved in PMN recruitment in ALI (53). Macrophage inflammatory protein-2
(MIP-2) is another rat C-X-C chemokine generated by macrophages in
response to LPS (46) and also is implicated in the
pathogenesis of ALI (7). Blockade with anti-CINC and
anti-MIP antibodies significantly reduces alveolar PMN accumulation in
animal models of ALI (7, 49). Together, CINC-1 and MIP-2
are important effectors of PMN emigration into lung tissue.
Nitric oxide (NO) prevents PMN accumulation in tissue (16, 20, 45). This effect can occur by the downregulation of cell adhesion molecules on the endothelium (29) and PMNs (30) or by affecting the production of chemokines (8).
Sheridan et al. (51) previously found that
L-arginine (L-Arg), the substrate for NO
production, decreases LPS-induced PMN accumulation and attenuates ALI
in a rat model of endotoxemia. In the same animal model, exogenous
administration of L-Arg decreased alveolar macrophage
production of tumor necrosis factor- (TNF-
) and IL-1
by a NO
synthase (NOS)-dependent mechanism (34). Angele et al.
(2) also found that L-Arg decreased plasma
IL-6 levels and preserved splenocyte function in a rat model of
hemorrhagic shock-resuscitation.
NO may be protective in ALI by serving as a control mechanism for
modulating the expression of nuclear factor-B (NF-
B)-responsive genes such as those of the chemokine superfamilies (33,
43). NF-
B is one described transcriptional regulator of
CINC-1 and MIP-2 (6). It is likely that modulation of
NF-
B by NO may inhibit CINC-1 and MIP-2 production and attenuate
LPS-induced ALI. In the resting state, NF-
B is bound to inhibitory
factor
B-
(I
B-
), which confines the complex to the
cytoplasm. Inflammatory stimuli such as LPS or ischemia lead to
dissociation of I
B-
from NF-
B; the latter element is able to
translocate to the nucleus and induce transcription. We hypothesized
that in vivo administration of L-Arg would prevent the
LPS-induced decrease in I
B-
levels and prevent NF-
B-mediated
expression of CINC-1 and MIP-2. The purpose of this study was to
determine the effect of L-Arg on LPS-induced CINC-1 and
MIP-2 protein and mRNA expression, NF-
B DNA binding, and I
B-
protein in the lung. We found that treatment with L-Arg
before LPS attenuated both CINC-1 and MIP-2 protein and mRNA production
and NF-
B DNA binding. This mechanism was mediated through
preservation of I
B-
protein levels after LPS.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals. Male Sprague-Dawley rats (body weight 300-350 g) were quarantined and maintained on a standard pellet diet for 2 wk before the initiation of experimental protocols. All animal experiments were approved by the University of Colorado Health Sciences Center Institutional Animal Care and Use Committee. Animals received humane care in accordance with the revised Guide for the Care and Use of Laboratory Animals (7th ed.; Washington, DC: Natl. Acad. Press, 1996).
Experimental protocol.
Awake rats were administered saline or LPS (0.5 mg/kg LPS;
Salmonella typhimurium, lot no. 47H4093, Sigma, St. Louis,
MO) freshly prepared in 0.9% saline via intraperitoneal injection. The
experimental design included the following groups: 1)
control treated with normal saline; 2) LPS alone;
3) 300 mg/kg ip of L-Arg given 30 min before LPS
(L-Arg-LPS); 4) 300 mg/kg ip of
D-Arg given 30 min before LPS (D-Arg-LPS);
5) 0.2 mg/kg ip of a NOS inhibitor,
2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), 30 min before L-Arg and LPS (AMT-L-Arg-LPS); and
6) AMT alone (AMT). Animals were anesthetized with
pentobarbital sodium (0.5 mg/kg ip) at 0.5, 1, 2, and 4 h after
LPS or saline administration. Lungs were then harvested, rinsed with
normal saline, blotted dry, snap-frozen in liquid nitrogen, and stored
at 70°C until analysis. Plasma was obtained by centrifugation (750 g for 15 min at 4°C) of heparinized whole blood taken from
the rats after treatment.
Measurement of L-Arg levels. L-Arg concentrations in rat plasma were determined with a Beckman 6300 amino acid analyzer that employed a 3-lithium-citrate buffer system according to the manufacturer's instructions (Beckman-Coulter, Fullerton, CA). L-Arg concentrations were reported in nanamoles per milliliter of plasma.
Nitrite/nitrate assay. Nitrite plus nitrate (NOx) production was measured in rat plasma as previously described (39) with a kit provided by R&D Systems (Minneapolis, MN). Briefly, the plasma was first diluted (1:2) in HEPES buffer and ultrafiltered to exclude proteins >10 kDa. The nitrate component of the filtrate was then reduced to nitrite by incubation with nitrate reductase and NADH at 37°C for 30 min. The total nitrite concentration in the samples was then measured by the addition of 100 µl of the Griess reagent (0.1% naphthylethylenediamide dihydrochloride in H2O and 1% sulfanilamide in 5% concentrated HCl) to 50 µl of sample. The optical density at 540 nm was determined spectrophotometrically with the use of a microplate reader (Bio-Rad, Hercules, CA) and plotted against a standard curve. Results are expressed in micromoles.
Nuclear and cytoplasmic protein isolation.
Nuclear extracts from lung tissue were prepared with a
modification of a previously published technique (47).
