Center for Surgical Research and Department of Surgery, Brown University School of Medicine and Rhode Island Hospital, Providence, Rhode Island 02903
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Regional hypoxia,
associated with hemorrhage, is thought to induce a variety of
alterations in immune cell function, including upregulation of
macrophage-inducible nitric oxide synthase (iNOS) expression and
activity (NO production). Furthermore, NO may cause immune cell
dysfunction similar to that associated with hemorrhagic shock. However,
it remains unknown whether hypoxia per se in the absence of any blood
loss is a sufficient stimulus to cause iNOS expression and NO
production by macrophages. To study this, male Sprague-Dawley rats
(275-325 g) were placed in a plastic box flushed with a gas
mixture containing 5% O2-95%
N2 for 60 min. Peritoneal and
splenic macrophages were isolated 0-5.5 h thereafter, and blood
samples were obtained. Nitrite and nitrate (stable degradation products
of NO) production by splenic and peritoneal macrophages cultured for 48 h was significantly increased 3 and 5.5 h after hypoxemia. The increase
in NO production by macrophages was preceded by elevated expression of
iNOS mRNA at 1.5 h after hypoxia. Additionally, interferon-
(IFN-
) levels in plasma from rats subjected to hypoxemia were
significantly elevated soon after the insult (0-1.5 h
posthypoxemia), suggesting a causal relationship between IFN-
production and upregulation of iNOS activity. We propose that a
hypoxemia-induced increase in macrophage iNOS activity following
hemorrhage may in part be responsible for the observed immune
dysfunction. Thus attempts to suppress macrophage iNOS activity after
this form of trauma may be helpful in improving immune function under
those conditions.
inducible nitric oxide synthase; nitric oxide; interferon; hemorrhagic shock; splenic macrophages; peritoneal macrophages
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
STUDIES INDICATE THAT trauma and hemorrhagic shock lead
to profound depression of both immunoresponsiveness and organ function (9, 38). Additionally, it has been demonstrated that decreased tissue
perfusion during hemorrhagic shock significantly reduces tissue
oxygenation, leading to decreased intracellular ATP levels under such
conditions (20). Furthermore, there is evidence that regional hypoxia
is responsible for initiating the cascade of events leading to the
observed alterations in cellular and organ metabolism following
trauma-hemorrhage (9). In this regard, hypoxemia in the absence of
blood loss or significant hypotension has been demonstrated to induce
release of proinflammatory cytokines [tumor necrosis factor-
(TNF-
), interleukin (IL)-1
, and IL-6] by macrophages (13).
Increased synthesis of these proinflammatory cytokines has been
implicated in both the pathophysiology and the mortality associated
with traumatic and thermal injury (12, 28, 36). Thus, after injury,
tissue hypoxia may in itself be a sufficient stimulus to induce
activation of macrophage proinflammatory cytokine synthesis, which in
turn contributes to the systemic inflammatory response.
In addition to proinflammatory cytokines, macrophages are also a major
cellular source of nitric oxide (NO) (18). These cells synthesize the
free radical NO from the guanidino group of
L-arginine via the inducible
form of nitric oxide synthase (iNOS) that is upregulated by
proinflammatory stimuli such as TNF-, interferon-
(IFN-
), or
lipopolysaccharide (LPS). Recent studies indicate that plasma nitrite
and nitrate
(NO
2/NO
3; the stable end products of NO) are significantly increased following adverse circulatory conditions, such as trauma-hemorrhage, sepsis, and
thermal injury (6, 11, 25, 31, 34). Additionally, increased iNOS
expression has been reported under these pathophysiology conditions
(16), suggesting that the increased production of NO is of iNOS origin.
Elevated NO production has been implicated in cellular and organ
dysfunction seen under these conditions (6, 11, 25, 27, 34). However,
it remains unknown whether hypoxemia encountered during and after
hemorrhagic shock causes the induction of iNOS expression and activity.