Lungs were homogenized in 5 volumes of homogenate buffer (10 mM HEPES,
10 mM KCl, 0.5 M sucrose, 0.1 mM EGTA, and 1 mM dithiothreitol). Homogenates were then centrifuged at 750 g for 10 min at
4°C to isolate crude nuclei. The supernatants, which contained the
cytosolic fraction, were stored at 70°C and used for CINC-1 and
MIP-2 protein determination. The crude nuclear pellet was then
resuspended in 100 µl of ice-cold nuclear extraction buffer (20 mM
HEPES, 0.4 M NaCl, 1 mM EGTA, and 1 mM dithiothreitol). Samples were
incubated on ice for 30 min with brief, gentle vortexing every
5-10 min. The nuclear extract was then centrifuged at 12,000 g for 5 min at 4°C, and the supernatant was collected and
stored at
70°C. Protein was quantified in both cytosolic and
nuclear extracts with the Coomassie Plus protein assay kit (Pierce,
Rockford, IL).
Electrophoretic mobility shift assay.
NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3',
binding site underlined) consensus oligonucleotide was 5'-end labeled
with [
-32P]dATP with T4 polynucleotide kinase.
Unincorporated nucleotide was removed with a NucTrap probe purification
column (Stratagene, La Jolla, CA). Five micrograms of nuclear protein
were incubated with labeled oligonucleotide [100,000-200,000
counts/min (cpm)] in binding buffer [10 mM Tris · HCl, pH
7.5, 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl2, 0.5 µg of
poly(dI-dC) · poly(dI-dC), 1% Nonidet P-40, and 4% glycerol]
for 25 min at room temperature in a final volume of 25 µl. Free
oligonucleotide and oligonucleotide-bound proteins were then separated
by electrophoresis on a native 4% polyacrylamide gel. The gel was
subsequently dried and exposed to X-ray film between two intensifying
screens overnight at
70°C. For supershift studies, antibody (5 µg) to the p65 subunit of NF-
B (Santa Cruz Biotechnology, Santa
Cruz, CA) was added before the addition of the labeled
oligonucleotide. Binding of the antibody to the DNA-protein complex was
indicated by a supershift in the electrophoretic mobility shift assay
(EMSA). To further demonstrate specificity, excess unlabeled (cold
probe) oligonucleotide was used as a specific competitor.
Immunoblotting.
Lung tissue was homogenized with a tissue homogenizer (Virtishear
homogenizer; Virtis, Gardner, NY) in 5 volumes of homogenization buffer
(25 mM Tris · HCl, 2 mM EGTA, 1 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4). After centrifugation (3,000 g at 4°C for 20 min), the supernatant was collected. Total
protein concentration was determined with the Coomassie Plus protein
assay. Samples (20 µg of crude protein) were mixed with an equal
volume of sample buffer (100 mM Tris · HCl, 2% SDS, 0.02%
bromphenol blue, and 10% glycerol) and boiled for 5 min.
Electrophoresis was performed on a 4-20% linear gradient
SDS-polyacrylamide gel. Proteins were then electrophoretically
transferred onto a nitrocellulose membrane (Bio-Rad). Membranes were
blocked for 1 h at room temperature with antibody buffer (PBS
containing 0.1% Tween 20 and 5% nonfat dried milk) and then incubated
with rabbit polyclonal anti-IB-
(1:500 dilution with antibody
buffer) for 1 h at room temperature. Membranes were washed three
times in PBS containing 0.1% Tween 20 and then incubated in
peroxidase-labeled goat anti-rabbit IgG (1:10,000 dilution with
antibody buffer) for 1 h at room temperature. Membranes were then
washed three times, and antigen-antibody complexes were revealed by the
enhanced chemiluminescence method (Amersham, Arlington Heights, IL).
Quantification of the immunoblot was performed by computer-assisted
densitometry (NIH application 1.599b4). Density values are expressed as
a percentage of the saline control level of each experiment. All
densities are means ± SE of three separate experiments.
Northern blotting.
Northern blotting was performed as described by Henderson et al.
(22). Lung tissue was homogenized with a tissue
homogenizer (Virtishear, Virtis) in 10 volumes of TRI Reagent
(Molecular Research Center, Cincinnati, OH). Total RNA was extracted
with the guanidinium isothiocyanate method (11).
Quantification of RNA was performed spectrophotometrically by reading
the absorbency at 260 nm. Twelve micrograms of total RNA from each
sample were denatured and electrophoresed on a 1.2% agarose-6.6%
formaldehyde gel containing 10 mg/ml of ethidium bromide. Gels were
soaked in 10× SSPE for 1 h before overnight capillary transfer
(GIBCO BRL, Life Technologies, Gaithersburg, MD) in 10× SSPE onto a
nylon membrane (Stratagene). After transfer, the membrane was
ultraviolet (UV) cross-linked with a UV Stratalinker (Stratagene), and
rRNA was visualized and photographed under UV light to assess RNA
loading. Membranes were then wet with 2× SSPE and placed in a Mini-6
hybridization oven (Hybaid, Franklin, MA). All prehybridization,
hybridization, and high-stringency wash temperatures were calculated
based on a previously published method (22) that employed
the equation Tm50 = 16.6 (log
[Na+]) + 81.5 + 0.41 (%GC content) 675 (no. of bases in probe), where Tm50 is the temperature at
which 50% of the DNA probe-RNA hybrids melt, [Na+] is
the molar concentration of Na+ in the hybridization
solution and %GC is the percentage of guanidine and cytosine
residues in the probe. The hybridization solution consisted of 1×
SSPE, 2× Denhardt's solution, 1% nonfat dry milk, 10% dextran
sulfate, 2% SDS, 200 µg/ml of salmon sperm DNA, 200 µg/ml of yeast
tRNA, and 200 µg/ml of polyadenylic acid. Prehybridization was
carried out at the probe-appropriate temperature for 8-12 h. The
prehybridization solution was then decanted, and 10 ml of hybridization
solution were added and incubated for ~18 h. The hybridization
solution was identical to the prehybridization solution except for the
addition of labeled probe at a final concentration of 5.0 × 106 cpm/ml. Both CINC-1 and MIP-2 probes were single-strand
30-mer DNA oligonucleotides synthesized by GIBCO BRL. The CINC-1 probe sequence (5'-GCGGCATCACCTTCAAACTCTGGATGTTCT-3') was complementary to
nucleotides 170-199 of the CINC-1 cDNA (14). The
MIP-2 probe had the sequence
5'-GTTAGCCTTGCCTTTGTTCAGTATCTTTTG-3' and was complementary to rat
MIP-2 cDNA nucleotides 304-333. Oligonucleotide probes were
labeled by terminal deoxynucleotidyltransferase (TdT). The labeling
reaction was set up by adding 2 µl of a 10 ng/µl solution of
oligonucleotide, 2 µl of 1 mg/ml of bovine serum albumin, 4 µl of
5× TdT buffer (GIBCO BRL), 10 µl (33 pmol) of
[
-32P]dATP (DuPont NEN, Boston, MA), and 2 µl of 15 U/µl of recombinant TdT (GIBCO BRL). The reaction was incubated for
1 h at 37°C. Unincorporated nucleotide was removed by a NucTrap
probe purification column (Stratagene). The activity of the probe was
determined by scintillation.
Rat chemokine protein ELISA. CINC-1 and MIP-2 were measured in lung homogenates (cytoplasmic fraction as described in Nuclear and cytoplasmic protein isolation) with a rat-specific ELISA system. Kits were obtained from ImmunoBiological Laboratories (CINC-1; Fujioka City, Japan) and BioSource International (MIP-2; Camarillo, CA). The absorbency of standards and samples was determined spectrophotometrically with a microplate reader (Bio-Rad). Results were recorded as optical densities and plotted against the linear portion of the standard curve.
Reagents.
All antibodies for immunoblotting and EMSA were obtained from Santa
Cruz Biotechnology. The IB-
antibody was a rabbit polyclonal IgG
against the carboxy terminus of human I
B-
that cross-reacts with
rat I
B-
. The NF-
B antibodies were both goat polyclonal IgGs
raised against the carboxy terminus of the human p65 subunit of
NF-
B, and both cross-reacted with the rat NF-
B subunits. All
other chemicals, unless stated, were obtained from Sigma.
Statistical analysis. Statistical analyses were performed with StatView software (Brain Power, Calabasas, CA). Data are presented as means ± SE. Statistical evaluation used standard one-way ANOVA with post hoc Bonferroni-Dunn test. Significance was accepted at a confidence limit of 95%.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
L-Arg plasma determinations. To determine the effect of L-Arg supplementation and LPS on plasma L-Arg concentrations, plasma was harvested from rats 1 h after saline, LPS, or L-Arg treatment and analyzed for L-Arg (n = 3 rats/group). Intraperitoneal L-Arg supplementation resulted in an increase in plasma L-Arg compared with saline control (L-Arg, 660.23 ± 129.29 nmol/ml vs. saline, 166.97 ± 9.05 nmol/ml; P < 0.05). LPS led to a decrease in circulating plasma L-Arg levels compared with saline control (saline, 166.97 ± 9.05 nmol/ml vs. LPS: 83.80 ± 14.43 nmol/ml; P < 0.05).
Nitrate/nitrite production.
To determine the effect of LPS, supplemental L-Arg
administration, and NOS inhibition on plasma nitrate and nitrite
production, rat plasma was assayed for total NOx 2 h
after saline or LPS (Fig. 1). Compared
with saline, LPS caused an increase in NOx (saline, 13.72 ± 1.00 µmol vs. LPS, 22.14 ± 0.21 µmol;
P < 0.05) 2 h after LPS administration. However,
L-Arg administration before LPS induced an even greater
increase in NOx compared with both saline and LPS
(L-Arg-LPS, 35.80 ± 1.34 µmol; P < 0.01). A NOS inhibitor (AMT) given before LPS abolished the LPS-induced
increase in NOx (AMT-LPS, 7.47 ± 0.23 µmol;
P < 0.05 compared with LPS) and had an inhibitory
effect on baseline NOx levels (AMT alone, 9.11 ± 0.25 µmol; P < 0.05 compared with saline).
|
Effect of L-Arg on lung CINC-1 and MIP-2 protein.
To determine the effect of L-Arg on chemokine protein
production after LPS, rat lung tissue was examined 2 h after LPS
for the presence of CINC-1 (Fig.
2A) and MIP-2 (Fig.
2B). Compared with saline treatment, LPS caused a
significant increase in both CINC-1 (saline, 27.1 ± 10.1 vs. LPS,
1,275.5 ± 239.3; P < 0.05) and MIP-2 (saline,
10.3 ± 2.7 vs. LPS, 109.3 ± 19.7; P < 0.05) in lung homogenate. L-Arg given before LPS attenuated
the LPS-induced increase in both CINC-1 (L-Arg-LPS,
257.7 ± 75.0; P < 0.05 vs. LPS) and MIP-2
(L-Arg-LPS, 17.7 ± 4.3; P < 0.05 vs.
LPS). D-Arg before LPS had no effect on the increase in
CINC-1 (D-Arg-LPS, 1,283 ± 83.3; P = 0.68 vs. LPS) and MIP-2 (D-Arg-LPS, 97.8 ± 24.2; P = 0.58 vs. LPS) production compared with LPS alone.
The NOS inhibitor AMT given before L-Arg and LPS abolished
the L-Arg-associated attenuation of CINC-1
(AMT-L-Arg-LPS, 1339.8 ± 251.6; P < 0.05 vs. L-Arg-LPS) and MIP-2 (AMT-L-Arg-LPS,
75.3 ± 6.8; P < 0.05 vs. L-Arg-LPS).
|
Effect of L-Arg on lung CINC-1 and MIP-2 mRNA.