Albina et al. (1) demonstrated that macrophages exposed to an in vitro
anoxic environment display increased iNOS expression and activity. In
view of this, it is possible that the regional hypoxemia during
hemorrhagic shock (in vivo) may be a sufficient stimulus to induce iNOS
expression and activity in macrophages. The aim of the present study,
therefore, was to determine whether hypoxemia per se increases NO
production by macrophages in vivo and, if so, whether this increase was
due to elevated iNOS expression.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals. In these studies, male Sprague-Dawley rats (260-325 g; Charles River Laboratories, Wilmington, MA) were utilized. All animals were fasted for 12 h before the experiment but were allowed water ad libitum. All procedures were carried out in accordance with the guidelines set forth in the Animal Welfare Act and the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the project was approved by the Institutional Animal Care and Use Committee of Rhode Island Hospital and Brown University.
Hypoxemia model. Hypoxemia was induced in the rats according to the procedure previously described by Ertel et al. (13), with some modifications. In brief, animals were placed in a plastic box (22 × 23 × 30 cm) with an inlet and an outlet through which the hypoxic gas mixture or room air flowed. Hypoxemia was induced by flushing the plastic box with a gas mixture of 5% O2-95% N2 bubbled through distilled water at a flow rate of ~10 l/min via a bottle containing distilled water. The animals were exposed to this hypoxic environment for 1 h, after which they were returned to room air. Previous studies from our laboratory demonstrated that this model of systemic hypoxemia results in a drop of the arterial PO2 to 40 mmHg after 10 min of hypoxemia (13). The arterial PO2 remained between 30 and 40 mmHg until hypoxemia was stopped, and it returned to baseline values within 10 min after the hypoxemia period (13). The blood pressure slowly decreased by 14 mmHg, and values returned to normal immediately after hypoxemia. No mortality was observed in the group of animals subjected to hypoxemia (13). Sham animals underwent the same procedure; however, the plastic box was flushed with room air. The animals (6 or 7 animals/time point) were killed at the end of hypoxia (0 h), 1.5, 3, or 5.5 h after completion of the hypoxic period by methoxyflurane overdose (Metofane, Pitman-Moore, Mundelein, IL). Peritoneal macrophages were harvested by peritoneal lavage, spleens were removed, and blood was obtained.
Plasma collection and storage.
Whole blood was obtained by cardiac puncture and placed in
microcentrifuge tubes (Microtainer, Becton Dickinson, Rutherford, NJ).
The tubes were then centrifuged at 16,000 g for 15 min at 4°C. Plasma was
separated, placed in pyrogen-free microcentrifuge tubes, immediately
frozen, and stored at 80°C until assayed.
Preparation of peritoneal and splenic macrophages.
Resident peritoneal macrophages were obtained from rats by peritoneal
lavage with PBS, and monolayers were established as previously
described (4, 41). The spleens were removed aseptically and placed in
separate petri dishes containing cold (4°C) PBS. The spleens were
dissociated by grinding, suspended in medium, and used to establish a
macrophage culture as previously described (41). The macrophage
monolayers were cultured in Click's medium containing 10% FCS for 48 h (at 37°C, 5% CO2, and 90%
humidity) without LPS and in the presence of LPS (10 µg/ml). Click's
medium contains 1,800 µM
L-arginine, compared with 225 µM L-arginine in rat plasma
(2). The availability of
L-arginine is therefore not
limited in the culture medium in vitro. Cell-free culture supernatants
were collected, aliquoted, and stored at 80°C until assayed
for
NO
2/NO
3 levels.
Determination of NO production.
Macrophage NO production was determined by measuring the concentration
of the stable NO degradation products
NO2/NO
3 in the supernatants using a colorimetric assay kit (Cayman Chemicals, Ann Arbor, MI) as previously described (31). Briefly, supernatant samples were thawed, and nitrate in the samples was converted to
nitrite by the addition of nitrate reductase. Nitrite concentration was
then detected by the addition of Griess reagent and quantitatively measured by analysis with a spectrophotometer (Bio-Tek ELK 311 microplate autoreader, Bio-Tek, Winooski, VT) at 550 nm.