To examine the effect of L-Arg on chemokine mRNA transcript
expression after LPS, rat lung tissue was examined 2 h after LPS for the presence of CINC-1 (Fig. 3,
A and B) and MIP-2 (Fig. 3, C and
D) mRNAs with Northern blotting. Compared with saline
control, LPS caused a significant increase in both CINC-1 and
MIP-2 mRNAs in lung homogenate. L-Arg attenuated the
LPS-induced increase in both CINC-1 and MIP-2 messages.
D-Arg had no effect on the LPS-induced increase in either
CINC-1 or MIP-2 message. Similar to the results obtained for chemokine
protein, NOS inhibition (AMT) before L-Arg and LPS
abolished the L-Arg-associated attenuation of CINC-1 and
MIP-2 mRNA expression.
|
Effect of L-Arg on lung IB-
.
Compared with saline, LPS caused a significant decrease in I
B-
within 1 h, which returned to saline control levels by 4 h
(Fig. 4, A and B).
Densitometry of Western blots from three separate experiments revealed
no significant difference between the 0.5- and 4-h time points in the
LPS-treated animals and no difference between saline control and the
LPS-treated 4-h time point (data not shown). Therefore, to determine
the effect of L-Arg on I
B-
after LPS, rat lung tissue
was examined for I
B-
1 h after LPS. L-Arg before
LPS attenuated the LPS-induced decrease in I
B-
at 1 h (Fig.
4, C and D). D-Arg had no effect on
the level of I
B-
protein after LPS. NOS inhibition
prevented the effects of L-Arg on I
B-
levels after
LPS. AMT alone had a similar effect to saline-treated animals (data not
shown).
|
Effect of L-Arg on lung NF-B DNA binding.
To determine the effect of L-Arg on NF-
B DNA binding
after LPS, rat lung tissue was examined 1 h after LPS for NF-
B
DNA binding by EMSA (Fig. 5). Compared
with saline treatment, LPS challenge caused a significant increase in
NF-
B DNA binding within 1 h. L-Arg given before LPS
attenuated the LPS-induced NF-
B DNA binding. D-Arg had
no effect on LPS-induced NF-
B DNA binding. Treatment with AMT before
L-Arg and LPS abolished the effect of L-Arg.
Administration of AMT alone had a similar effect to that in
saline-treated animals.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sheridan et al. (51) previously found that
L-Arg decreases lung PMN accumulation and pulmonary
vascular injury after LPS (51). Our present studies tested
the effect of L-Arg treatment before LPS on chemokine
production in the lung. Northern blot and ELISA analysis of lung
homogenates confirmed that a low dose of LPS caused an increase in lung
CINC-1 and MIP-2 proteins and mRNAs as early as 2 h after LPS.
L-Arg attenuated both chemokine mRNA expression and protein
production in the lung after LPS. Furthermore, we attempted to
determine the mechanism whereby L-Arg decreases chemokine
production in this model. The attenuation of chemokine production by
L-Arg was associated with maintenance of IB-
levels
and a decrease in NF-
B DNA binding. Finally, inhibition of NOS
abolished the effects of L-Arg on LPS-induced chemokine
mRNA and protein production, I
B-
levels, and NF-
B DNA binding.
L-Arg is the sole substrate for NO synthesis in mammalian
systems. The protective anti-inflammatory role of L-Arg or
NO in the lung has been evaluated in a number of studies. In animals, previous investigations have demonstrated that L-Arg or NO
alleviates the pulmonary hypertension seen after mesenteric
ischemia-reperfusion (16). Furthermore,
L-Arg or NO decreases alveolar macrophage proinflammatory
cytokine (tumor necrosis factor-, IL-1
) production (34), prevents alveolar edema (25), and
attenuates PMN accumulation after LPS (20, 51). Inhaled NO
also decreases PMN recruitment and attenuates lung injury in severe
experimental hyaline membrane disease (27). Neutrophil
accumulation in tissues is a complex process involving the production
of chemokines (7, 13, 21) as well as PMN-endothelial cell
interactions mediated by adhesion molecules (3). In vitro
studies suggest that NO decreases chemokine production by the
endothelium (15), vascular smooth muscle
(56), and alveolar macrophages (34, 54).
Additional in vitro data demonstrate that NO reduces PMN
2-integrin (45) and endothelial cell
P-selectin and intercellular adhesion molecule (ICAM)-1 (4, 32) expression. In patients with acute respiratory distress syndrome (ARDS), inhaled NO decreases PMN
2-integrin
(CD11b/CD18) expression and bronchoalveolar lavage fluid IL-8 levels
(10). The in vivo physiological implications of these data
are not completely understood. Thus data from the current study,
previous work by this laboratory (16, 51), and that of
other investigators (20, 45) support the
hypothesis that the L-Arg/NO pathway regulates PMN tissue
sequestration by downregulation of both chemokine production and
adhesion molecule expression.
Despite the beneficial role of NO in ALI, NO may contribute to lung
injury by generation of oxidant by-products. Generation of
peroxynitrite from the reaction of NO and superoxide can contribute to
tissue damage after sepsis (18, 44, 58). Specifically, there is evidence to suggest that the liberation of NO by inducible NOS
(iNOS) leads to tissue damage and contributes to lung injury (24). In fact, iNOS inhibition attenuates LPS-induced ALI
in rabbits (36) and dogs (41) and hemorrhagic
shock-induced ALI in mice and rats (24). However,
experiments utilizing iNOS knockout [(/
)] mice to substantiate
the pathological role of iNOS in systemic inflammation give conflicting
results. In support of iNOS as an effector of sustained inflammation
and tissue damage, iNOS(
/
) mice demonstrate reduced levels of
NF-
B and signal transducer and activation of transcription-3 (STAT3)
activation in the lung after hemorrhagic shock (24).
Furthermore, Kristof et al. (28) found that LPS injection
in iNOS (
/
) mice elicited no significant change in lactate
dehydrogenase activity, lung wet-to-dry weight ratio, or pulmonary
nitrotyrosine staining compared with wild-type mice (28).
On the other hand, iNOS inhibition or gene deletion may increase tissue
PMN accumulation. Hickey et al. (23) found that leukocyte
accumulation in the lung was elevated in iNOS(/
) mice given
systemic LPS, suggesting that induction of iNOS is a homeostatic regulator for leukocyte recruitment. Thus, early in the course of
LPS-induced pulmonary inflammation, NO may act by a negative feedback
mechanism to inhibit the production of chemokines and thereby attenuate
lung PMN accumulation. Indeed, one group of investigators
(1) has evaluated the role of iNOS in PMN accumulation in
a murine model of peritonitis induced by zymosan. In iNOS(
/
) mice,
increased PMN accumulation after zymosan injection was associated with
increased peritoneal levels of MIP-2 and KC (a murine C-X-C chemokine)
(1). Furthermore, in a murine model of endotoxin-induced ALI, NOS inhibition increased proinflammatory cytokine protein and mRNA
expression and NF-
B DNA binding (57).
The data in this study suggest that the anti-inflammatory effects of
L-Arg are mediated by NOS because NOS inhibition in the presence of supplemental L-Arg prevented the attenuation of
chemokine production after LPS. We chose AMT given its reported 20-fold selectivity for iNOS inhibition over endothelial NOS (eNOS)
(55). However, the constitutive NOS enzymes may regulate
PMN accumulation after a proinflammatory stimulus. Indeed, Lefer
et al. (31) found that transmigration of PMNs into the
peritoneum of neuronal NOS(/
) and eNOS(
/
) mice was increased
after thioglycolate injection. Additionally, NO derived from eNOS may
play a protective role in LPS-induced ALI (19). Despite
the reported high degree of selectivity of AMT for iNOS, it is likely
that AMT also inhibited the constitutive NOS isoform. This is
further substantiated by the fact that AMT inhibited the production of
NO in the saline control animals.
Transcription factor activation is a pivotal step in the
pathophysiology of ARDS. Indeed, consensus sequences for NF-B have been identified in the promoter regions of several genes (TNF-
, IL-1, and ICAM-1) that have been implicated in the pathogenesis of ARDS
(37). NF-
B is activated and bound to DNA in alveolar macrophages from patients with ARDS (37, 48). Furthermore, inhibition of NF-
B DNA binding has been shown to be protective against lung injury in animal studies (40). I
B-
regulates the activation of NF-
B by preventing the latter from
translocating into the nucleus and affecting gene transcription. Loss
of I
B-
has been associated with NF-
B activation and
proinflammatory gene transcription in models of ALI (38).
Previous studies have demonstrated that transcription of both CINC-1
(5) and MIP-2 (42) are NF-
B dependent.
Multiple in vitro studies have investigated the inhibitory effect of NO
on the IB-
-NF-
B complex. First, endogenously produced NO may
act to inhibit the degradation of I
B-
. In vitro, NO donors inhibit NF-
B activation by blocking I
B-
phosphorylation (and subsequent degradation) in rat vascular smooth muscle cells (26, 43). Second, NO may act to induce an increase in I
B-
mRNA transcription independent of NF-
B activation. Despite attenuation of
NF-
B DNA binding in human vascular smooth muscle cells after TNF-
in the presence of NO donors, I
B-
mRNA expression was increased
(43). Third, NO may enhance I
B-
promoter activity. NO, but not antioxidants, induced human endothelial cell I
B-
promoter activity in vitro (52). And finally, NO may act
to inhibit nuclear translocation of NF-
B independent of I
B-
degradation. In murine macrophages, NO leads to S-nitrosylation of the
NF-
B p50 subunit and inhibition of NF-
B-dependent DNA binding,
promoter activity, and gene transcription (12). This
regulatory action of NO is revealed by in vivo NOS inhibition after
systemic LPS, which increases proinflammatory cytokine protein and mRNA
expression by a NF-
B-dependent mechanism (57). Our
results suggest that, in vivo, supplemental L-Arg acts to
inhibit chemokine gene expression by prevention of NF-
B DNA binding
and preservation of normal I
B-
levels early in the course of sepsis.
In summary, this work demonstrates that in vivo provision of the NO
substrate L-Arg in a rat model of lung injury prevents chemokine production by the maintenance of IB-
levels and the attenuation of NF-
B DNA binding. Our findings corroborate previously reported in vitro data (34) regarding the effects
of NO on the production of proinflammatory mediators and elucidate a
mechanism for the attenuation of PMN accumulation in this model of
LPS-induced lung injury. Provision of L-Arg early in the
course of systemic inflammation may act to downregulate the
inflammatory response via a NO-dependent mechanism before the tissue
damage that occurs as the result of PMN accumulation and free radical production.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Lihua Ao for technical assistance with immunoblotting and Alex Poole for assistance in Northern blotting.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of General Medical Sciences Grant GM-4922 and National Institute of Child Health and Human Development Grant R03-HD-36256-01.
Casey M. Calkins is the Kiwanis Trauma Research Fellow.
Address for reprint requests and other correspondence: R. C. McIntyre, Jr., Dept. of Surgery, Univ. of Colorado Health Sciences Center, 4200 East Ninth Ave., Campus Box C-313, Denver, CO 80262 (E-mail: robert.mcintyre{at}uchsc.edu).