RNA isolation and Northern blot analysis.
Total RNA from peritoneal macrophages was isolated using a single-step
liquid phase separation by TriPure isolation reagent (Boehringer
Mannheim, Mannheim, Germany) according to the manufacturer's recommendations. In brief, peritoneal macrophages were washed after 2 h
of incubation, 1 ml of TriPure reagent per 5 × 106 cells was added to the tissue
culture well, and the adherent cells were removed by scraping them from
the well into the reagent to lyse them. The cellular lysate was
homogenized by passing it through a pipette several times, transferred
into a polypropylene tube, and stored at 70°C until the
isolation process was continued. RNA was isolated from the thawed
samples by the addition of chloroform. All RNA samples had a ratio of
absorbance at 260 nm to absorbance at 280 nm that was
>1.5, and RNA content in all samples was found to be
similar by electrophoresis of 2 µg of total RNA in an agarose gel and
staining with ethidium bromide.
Cell line maintenance. The macrophage cell-line RAW 264.7 was obtained from the American Type Culture Collection and maintained according to their recommendations.
Determination of plasma IFN- levels.
IFN-
levels in plasma were determined using a specific bioassay
(23). Serial dilutions of plasma were added to RAW 264.7 cells and
incubated at 37°C in 5% CO2
for 24 h. Cell-free supernatants were collected, and nitrite
concentration was determined by the Griess reaction. The light
absorbance was measured at 550 nm with an automated microplate reader
(EL-311, Bio-Tek). The IFN-
concentration in the samples was
determined from a standard curve generated with recombinant IFN-
(Genzyme, Cambridge, MA).
Statistical analysis. Data are presented as means ± SE of six or seven animals per group unless otherwise indicated. One-way ANOVA followed by the Student-Newman-Keuls test as a post hoc test for multiple comparisons was used to determine the significance of the differences between experimental means. P < 0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hypoxemia induces NO production by macrophages.
The
NO2/NO
3
concentrations in the supernatants of nonstimulated peritoneal
macrophages (Fig.
1A)
harvested at the end of hypoxia (0 h) and 1.5 h after the end of
hypoxia were comparable to those of sham animals. However, peritoneal
macrophages harvested 3 or 5.5 h after the end of hypoxia produced
significantly greater levels of
NO
2/NO
3 than macrophages from sham animals.
NO
2/NO
3 concentrations in the supernatants of macrophages harvested from rats 3 h after the end of hypoxia were increased 1.25-fold compared with shams
(P < 0.05), whereas supernatants
from macrophages harvested 5.5 h posthypoxia displayed a 1.6-fold
increase in
NO
2/NO
3 concentration (P < 0.05 compared
with shams). Although there was a trend toward a higher concentration
of
NO
2/NO
3 in the supernatants of macrophages harvested 5.5 h posthypoxia compared
with 3 h posthypoxia, the values were not significantly different.
|
|
Macrophage iNOS mRNA is expressed following hypoxemia.
The levels of iNOS mRNA in nonstimulated peritoneal macrophages was
determined by Northern blot analysis. mRNA for iNOS was not expressed
in macrophages from sham-treated rats or macrophages harvested
immediately after the end of hypoxia (Fig.
3). However, peritoneal macrophages
harvested 1.5 or 3 h after the end of hypoxia showed increased
expression of mRNA for iNOS at 130.5 kb (comparable to the positive
control).
|
Hypoxia induces increased levels of circulating
IFN-.