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. Section 1734 solely to indicate this fact.
Received 8 May 2000; accepted in final form 20 September 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ajuebor, MN,
Virag L,
Flower RJ,
Perretti M,
and
Szabo C.
Role of inducible nitric oxide synthase in the regulation of neutrophil migration in zymosan-induced inflammation.
Immunology
95:
625-630,
1998[ISI][Medline].
2.
Angele, MK,
Smail N,
Knoferl MW,
Ayala A,
Cioffi WG,
and
Chaudry IH.
L-Arginine restores splenocyte functions after trauma and hemorrhage potentially by improving splenic blood flow.
Am J Physiol Cell Physiol
276:
C145-C151,
1999
3.
Barnett, CC, Jr,
Moore EE,
Mierau GW,
Partrick DA,
Biffl WL,
Elzi DJ,
and
Silliman CC.
ICAM-1-CD18 interaction mediates neutrophil cytotoxicity through protease release.
Am J Physiol Cell Physiol
274:
C1634-C1644,
1998
4.
Biffl, WL,
Moore EE,
Moore FA,
and
Barnett C.
Nitric oxide reduces endothelial expression of intercellular adhesion molecule (ICAM)-1.
J Surg Res
63:
328-332,
1996[ISI][Medline].
5.
Blackwell, TS,
Blackwell TR,
Holden EP,
Christman BW,
and
Christman JW.
In vivo antioxidant treatment suppresses nuclear factor-kappa B activation and neutrophilic lung inflammation.
J Immunol
157:
1630-1637,
1996[Abstract].
6.
Blackwell, TS,
Holden EP,
Blackwell TR,
DeLarco JE,
and
Christman JW.
Cytokine-induced neutrophil chemoattractant mediates neutrophilic alveolitis in rats: association with nuclear factor kappa B activation.
Am J Respir Cell Mol Biol
11:
464-472,
1994[Abstract].
7.
Bless, NM,
Warner RL,
Padgaonkar VA,
Lentsch AB,
Czermak BJ,
Schmal H,
Friedl HP,
and
Ward PA.
Roles for C-X-C chemokines and C5a in lung injury after hindlimb ischemia-reperfusion.
Am J Physiol Lung Cell Mol Physiol
276:
L57-L63,
1999
8.
Brown, Z,
Robson RL,
and
Westwick J.
L-Arginine/nitric oxide pathway: a possible signal transduction mechanism for the regulation of the chemokine IL-8 in human mesangial cells.
Adv Exp Med Biol
351:
65-75,
1993[Medline].
9.
Carey, LA,
Perkowski SZ,
Lipsky CL,
Cirelli RA,
Spath JA, Jr,
and
Gee MH.
Neutrophil recruitment as a factor limiting injury or promoting recovery from acute lung injury.
Am J Physiol Heart Circ Physiol
272:
H279-H289,
1997
10.
Chollet-Martin, S,
Gatecel C,
Kermarrec N,
Gougerot-Pocidalo MA,
and
Payen DM.
Alveolar neutrophil functions and cytokine levels in patients with the adult respiratory distress syndrome during nitric oxide inhalation.
Am J Respir Crit Care Med
153:
985-990,
1996[Abstract].
11.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
12.
DelaTorre, A,
Schroeder RA,
Punzalan C,
and
Kuo PC.
Endotoxin-mediated S-nitrosylation of p50 alters NF-kappa B-dependent gene transcription in ANA-1 murine macrophages.
J Immunol
162:
4101-4108,
1999
13.
Donnelly, SC,
Strieter RM,
Kunkel SL,
Walz A,
Robertson CR,
Carter DC,
Grant IS,
Pollok AJ,
and
Haslett C.
Interleukin-8 and development of adult respiratory distress syndrome in at-risk patient groups.
Lancet
341:
643-647,
1993[ISI][Medline].
14.
Fan, J,
Marshall JC,
Jimenez M,
Shek PN,
Zagorski J,
and
Rotstein OD.
Hemorrhagic shock primes for increased expression of cytokine-induced neutrophil chemoattractant in the lung: role in pulmonary inflammation after lipopolysaccharide.
J Immunol
161:
440-447,
1998
15.
Fowler, AA, III,
Fisher BJ,
Sweeney LB,
Wallace TJ,
Natarajan R,
Ghosh SS,
and
Ghosh S.
Nitric oxide regulates interleukin-8 gene expression in activated endothelium by inhibiting NF-kappaB binding to DNA: effects on endothelial function.
Biochem Cell Biol
77:
201-208,
1999[ISI][Medline].
16.
Fullerton, DA,
Eisenach JH,
McIntyre RC, Jr,
Friese RS,
Sheridan BC,
Roe GB,
Agrafojo J,
Banerjee A,
and
Harken AH.
Inhaled nitric oxide prevents pulmonary endothelial dysfunction after mesenteric ischemia-reperfusion.
Am J Physiol Lung Cell Mol Physiol
271:
L326-L331,
1996
17.
Fullerton, DA,
McIntyre RC, Jr,
Hahn AR,
Agrafojo J,
Koike K,
Meng X,
Banerjee A,
and
Harken AH.
Dysfunction of cGMP-mediated pulmonary vasorelaxation in endotoxin-induced acute lung injury.
Am J Physiol Lung Cell Mol Physiol
268:
L1029-L1035,
1995
18.
Gagnon, C,
Leblond FA,
and
Filep JG.
Peroxynitrite production by human neutrophils, monocytes and lymphocytes challenged with lipopolysaccharide.
FEBS Lett
431:
107-110,
1998[ISI][Medline].
19.