Plasma levels of IFN-
(Fig. 4) were
significantly increased at the end of the hypoxic period (0 h) compared
with sham animals (+202.7%, P < 0.05), which remained significantly elevated 1.5 h after the end of
hypoxia (P < 0.05 compared with
shams). By 5.5 h after the end of hypoxia, plasma IFN-
had returned
to sham levels.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The experiments reported here were conducted to investigate the role of
severe hypoxemia in vivo in modulating macrophage iNOS expression and
activity. Hypoxemia has been shown to induce the expression and release
of proinflammatory cytokines favoring the adaptation of the organism to
hypoxic conditions (13, 14). A similar elevation of circulatory
proinflammatory cytokines has been reported following hemorrhagic shock
(10), elective surgery (30), severe thermal injury (19), or traumatic
injury (35). Moreover, recent studies have shown that after hemorrhagic
shock there are increased plasma
NO2/NO
3 levels (31). This increase in plasma
NO
2/NO
3 appears to be due to induction of iNOS, since iNOS expression has been
shown in a model of prolonged hemorrhagic shock 5 h after the beginning
of the hypotensive period (16). However, it remains unknown which
factor(s) mediate the induction of iNOS expression and activity
following hemorrhagic shock. In light of the observations that
macrophages exposed to hypoxic conditions in vitro express iNOS
activity (1) and since regional hypoxia is a hallmark of hemorrhagic
shock, we postulated that hypoxia in vivo, in the absence of blood
loss, induces iNOS activity in macrophages and therefore represents a
potential mechanism by which iNOS expression and activity is
upregulated following hemorrhagic shock.
The results of the present study indicate that expression of iNOS mRNA
in macrophages was detectable after in vivo hypoxia, leading to
increased NO production in vitro. Furthermore, our findings also
indicate that hypoxemia caused an increase in circulating IFN- soon
after the insult and before the expression of iNOS mRNA, thus
suggesting a causal relationship. All animals in the present study were
subjected to severe hypoxia for 1 h; therefore, it remains unknown what
degree or duration of hypoxia is required to induce iNOS expression and
increase macrophage NO production.
Several in vitro studies have examined the effect of hypoxia on iNOS
expression by macrophages (1, 22). These in vitro studies show that
hypoxia alone does not induce iNOS expression and indicate that
reoxygenation is required, along with a secondary stimulus such as
IFN- or LPS, for the induction of iNOS. The results of the study
presented here are consistent with these in vitro findings, since iNOS
mRNA expression was not observed in macrophages isolated immediately
following hypoxemia but was present 1.5 h posthypoxemia. This would
suggest that reoxygenation is necessary for the induction of iNOS mRNA
expression. Additionally, the elevated IFN-
levels in the plasma
during the early posthypoxic period may provide the secondary stimuli
required for the induction of iNOS, as indicated by in vitro
experiments (22).
Recent studies of Melillo et al. (21) have shown that the iNOS gene
promoter sequence contains a hypoxia response element that in
combination with IFN- leads to the activation of iNOS gene
transcription. In this regard, hypoxia in combination with IFN-
may
have induced iNOS transcription and mRNA expression in the present
study. These results further support the hypothesis that the increased
plasma IFN-
levels observed early posthypoxemia may contribute to
the induction of iNOS expression and activity in the present study.
Furthermore, the identification of a functional hypoxia-responsive
sequence in the iNOS promoter suggests that iNOS is a hypoxia-inducible
gene (21).
The nuclear transcription factor NF-B is also likely to be important
to the induction of iNOS expression following hypoxemia, as it has been
shown to be required for activation of iNOS gene expression in
macrophages (40) and is also activated under hypoxic conditions (24).
Reactive oxygen species that are generated during hypoxemia can
activate NF-
B translocation to the nucleus by inducing the
disassociation of the inhibitory subunit I-
B by a tyrosine
kinase-dependent process (3, 33, 39). Furthermore, recent studies have
demonstrated that hemorrhagic shock also induces the activation of
NF-
B in immune cells (15, 26, 29). Although the present study did
not examine NF-
B, it is likely that it plays an important role in
the induction of iNOS expression following hypoxemia. Recent studies
indicate that NO inactivates NF-
B expression in endothelial cells,
which might reflect an adaptive negative feedback mechanism following
cell activation (32). A similar control mechanism has not yet been
identified in macrophages. Nonetheless, complete inhibition of NO might
be detrimental for the host following hypoxic conditions. This,
however, remains to be determined.
Because in vitro stimulation of macrophages with LPS induced similar
levels of NO production in all groups, the results presented here
suggest that the capacity of macrophages to produce NO in response to
LPS is not altered by hypoxemia. Furthermore, the observation that the
levels of NO production 3 and 5.5 h after hypoxemia, in the absence of
stimulation, were similar to LPS-stimulated levels of production
suggests that hypoxia is a sufficient stimulus to induce maximal NO
release. In contrast, Arya and Garcia (8) demonstrated both increased
constitutive and LPS-stimulated NO production by peritoneal macrophages
from animals that were subjected to hypoxia and reoxygenation 1 day
after an endotoxin challenge. However, the present observations that
the capacity of macrophages to produce NO is not altered by hypoxia
suggests that the effect is only at the level of iNOS expression rather
than involving altered enzyme kinetics. It remains unknown whether
endotoxin priming alters the kinetic parameters of iNOS or whether
hypoxia (in the absence of endotoxin) alters the capacity of
macrophages to produce NO at later times postinsult than those examined
in this study. Although previous studies have shown that hypoxia increases constitutive NO synthase (cNOS)-derived NO production (7),
macrophages do not express cNOS, and thus the increased NO2/NO
3
levels in the supernatants following hypoxia or LPS stimulation are
likely to be due to iNOS activity.
Melillo et al. proposed that hypoxia in combination with IFN-
provides signaling for the induction of iNOS gene expression (21),
whereas hypoxia alone is not sufficient to activate iNOS gene
expression. The present study supports these findings, as plasma
IFN-
levels were elevated early posthypoxemia, before iNOS
expression. However, the cellular source of the increased plasma
IFN-
is unknown. Studies by Klokker et al. (17) have demonstrated
that natural killer (NK) cell numbers and activity are increased
following hypoxia. NK cells are a major non-T lymphocyte source of
IFN-
(17). Studies to date have not addressed the effect of
hypoxemia on IFN-
production by T lymphocytes. It is, however,
possible that the increased levels of IFN-
observed in the present
study might be of NK cell origin as previously demonstrated by Klokker
et al. (17). Nonetheless, it remains unknown whether decreased
PO2 levels during hypoxemia directly
increase IFN-
release, leading to the induction of iNOS expression
in macrophages, or whether other factors are important. For example,
decreased intracellular ATP levels following hemorrhagic shock (20)
probably caused by regional hypoxia (9) may trigger a cascade of events
leading to iNOS expression. Thus the effect of hypoxemia on iNOS
expression might be mediated via decreased intracellular ATP levels.
Additionally, activation of other second messenger systems (i.e.,
cyclic nucleotides, protein kinase C, Ca2+) may also be important, as
they all have been shown to regulate iNOS activity (18). Further
experimental analysis is required to precisely identify the
mechanism(s) of iNOS induction following hypoxemia.
Recent studies from our laboratory indicate that PGE2 levels are increased after 1 h of severe hypoxia (37). PGE2 is known to be a potent vasodilator (37). The release of PGE2 therefore might be part of an adaptive response to hypoxemia, allowing more blood flow and oxygen delivery to the ischemic tissues. The release of cNOS-derived NO production also appears to be involved in the regulation of blood flow (5), whereas iNOS-derived NO production has been implicated in producing cellular and organ dysfunction (6, 11, 25, 27, 34). Because specific iNOS blockers are available, e.g., L-N6-(1-iminoethyl)lysine, administration of these agents might prevent the deleterious effects of iNOS-derived NO production without blocking the beneficial vasodilatory effects of cNOS-derived NO.
In summary, the results of the present study indicate that hypoxia per se in the absence of blood loss caused increased production of iNOS-derived NO by macrophages. Thus regional hypoxia during hemorrhagic shock may be responsible for the induction of iNOS under such conditions. Because elevated NO production has been implicated in producing cellular and organ dysfunction (6, 11, 25, 27, 34), therapeutic interventions that specifically depress iNOS activity following trauma and hemorrhagic shock may be helpful in decreasing morbidity and mortality after such conditions.