Gryglewski, RJ,
Wolkow PP,
Uracz W,
Janowska E,
Bartus JB,
Balbatun O,
Patton S,
Brovkovych V,
and
Malinski T.
Protective role of pulmonary nitric oxide in the acute phase of endotoxemia in rats.
Circ Res
82:
819-827,
1998
20.
Guidot, DM,
Hybertson BM,
Kitlowski RP,
and
Repine JE.
Inhaled NO prevents IL-1-induced neutrophil accumulation and associated acute edema in isolated rat lungs.
Am J Physiol Lung Cell Mol Physiol
271:
L225-L229,
1996
21.
Harada, A,
Sekido N,
Akahoshi T,
Wada T,
Mukaida N,
and
Matsushima K.
Essential involvement of interleukin-8 (IL-8) in acute inflammation.
J Leukoc Biol
56:
559-564,
1994[Abstract].
22.
Henderson, GS,
Conary JT,
Davidson JM,
Stewart SJ,
House FS,
and
McCurley TL.
A reliable method for Northern blot analysis using synthetic oligonucleotide probes.
Biotechniques
10:
190-197,
1991[ISI][Medline].
23.
Hickey, MJ,
Sharkey KA,
Sihota EG,
Reinhardt PH,
Macmicking JD,
Nathan C,
and
Kubes P.
Inducible nitric oxide synthase-deficient mice have enhanced leukocyte-endothelium interactions in endotoxemia.
FASEB J
11:
955-964,
1997
24.
Hierholzer, C,
Harbrecht B,
Menezes JM,
Kane J,
MacMicking J,
Nathan CF,
Peitzman AB,
Billiar TR,
and
Tweardy DJ.
Essential role of induced nitric oxide in the initiation of the inflammatory response after hemorrhagic shock.
J Exp Med
187:
917-928,
1998
25.
Hinder, F,
Stubbe HD,
Van Aken H,
Waurick R,
Booke M,
and
Meyer J.
Role of nitric oxide in sepsis-associated pulmonary edema.
Am J Respir Crit Care Med
159:
252-257,
1999
26.
Katsuyama, K,
Shichiri M,
Marumo F,
and
Hirata Y.
NO inhibits cytokine-induced iNOS expression and NF-B activation by interfering with phosphorylation and degradation of I
B-
.
Arterioscler Thromb Vasc Biol
18:
1796-1802,
1998
27.
Kinsella, JP,
Parker TA,
Galan H,
Sheridan BC,
Halbower AC,
and
Abman SH.
Effects of inhaled nitric oxide on pulmonary edema and lung neutrophil accumulation in severe experimental hyaline membrane disease.
Pediatr Res
41:
457-463,
1997[Abstract].
28.
Kristof, AS,
Goldberg P,
Laubach V,
and
Hussain SN.
Role of inducible nitric oxide synthase in endotoxin-induced acute lung injury.
Am J Respir Crit Care Med
158:
1883-1889,
1998
29.
Kubes, P,
Kurose I,
and
Granger DN.
NO donors prevent integrin-induced leukocyte adhesion but not P-selectin-dependent rolling in postischemic venules.
Am J Physiol Heart Circ Physiol
267:
H931-H937,
1994
30.
Lefer, AM,
and
Lefer DJ.
The role of nitric oxide and cell adhesion molecules on the microcirculation in ischaemia-reperfusion.
Cardiovasc Res
32:
743-751,
1996[ISI][Medline].
31.
Lefer, DJ,
Jones SP,
Girod WG,
Baines A,
Grisham MB,
Cockrell AS,
Huang PL,
and
Scalia R.
Leukocyte-endothelial cell interactions in nitric oxide synthase-deficient mice.
Am J Physiol Heart Circ Physiol
276:
H1943-H1950,
1999
32.
Liu, P,
Xu B,
Hock CE,
Nagele R,
Sun FF,
and
Wong PY.
NO modulates P-selectin and ICAM-1 mRNA expression and hemodynamic alterations in hepatic I/R.
Am J Physiol Heart Circ Physiol
275:
H2191-H2198,
1998
33.
Matthews, JR,
Botting CH,
Panico M,
Morris HR,
and
Hay RT.
Inhibition of NF-B DNA binding by nitric oxide.
Nucleic Acids Res
24:
2236-2242,
1996
34.
Meldrum, DR,
McIntyre RC,
Sheridan BC,
Cleveland JC, Jr,
Fullerton DA,
and
Harken AH.
L-Arginine decreases alveolar macrophage proinflammatory monokine production during acute lung injury by a nitric oxide synthase-dependent mechanism.
J Trauma
43:
888-893,
1997[ISI][Medline].
35.
Meyrick, B,
and
Brigham KL.
Acute effects of Escherichia coli endotoxin on the pulmonary microcirculation of anesthetized sheep structure: function relationships.
Lab Invest
48:
458-470,
1983[ISI][Medline].
36.
Mikawa, K,
Nishina K,
Tamada M,
Takao Y,
Maekawa N,
and
Obara H.
Aminoguanidine attenuates endotoxin-induced acute lung injury in rabbits.
Crit Care Med
26:
905-911,
1998[ISI][Medline].
37.
Moine, P,
McIntyre R,
Schwartz MD,
Kaneko D,
Shenkar R,
Le Tulzo Y,
Moore EE,
and
Abraham E.
NF-B regulatory mechanisms in alveolar macrophages from patients with acute respiratory distress syndrome.
Shock
13:
85-91,
2000[ISI][Medline].
38.
Moine, P,
Shenkar R,
Kaneko D,
Le Tulzo Y,
and
Abraham E.
Systemic blood loss affects NF-B regulatory mechanisms in the lungs.
Am J Physiol Lung Cell Mol Physiol
273:
L185-L192,
1997
39.