![]() |
ACKNOWLEDGEMENTS |
---|
This investigation was supported by National Institute of General Medical Sciences Grant R01-GM-37127.
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: I. H. Chaudry, Center for Surgical Research, Rhode Island Hospital, Middle House II, 593 Eddy St., Providence, RI 02903.
Received 6 July 1998; accepted in final form 2 October 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Albina, J. E.,
W. L. Henry, Jr.,
B. Mastrofrancesco,
B. A. Martin,
and
J. S. Reichner.
Macrophage activation by culture in an anoxic environment.
J. Immunol.
155:
4391-4396,
1995[Abstract].
2.
Albina, J. E.,
C. D. Mills,
W. L. Henry, Jr.,
and
M. D. Caldwell.
Temporal expression of different pathways of L-arginine metabolism in healing wounds.
J. Immunol.
144:
3877-3880,
1990
3.
Anderson, M.,
F. Staal,
C. Gitler,
and
L. A. Herzenberg.
Separation of oxidant-initiated and redox-regulated steps in the NF-B signal transduction pathway.
Proc. Natl. Acad. Sci. USA
91:
527-531,
1994.
4.
Angele, M. K.,
A. Ayala,
B. A. Monfils,
W. G. Cioffi,
K. I. Bland,
and
I. H. Chaudry.
Testosterone and/or low estradiol: normally required but harmful immunologically for males after trauma-hemorrhage.
J. Trauma
44:
78-85,
1998[Medline].
5.
Angele, M. K.,
N. Smail,
P. Wang,
W. G. Cioffi,
K. I. Bland,
and
I. H. Chaudry.
L-Arginine restores the depressed cardiac output and regional perfusion following trauma-hemorrhage.
Surgery
124:
394-401,
1998[Medline].
6.
Aranow, J. S.,
J. Zhuang,
H. Wang,
V. Larkin,
M. Smith,
and
M. P. Fink.
A selective inhibitor of inducible nitric oxide synthase prolongs survival in a rat model of bacterial peritonitis: comparison with two nonselective strategies.
Shock
5:
116-121,
1996[Medline].
7.
Arnet, U. A.,
A. McMillan,
J. L. Dinerman,
B. Ballerman,
and
C. J. Lowenstein.
Regulation of endothelial nitric-oxide synthase during hypoxia.
J. Biol. Chem.
271:
15069-15073,
1996
8.
Arya, G.,
and
V. F. Garcia.
Hypoxia/reoxygenation affects endotoxin tolerance.
J. Surg. Res.
59:
13-16,
1995[Medline].
9.
Chaudry, I. H.,
and
A. Ayala.
Immune consequences of hypovolemic shock and resuscitation.
Curr. Opin. Oncol.
6:
385-392,
1993.
10.
Chaudry, I. H.,
A. Ayala,
W. Ertel,
and
R. N. Stephan.
Hemorrhage and resuscitation: immunological aspects.
Am. J. Physiol.
259 (Regulatory Integrative Comp. Physiol. 28):
R663-R678,
1990
11.
Cuzzocrea, S.,
B. Zingarelli,
and
L. Sautebin.
Multiple organ failure following zymosan-induced peritonitis.
Shock
8:
268-275,
1998.
12.
Damas, P.,
D. Ledoux,
M. Nys,
Y. Vrindts,
D. De Groote,
P. Franchimont,
and
M. Lamy.
Cytokine serum level during severe sepsis in human IL-6 as a marker of severity.
Ann. Surg.
215:
356-362,
1992[Medline].
13.
Ertel, W.,
M. H. Morrison,
A. Ayala,
and
I. H. Chaudry.
Hypoxemia in the absence of blood loss or significant hypotension causes inflammatory cytokine release.
Am. J. Physiol.
269 (Regulatory Integrative Comp. Physiol. 38):
R160-R166,
1995
14.