Moncada, S,
Palmer RM,
and
Higgs EA.
Biosynthesis of nitric oxide from L-arginine. A pathway for the regulation of cell function and communication.
Biochem Pharmacol
38:
1709-1715,
1989[ISI][Medline].
40.
Nathens, AB,
Bitar R,
Davreux C,
Bujard M,
Marshall JC,
Dackiw AP,
Watson RW,
and
Rotstein OD.
Pyrrolidine dithiocarbamate attenuates endotoxin-induced ALI.
Am J Respir Cell Mol Biol
17:
608-616,
1997
41.
Numata, M,
Suzuki S,
Miyazawa N,
Miyashita A,
Nagashima Y,
Inoue S,
Kaneko T,
and
Okubo T.
Inhibition of inducible nitric oxide synthase prevents LPS-induced acute lung injury in dogs.
J Immunol
160:
3031-3037,
1998
42.
Ouaaz, F,
Li M,
and
Beg AA.
A critical role for the RelA subunit of nuclear factor kappaB in regulation of multiple immune-response genes and in Fas-induced cell death.
J Exp Med
189:
999-1004,
1999
43.
Peng, HB,
Libby P,
and
Liao JK.
Induction and stabilization of I kappa B alpha by nitric oxide mediates inhibition of NF-B.
J Biol Chem
270:
14214-14219,
1995
44.
Pulido, EJ,
Shames BD,
Selzman CH,
Barton HA,
Banerjee A,
Bensard DD,
and
McIntyre RC, Jr.
Inhibition of PARS attenuates endotoxin-induced dysfunction of pulmonary vasorelaxation.
Am J Physiol Lung Cell Mol Physiol
277:
L769-L776,
1999
45.
Sato, Y,
Walley KR,
Klut ME,
English D,
D'Yachkova Y,
Hogg JC,
and
van Eeden SF.
Nitric oxide reduces the sequestration of polymorphonuclear leukocytes in lung by changing deformability and CD18 expression.
Am J Respir Crit Care Med
159:
1469-1476,
1999
46.
Schmal, H,
Shanley TP,
Jones ML,
Friedl HP,
and
Ward PA.
Role for macrophage inflammatory protein-2 in lipopolysaccharide-induced lung injury in rats.
J Immunol
156:
1963-1972,
1996[Abstract].
47.
Schreiber, E,
Matthias P,
Muller MM,
and
Schaffner W.
Rapid detection of octamer binding proteins with `mini-extracts' prepared from a small number of cells.
Nucleic Acids Res
17:
6419,
1989[ISI][Medline].
48.
Schwartz, MD,
Moore EE,
Moore FA,
Shenkar R,
Moine P,
Haenel JB,
and
Abraham E.
Nuclear factor-B is activated in alveolar macrophages from patients with acute respiratory distress syndrome.
Crit Care Med
24:
1285-1292,
1996[ISI][Medline].
49.
Shanley, TP,
Schmal H,
Warner RL,
Schmid E,
Friedl HP,
and
Ward PA.
Requirement for C-X-C chemokines (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant) in IgG immune complex-induced lung injury.
J Immunol
158:
3439-3448,
1997[Abstract].
50.
Sheridan, BC,
McIntyre RC,
Agrafojo J,
Meldrum DR,
Meng X,
and
Fullerton DA.
Neutrophil depletion attenuates endotoxin-induced dysfunction of cGMP-mediated pulmonary vasorelaxation.
Am J Physiol Lung Cell Mol Physiol
271:
L820-L828,
1996
51.
Sheridan, BC,
McIntyre RC, Jr,
Meldrum DR,
and
Fullerton DA.
L-Arginine prevents lung neutrophil accumulation and preserves pulmonary endothelial function after endotoxin.
Am J Physiol Lung Cell Mol Physiol
274:
L337-L342,
1998
52.
Spiecker, M,
Darius H,
Kaboth K,
Hubner F,
and
Liao JK.
Differential regulation of endothelial cell adhesion molecule expression by nitric oxide donors and antioxidants.
J Leukoc Biol
63:
732-739,
1998[Abstract].
53.
Tang, WW,
Yi ES,
Remick DG,
Wittwer A,
Yin S,
Qi M,
and
Ulich TR.
Intratracheal injection of endotoxin and cytokines. IX. Contribution of CD11a/ICAM-1 to neutrophil emigration.
Am J Physiol Lung Cell Mol Physiol
269:
L653-L659,
1995
54.
Thomassen, MJ,
Buhrow LT,
Connors MJ,
Kaneko FT,
Erzurum SC,
and
Kavuru MS.
Nitric oxide inhibits inflammatory cytokine production by human alveolar macrophages.
Am J Respir Cell Mol Biol
17:
279-283,
1997
55.
Tracey, WR,
Nakane M,
Basha F,
and
Carter G.
In vivo pharmacological evaluation of two novel type II (inducible) nitric oxide synthase inhibitors.
Can J Physiol Pharmacol
73:
665-669,
1995[ISI][Medline].
56.
Tsao, PS,
Wang B,
Buitrago R,
Shyy JY,
and
Cooke JP.
Nitric oxide regulates monocyte chemotactic protein-1.
Circulation
96:
934-940,
1997
57.
Walley, KR,
McDonald TE,
Higashimoto Y,
and
Hayashi S.
Modulation of proinflammatory cytokines by nitric oxide in murine acute lung injury.
Am J Respir Crit Care Med
160:
698-704,
1999
58.
Wizemann, TM,
Gardner CR,
Laskin JD,
Quinones S,
Durham SK,
Goller NL,
Ohnishi ST,
and
Laskin DL.
Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia.
J Leukoc Biol
56:
759-768,
1994[Abstract].