Helfman, T.,
and
V. Falanga.
Gene expression in low oxygen tension.
Am. J. Med. Sci.
306:
37-42,
1993[Medline].
15.
Hierholzer, C.,
B. Harbrecht,
J. M. Menezes,
J. Kane,
J. MacMicking,
C. E. Nathan,
A. B. Peitzman,
T. R. Billiar,
and
D. J. Tweardey.
Essential role of induced nitric oxide in the initiation of the inflammatory response after hemorrhagic shock.
J. Exp. Med.
187:
917-928,
1998
16.
Kelly, E.,
N. S. Shah,
N. N. Morgan,
S. C. Watkins,
A. B. Peitzman,
and
T. R. Billiar.
Physiologic and molecular characterization of the role of nitric oxide in hemorrhagic shock: evidence that type II nitric oxide synthase does not regulate vascular decompensation.
Shock
7:
157-163,
1997[Medline].
17.
Klokker, M.,
A. Kharazmi,
H. Galbo,
I. Bygbjerg,
and
B. K. Pedersen.
Influence of in vivo hypobaric hypoxia on function of lymphocytes, neutrocytes, natural killer cells, and cytokines.
J. Appl. Physiol.
74:
1100-1106,
1993[Abstract].
18.
MacMicking, J.,
Q. W. Xie,
and
C. Nathan.
Nitric oxide and macrophage function.
Annu. Rev. Immunol.
15:
323-350,
1997[Medline].
19.
Marano, M. A.,
Y. Fong,
L. L. Moldawer,
H. Wei,
S. E. Calvano,
K. J. Tracey,
P. S. Barie,
K. Manogue,
A. Cerami,
G. T. Shires,
and
S. F. Lowry.
Serum cachectin/tumor necrosis factor in critically ill patients with burns correlates with infection and mortality.
Surg. Gynecol. Obstet.
170:
32-38,
1990[Medline].
20.
Meldrum, D. R.,
A. Ayala,
and
I. H. Chaudry.
Energetics of defective macrophage antigen presentation following hemorrhage as determined by ultraresolution 31P nuclear magnetic resonance spectrometry: restoration with ATP-MgCl2.
Surgery
112:
150-158,
1992[Medline].
21.
Melillo, G.,
T. Musso,
A. Sica,
L. S. Taylor,
G. W. Cox,
and
L. Varesio.
A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter.
J. Exp. Med.
182:
1683-1693,
1995[Abstract].
22.
Melillo, G.,
L. S. Taylor,
A. Brooks,
G. W. Cox,
and
L. Varesio.
Regulation of inducible nitric oxide synthase expression in IFN-treated murine macrophages cultured under hypoxic conditions.
J. Immunol.
157:
2638-2644,
1996[Abstract].
23.
Migliorini, P.,
G. Corradin,
and
S. B. Corradin.
Macrophage NO2 production as a sensitive and rapid assay for the quantitation of IFN-
.
J. Immunol. Methods
139:
107-114,
1991[Medline].
24.
Muraoka, K.,
K. Shimizu,
X. Sun,
Y. K. Zhang,
T. Tani,
T. Hashimoto,
M. Yagi,
I. Miyazaki,
and
K. Yamamoto.
Hypoxia, but not reoxygenation, induces interleukin-6 gene expression through NF-kappa B activation.
Transplantation
63:
466-470,
1997[Medline].
25.
Salzman, A. L.,
M. J. Menconi,
N. Unno,
R. M. Ezzell,
D. M. Casey,
P. K. Gonzalez,
and
M. P. Fink.
Nitric oxide dilates tight junctions and depletes ATP in cultured Caco-2BBe intestinal epithelial monolayers.
Am. J. Physiol.
268 (Gastrointest. Liver Physiol. 31):
G361-G373,
1995
26.
Samy, T. S. A.,
A. Ayala,
R. A. Catania,
and
I. H. Chaudry.
Trauma-hemorrhage activates signal transduction pathways in mouse splenic T-cells.
Shock
9:
443-450,
1998[Medline].
27.
Schwacha, M. G.,
and
S. D. Somers.
Thermal injury-induced immunosuppression in mice: the role of macrophage-derived reactive nitrogen intermediates.
J. Leukoc. Biol.
63:
51-58,
1998[Abstract].
28.
Shalaby, M. R.,
A. Waage,
and
T. Espevik.
Endotoxin, tumor necrosis factor-alpha and interleukin 1 induce interleukin 6 production in vivo.
Clin. Immunol. Immunopathol.
53:
488-498,
1989[Medline].
29.
Shenker, R.,
and
E. Abraham.
Hemorrhage induces rapid in vivo activation of NF-B in murine intra-parenchymal lung mononuclear cells.
Am. J. Respir. Cell Mol. Biol.
16:
145-152,
1997[Abstract].
30.
Shenkin, A.,
W. D. Fraser,
J. Series,
F. P. Winstanley,
A. C. McCartney,
H. J. Burns,
and
J. VanDamme.
The serum interleukin 6 response to elective surgery.
Lymphokine Res.
8:
123-127,
1989[Medline].
31.
Smail, N.,
R. A. Catania,
P. Wang,
W. G. Cioffi,
K. I. Bland,
and
I. H. Chaudry.
Gut and liver: the organs responsible for increased nitric oxide production after trauma-hemorrhage and resuscitation.
Arch. Surg.
133:
399-405,
1998
32.
Spiecker, M.,
H. Darius,
K. Kaboth,
F. Hubner,
and
J. K. Liao.
Differential regulation of endothelial cell adhesion molecule expression by nitric oxide donors and antioxidants.
J. Leukoc. Biol.
63:
732-739,
1998[Abstract].
33.
Suzuki, Y. J.,
M. Mizuno,
and
L. Packer.
Signal transduction for nuclear factor-B activation: proposed location of antioxidant-inhibitable step.
J. Immunol.
153:
5008-5015,
1994
34.
Szabo, C.,
and
C. Thiemermann.
Role of nitric oxide in hemorrhagic, traumatic, and anaphylactic shock and thermal injury.
Shock
2:
145-155,
1994[Medline].
35.
Takayama, T. K.,
C. Miller,
and
G. Szabo.
Elevated tumor necrosis factor production concomitant to elevated prostaglandin E2 production by trauma patients' monocytes.
Arch. Surg.
125:
29-35,
1990[Abstract].
36.
Waage, A.,
P. Brandtzaeg,
A. Halstensen,
P. Kierulf,
and
T. Espevik.
The complex pattern of cytokines in serum from patients with meningococcal septic shock. Association between interleukin 6, interleukin 1, and fatal outcome.
J. Exp. Med.
169:
333-338,
1989[Abstract].
37.
Wang, P.,
Z. F. Ba,
and
I. H. Chaudry.
Severe hypoxemia in the absence of blood loss depresses hepatocellular function and up-regulates IL-6 and PGE2.
Biochim. Biophys. Acta
1361:
42-48,
1997[Medline].
38.
Wang, P.,
G. Singh,
M. W. Rana,
Z. F. Ba,
and
I. H. Chaudry.
Preheparinization improves organ function after hemorrhage and resuscitation.
Am. J. Physiol.
259 (Regulatory Integrative Comp. Physiol. 28):
R645-R650,
1990
39.
West, M. A.,
and
C. Wilson.
Hypoxic alterations in cellular signal transduction in shock and sepsis.
New Horiz.
4:
168-178,
1996[Medline].
40.
Xie, Q. W.,
Y. Kashiwabara,
and
C. Nathan.
Role of the transcription factor NF-B/Rel in induction of nitric oxide synthase.
J. Biol. Chem.
269:
4705-4708,
1994
41.
Zellweger, R.,
A. Ayala,
C. M. DeMaso,
and
I. H. Chaudry.
Trauma-hemorrhage causes prolonged depression in cellular immunity.
Shock
4:
149-153,
1995[Medline].